Hindawi Publishing CorporationISRN BiochemistryVolume 2013 Article ID 463527 13 pageshttpdxdoiorg1011552013463527

Review ArticleCav12 Cell Proliferation and New Target in Atherosclerosis

Nikolai M Soldatov

Humgenex Inc Kensington MD 20895 USA

Correspondence should be addressed to Nikolai M Soldatov soldatovnhumgenexverizonnet

Received 25 February 2013 Accepted 20 March 2013

Academic Editors I de la Serna and H Yonekawa

Copyright copy 2013 Nikolai M SoldatovThis is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Cav12 calcium channels are the principal proteins involved in electrical mechanical andor signaling functions of the cellCav12 couples membrane depolarization to the transient increase in intracellular Ca2+ concentration that is a trigger for musclecontraction and CREB-dependent transcriptional activation The CACNA1C gene coding for the Cav12 pore-forming 120572

1C subunitis subject to extensive alternative splicingThis review is the first attempt to follow the association between cell proliferation Cav12expression and splice variation and atherosclerosis Based on insights into the association between the atherosclerosis-inducedmolecular remodeling of Cav12 proliferation of vascular smoothmuscle cells and CREB-dependent transcriptional signaling thisreview will give a perspective outlook for the use of the CACNA1C exon skipping as a new potential gene therapy approach toatherosclerosis

1 Introduction

It has been long known that Cav12 calcium channel blockersinhibit human brain tumor [1] pancreatic cancer [2 3]breast cancers [4] and small cell lung cancer [5] becausethey inhibit cell proliferation and DNA synthesis Correla-tion between the oncogenic transformation and expressionof both Cav12 and Cav3 channels was demonstrated inspontaneously immortalized 3T3 fibroblasts [6 7] suggestingthat both types of calcium channels may play a role incell proliferation Indeed studies showed that Cav3 (T-type) calcium channels regulate proliferation for exampleof BC

3H1 cells [8] vascular smooth muscle (VSM) cells [9]

and glioma neuroblastoma and neuroblastoma times gliomahybrid cells [10] Unlike the majority of cells including thelisted ones normal human fibroblasts express only Cav12[11]The pore-forming 120572

1C subunit of this channel was clonedfrom human fibroblasts and identified as a ldquoshortrdquo (exon 1)isoform of the 120572

1C-coding gene CACNA1C [12] A variety ofCav12 calcium channel blockers including dihydropyridines(DHPs) nifedipine and nicardipine as well as diltiazemand verapamil inhibit cell proliferation and DNA synthesisin fibroblasts [13] Thus human fibroblast is an excellentcell type to study the roles of Cav12 in proliferation notcomplicated by expression of other Cav genetic variants celltransformation andor differentiation

2 Cak12 and Proliferation of NormalHuman Fibroblasts

Our earlier studies performed on normal human diploidfibroblasts have revealed a number of important features thatpoint to the plasticity of the Cav12 expression in response tocell culture conditions including cell-cell contact inhibitionpresence ofmitogens and secondmessengersThe expressionof Cav12 in the plasma membrane was measured using theDHP radioligand binding assay DHPs bind to Cav12 withhigh affinity in equimolar ratio and are excellent probesfor the expression of total (functional and dormant) Cav12in the plasma membrane The test system was based onthe measurement of specific binding of 26-dimethyl-3-methoxycarbonyl-5-([23-3H

2]-n-propoxycarbonyl)-4-(21015840-

difluoromethoxyphenyl)-14-dihydropyridine ([3H]PMDNCBI PubChem CID 14267917) [15] to human embryonicdiploid fibroblasts grown in Eaglersquos medium supplementedwith 10 serum Under standard conditions of incubation(1 h at room temperature in Tris-buffered saline) [3H]PMDinteracted with 119870

119889asymp 39 nM with a single class of DHP

receptors that were present at themaximumdensity (119861max) ofasymp12 pmol106 cells in a sparse culture of fibroblasts (asymp3ndash5 times1000cm2) [16] The turnover rate of DHP receptors isapproximately exponential with a half-life of asymp12 h as it was

2 ISRN Biochemistry

10 serum

Serum-free

0 04 08 12 16

Confluent monolayer

Nonconfluent monolayer10 serum 2 (control)

1

3

4

5

6

lowast

lowast

lowast

lowast

lowast

[3H]PMD binding

+cAMP

+Ca2+

+diltiazem

(a)

10 serum (control)

Serum-free 10 serum

05

1

15

0 1 2 3 4 5 6 7Time (d)

[3H

]PM

D b

indi

ng

(b)

Figure 1 Effect of serum and second messengers on the density of DHP receptors in normal human fibroblasts (a) On day 2 after theplating of fibroblasts at high (bar 1) or low density (bars 2ndash6) the growth medium was supplemented with 1mM 8Br-cAMP (bar 3) 1120583MCa2+ ionophore A23187 (bar 4) 1120583M D-cis-diltiazem (bar 5) or growth medium was replaced by serum-free Eaglersquos medium containing01 BSA (bar 6) After 4 days the density of DHP receptors was measured at 2 nM [3H]-PMD as pmol [3H]PMD specifically bound per 106cells of confluent (bar 1 gt2 times 104 cellscm2) or nonconfluent monolayers (bars 2ndash6 3ndash5 times 103 cellscm2) Mean of 5-6 measurements plusmn SEMlowast

119875 lt 005 (b) Effect of serum deprivation on the density of DHP receptors in fibroblasts On day 2 after plating sparse cultures of fibroblastswere incubated for up to 4 days in serum-free Eaglersquos medium supplemented with 01 BSA The horizontal bars indicate when serum-freemedium was replaced by standard growth medium containing 10 serum The time course of changes in the density of DHP binding siteswas measured as in (a) at different time points (not shown for simplicity) and compared with that in cells not subjected to serum-deprivation(blue line)

estimated from the rate of loss of [3H]PMD binding sitesin response to the net inhibition of protein synthesis bycycloheximide With progression to confluent monolayersthe 119870

119889value for [3H]PMD binding did not change but the

119861max value decreased asymp4 fold (Figure 1(a) compare bars 1and 2) suggesting that expression of Cav12 is responsiveto the arrest of fibroblasts proliferation by cell-cell contactinhibition

The involvement of Cav12 in proliferation of humanfibroblasts was further supported by the finding that con-centration of DHP receptors and respectively of Cav12is strongly affected by mitogens and second messengersDemonstrating a remarkable plasticity of the Cav12 expres-sion in normal human fibroblasts (Figure 1(b)) serum depri-vation induced a 2-fold increase in the density of DHP recep-tors that reached its maximum after 3-4 days of cultivationin the absence of serum (compare also bars 2 and 6 inFigure 1(a)) The elevation of the Cav12 expression wasfully reversible and highly sensitive to serum and othermitogens An addition of 10 serum reduced the densityof DHP receptors to the initial level with almost the sametime course (Figure 1(b)) Thus inhibition of cell growthproliferation and DNA synthesis in fibroblasts by serumdeprivation stimulates expression ofCav12with a time coursecomparable with the DHP receptor turnover rate

The response of fibroblasts to serum deprivation byboosting Cav12 expression is essentially identical to thatcaused by the inhibition of Cav12 by 1 120583M diltiazem

(Figure 1(a) compare bars 5 and 6) the calcium channelblocker that does not compete with DHPs for binding withthe channel In fact diltiazem was present in the DHP bind-ing assay medium throughout all experiments to enhancethe affinity of the DHP probe to the channel receptor [17]Thus it is reasonable to suggest that by boosting the Cav12expression the cell recruits more routes for the Ca2+ entrythrough the plasma membrane to overcome the lack of mito-gens in serum deprivation or lack of conducting channelsin the presence of diltiazem both aimed at supporting cellproliferation until cell-cell contact inhibition terminates itand turns the cells into a quiescent state

If this hypothesis is true then the addition of DHP-insensitive routes for Ca2+ entry through the plasma mem-brane should eliminate the need in higher level of Cav12expression This observation exactly has been made whenthe serum-stimulated cells were supplemented with Ca2+ionophore A23187 (Figure 1(a) bar 4) The A23187-inducedCa2+ entry dramatically reduced the cellular expression ofCav12 A similar effect was observed with 8Br-cAMP theplasma membrane-permeable derivative of cAMP showingthat stimulation of the alternative cAMP-dependent cellsignaling pathway may reduce the needs in Cav12 in prolif-eration of fibroblasts

Stimulation of Cav12 expression in fibroblasts arrested inthe quiescent state by serum deprivation strongly dependson cell proliferation The measurement of [14C]thymidineincorporation as an assay for DNA synthesis in cells showed

ISRN Biochemistry 3

Synthesis of DNA ( of control)

bFGF

EGF

Insulin

Serum-free

10 serum

0 50 100 150 200

0 05 1 15 2

Control

[3H]PMD binding

Figure 2 Effect of mitogens on the density of DHP receptors (openbars) and cell proliferation (dark bars) in normal human fibroblastsOn day 2 after cell plating the growth medium was replaced for4 days with serum-free Eaglersquos medium supplemented with 01BSA Cells were incubated for 4 days in the presence of 10 serum(control) 01 BSA (serum-free) epidermal growth factor (EGF15 ngmL) insulin (10 120583gmL) or basic fibroblast growth factor(bFGF 2 ngmL) The density of DHP receptors was measuredat 2 nM [3H]-PMD as pmol [3H]-PMD specifically bound per106 cells of nonconfluent monolayers (3ndash5 times 103 cellscm2) Cellproliferation was estimated from the synthesis of DNAmeasured by[14C]thymidine incorporation

(Figure 2 dark bars) that individual mitogens stimulated cellproliferation in the order bFGF gt insulin gt EGF In contrastin that very order the same mitogens suppressed expressionof Cav12 in fibroblasts as evidenced by the measurement ofDHP receptor binding (open bars) Consistent with relativelylow cell toxicity of dihydropyridines blockade of Cav12attenuated entry of cells into the S phase of cell cycle [18]Thus the plasticity of Cav12 in fibroblast proliferation is asso-ciated with transient Cav12 expression responsive to bothprimary effectors and secondmessengers of cell proliferation

3 Cak12 Variability

One of themost important features of Cav12 is its remarkablemolecular diversityThe channel consists of the pore-forming1205721C protein which binds calcium channel blockers Three

types of regulatory subunits 120573 1205722120575 and calmodulin (CaM)

are constitutively tethered to 1205721C All these subunits are

products of different genes that are located on differentchromosomes Cav12 shares the accessory subunits withother Cav1 and Cav2 channels Moreover 120572

1C 120573 and 1205722120575 aresubject to individual alternative splicing that generates a largediversity of Cav12 channel complexes The functional signif-icance of the Cav12 splice variation is poorly understood not

only because of the difficulties in identification of individualsplice variants in the naturally occurring oligomeric proteinsThe tendency of Cav12 to form large clusters in the plasmamembrane [19ndash23] as well as homo- and heterooligomeriza-tion of 120573 subunits [24] create additional major challenges forthe investigation of Cav12 in regulation of cell proliferationdifferentiation and other functionsmdashnot to speak of anadequate elucidation of their roles in disease and search fornew therapeutic approaches

Molecular cloning showed that 1205721C transcripts in human

fibroblasts are composed of exons 1ndash50 [14] with alternativesplicing of exons 2122 and 3132 and constitutive splicingof exons 33 and 45 (Figure 3(a)) [12] However variabilityassociated with exons 1 7 8 9 34 41 and 42 that were laterfound in human hippocampus heart and VSM cells [25ndash30]was not observed in fibroblasts

It is not known which Cav12 splice variants are expressedin fibroblasts in response to serum deprivation and whetherthey are structurally different from those present in pro-liferating cells However electrophysiological properties ofthe three of fibroblast 120572

1C splice variants (coexpressed with1205731a and 1205722120575-1) were compared in Xenopus oocyte expression

system [31] Characteristics of the voltage-dependence andkinetics of inactivation of the barium current through the1205721C70 channel encoded by the CACNA1C transcript lacking

exons 22 31 41A and 45 were found to be very similar tothose recorded with 120572

1C77 (lacking exons 21 31 41A and45) and 120572

1C76 (also lacking exon 33) (Figure 3(b)) Howevervoltage dependence of the DHP inhibition of the currentwas significantly different in the 120572

1C70 and 1205721C77 channelsthe IC

50values for the concentration dependence of the

barium current inhibition by (+)isradipine almost identical(62 and 73 nM resp) at minus40mV were significantly differentat minus90mV (680 and 79 nM for 120572

1C70 and 1205721C77 resp) Whilesuch a difference in the pharmacological properties of theexon 21- and exon 22-splice variants is deemed unimportantin the case of fibroblasts it may significantly contribute tothe tissue specificity of this major class of calcium channelblockers in cardiac vascular and other responsive cellsaffected by cardiovascular diseases

4 Cak12 in Atherosclerosis

Atherosclerosis is perhaps the single most deadly diseaseleading to about 600000 deaths annually in the USA mostof these due to the progression of the disease to heartattack or stroke [32] In spite of significant efforts themolecular mechanisms of atherosclerosis are not currentlywell understood and effective molecular targets for preven-tion and treatment are not elaborated Atherosclerosis is aninflammatory process in medium and large size arteries thatcauses endothelial perturbation and local release of cytokinesas well as dedifferentiation proliferation and migration ofVSM cells [33] Arterial VSM cells constitute the media of theartery and play a crucial role in its elasticity and contractilityMigration of VSM cells from the media to the intima of thearterial wall and proliferation of intimal smooth muscle cellsare the major early events in the formation of atheroscleroticlesions Recent advances in molecular genetics studies have

4 ISRN Biochemistry

II III IV

2

3

4

5

6

788A

9

10

1125

12 13 14 15

16

17 18

19

26

23

24 28

27

2934

36

2122

Plasmamembrane

Out

In

33

37+

+

38

39

40

HOOC

4443

454647484950

11A3132 41 4241A

9A

34A30

I

LA IQ

AID

65

++++4

++++

3 51 4

++++

3 51 2 4 26

++++

3 51 2 4

2035

6 6 31 2

H2N

(a)

20 2321 22 30 31 32 33 34

20 2321 22 30 31 32 33 34

20 2321 22 30 31 32 33 34

40 41A 41 44 45 46

40 41A 41 44 45 46

40 41A 41 44 45 46

20 2321 22 30 31 32 33 34

20 2321 22 30 31 32 33 34 40 41A 41 44 45 46

40 41A 41 44 45 46

20 2321 22 30 31 32 33 34 40 41A 41 44 45 46

1205721C69

1205721C70

1205721C71

1205721C73

1205721C76

1205721C77

(b)

Figure 3 Molecular diversity of Cav12 (a) Transmembrane topology of the Cav12 1205721C subunit in schematic diagram Four regions ofhomology (IndashIV) each composed of 6 transmembrane segments (numbered) are folded around the central pore The polypeptide sequenceis segmented by black bars and sequentially numbered (1ndash50) according to the CACNA1C genomic map [14] The segments correspondingto the invariant (blackgray) and alternative (blue) exons are outlined 120572-interaction domain (AID) of a constitutive 120573-binding is shown ingreen In the CaM-binding domain (CBD) apo-CaM and Ca2+-CaM are shared between LA and IQmotifs (red) respectively (b) Alternativesplicing of human fibroblast CACNA1C in the region of exons 20ndash46 The following splice variants were studied 120572

1C69 (GenBank z34809splice variant lacking alternative exons (Δ) 21 32 41A and 45) 120572

1C70 (z34810 Δ 22 31 41A and 45) 1205721C71 (z34811 Δ 22 31 and 45) 120572

1C73(z34812 Δ 22 31 33 and 45) 120572

1C76 (z34814 Δ 21 31 33 41A and 45) and 1205721C77 (z34815 Δ 21 31 41A and 45)

revealed that genetic polymorphisms significantly influencesusceptibility to atherosclerotic vascular diseases [34] How-ever none of the discovered susceptibility genes was directlyimplicated for proliferation and migration of VSM cells oneof the major pathophysiological responses to atherosclerosisat the cellular level

The presence and activity of Cav12 calcium channels inVSM cells has been established both in patch clamp andmolecular cloning experiments [29 35ndash38] Cav12 calciumchannels play a major role in atherosclerosis because they

are essential for Ca2+ signal transduction in VSM cellsContraction of VSM cells is triggered by the Ca2+ current(119868Ca) through Cav12 and thus is affected by Ca2+ channelblockers Since the 1990s it is known that DHPs particularlythe charged 2-aminoethoxymethyl DHP derivative amlodip-ine [39] exert a number of vasoprotective effects includingpotent antiatherogenic action [40] inhibition of migrationof VSM cells [41] and reduction of arterial intimal-medialthickening and plaque formation [42 43] Although it istempting to link this activity in part to pleiotropic effects of

ISRN Biochemistry 5

calcium channel blockers [44] it is the inhibition of prolifer-ation that essentially underlies it [45] The density of DHPreceptors was found to depend on VSM cell proliferation[46] Association of Cav12 with mitogenesis in VSM cells issupported by the findings that DHPs reduced DNA synthesisstimulated by serum and PdGF [47ndash49] serotonin [50] EGF[51] and H

2O2(in mesangial cells) [52] Expression of Cav12

in VSM cells was shown to be cell cycle dependent with thehighest calcium current density in G

1phase [53] Whether

these changes are reflected in the molecular repertoire ofthe Cav12 splice variants is the issue particularly importantfor the elucidation of new therapeutic targets in diseasesleading to pathogenic proliferation of VSM cells such asatherosclerosis

Investigation of the 1205721C alternative splicing in VSM

cells [28 54 55] revealed the involvement of a number ofCACNA1C exons generating impressive diversity of humanvascular 120572

1C that includes possibly the VSM-specific splic-ing of exons 99a [35 55] and exon 34 [28] To establishan association between the disease and CACNA1C splicevariation at the level of cell we have completed [26] thesingle-gene profiling of the 120572

1C molecular remodeling inVSM cells of an artery caused by atherosclerosis The VSMcells were identified in frozen sections of six surgical biopsysamples of femoral and carotid arteries by immunostainingwith an antibody against smoothmuscle 120572-actin [56] used asa marker for VSM cells The 120572-actin staining correlated withimmunostaining by an anti-120572

1C antibody in serial sectionsand was reduced in atherosclerotic regions (Figure 4) consis-tent with dedifferentiation of VSM cells [57 58]The reducedexpression of 120572

1C at the protein level was corroborated bythe quantitative RT-PCR data showing that the relative 120572

1CmRNA level in VSM cells (normalized to 18S RNA) wasreduced 37 plusmn 09 fold (mean plusmn SEM) in the atheroscleroticregion Overall the reduced expression of 120572

1C caused by thelocally elaborated cytokines in the atherosclerotic regions ofarteries resembles the reduced expression of DHP receptorsobserved in fibroblasts exposed to mitogens andor secondmessengers after serum deprivation (Figure 1)

To find out whether the altered expression of 1205721C

in atherosclerotic VSM cells is accompanied by changesin the CACNA1C alternative splicing pattern we isolatedthe immunohistochemically identified VSM cells by laser-capture microdissection from adjacent regions of arteriesaffected and not affected by atherosclerosis and identifiedthe CACNA1C splice variants by RT-PCR Our findingsrevealed an extended repertoire of the exon 21 120572

1C spliceisoforms in nonatherosclerotic VSM cells characterized bya complex splicing pattern of exons 9 9A 31ndash34 and41A including the electrophysiologically characterized 120572

1C71(GenBank z34811) 120572

1C73 (z34812) 1205721C125 (AY830711)

1205721C126 (AY830713) and 1205721C127 (AY830712) splice isoforms

However only the exon 22 isoform of 1205721C (1205721C77) was

identified in atherosclerotic VSM cells Thus the switch ofthe CACNA1C alterative splicing from exon 21 to exon 22(Figure 3(b)) is a molecular signature of the Cav12 remod-eling of VSM cells to a pathophysiological proliferating statein atherosclerosis The age gender ethnicity drug exposureand other co-morbid conditions did not appreciably affect

this common pattern of the 1205721C splice variation in VSM cells

in response to atherosclerosisCareful electrophysiological analysis exhibited a number

of differences in the properties of the ldquoatheroscleroticrdquo 1205721C77

channel as compared to the 1205721C isoforms in healthy VSM

cells The largest differences were found between the 1205721C77

and 1205721C127 channels (Figure 5) In response to step depolar-

ization applied from the holding potential of minus90mV bothchannels generate calcium currents (119868Ca) that inactivate withalmost identical kinetics (Figure 5(a)) However we foundthat 119868Ca through the1205721C77 channel recovers from inactivationsignificantly faster than that in 120572

1C127 (Figure 5(b)) and other1205721C isoforms present in healthy VSM cells This finding

suggests that alternative splicing in atherosclerosis may affectvascular tone as a result of the increase in the 119868Ca density inVSM cells A hyperpolarization shift of the activation curvefor the atherosclerotic 120572

1C77 channel variant as comparedto Cav12 in healthy VSM cells (Figure 5(c)) may also resultin an increase of calcium entry in VSM cells Howeverthe overall 3-4-fold reduction in the expression of Cav12in atherosclerotic VSM cells may scale down some of theobserved electrophysiological changes

5 CACNA1C Exon 22 as a New TherapeuticTarget in Atherosclerosis

Direct DNA sequencing of the crude PCR amplificationproducts indicated that the switch to the exon 22 isoform ofvascular 120572

1C was almost complete in atherosclerosis becauseno distortion of the nucleotide peaks in the region of exon2122 was seen when compared to the exon 20 invariantregion (Figure 6(a)) Thus Cav12 underwent almost quanti-tative exon 2122 remodeling in VSM cells of diseased arteryregions

Although the cellular mechanisms leading to theCACNA1C exon 2122 switch may be very complex theassociation with VSM cell proliferation is obvious Indeeda similar exon switch was observed in primary humanaortic cells in culture after the quiescent nonproliferatingcells containing predominantly exon 21 splice variantswere exposed to serum (Figure 6(b)) Unlike exon 21exon 22 contains the AvrII restriction site that allowsfor the assessment of its presence in PCR amplificationproduct of CACNA1C transcripts isolated from the cellsThe AvrII-sensitive exon 22 isoform of the 120572

1C transcriptwas not detected in the quiescent nonproliferating aorticcells (Figure 6(b) lane 2) However when 5 serum wasadded to the medium with nonconfluent aortic cellsDNA biosynthesis was activated while the level of the1205721C transcript decreased asymp3 fold and the presence of the

AvrII-sensitive exon 22 isoforms of 1205721C was easily detected

(Figure 6(b) lane 4) The isoform remodeling simulated inaortic primary cells in vitro was not complete as comparedto VSM cells in atherosclerotic regions of artery occludedwith heavy plaque burden which were selected for the1205721C molecular profiling However the cell culture results

demonstrate that in a different experimental system there isan obvious association between proliferation of VSM cells

6 ISRN Biochemistry

A Ki-67 NKi-67

50 12058350 120583

(a)

A N120572-actin 120572-actin

(b)

A N1205721C1205721C

(c)

Figure 4 Reduced expression of 1205721C in atherosclerotic (A) region of aorta as compared to the adjacent nonaffected (N) region From (a) to

(c) detection of proliferating cells in an exemplar biopsy of an artery by an antibody against human nuclear protein Ki-67 (a) Red arrowshighlight some proliferating nuclei (black staining) while blue arrows exemplify nonproliferating nuclei (light grey) Staining with antibodiesagainst smooth muscle 120572-actin (b) and against 120572

1C (c) Scale bars 50 120583m

downregulation of the CACNA1C expression and synthesisof the exon 22 120572

1C isoformRecent strategies targeting VSM cells to treat cardiovas-

cular diseases suggest indiscriminate disruption of Cav12[59ndash61] Is it possible to correct the described CACNA1Csplice defects induced by atherosclerosis without affectingthe transcripts of the gene lacking the rdquopathogenicrdquo exon22 Correction of defective genes responsible for diseasedevelopment is achieved by gene therapy Usually it requiresan insertion into the genome of a normal gene in place ofa defective one causing disease Such technique howeveris poorly controlled Currently one of the most promising

cutting-edge therapeutic approaches to correct defects asso-ciated with disease-induced expression of abnormal splicevariants is antisense-mediated exon skipping It is basedon the use of antisense oligonucleotides targeting specificexons to be removed The adenovirus-directed 120572

1C exon 22skipping-induced inhibition of VSM cell proliferation (andrespectively migration) can be used to rescue VSM cells fromremodeling in atherosclerosis The 120572

1C exon 22-skippingwill not alter the open reading frame of the 120572

1C transcriptbecause alternative exons 21 and 22 are of the equal size(60 nt)Themodified nonspliceosomal snRNAU7 gene alongwith its natural promoter and 31015840 elements exon 22-antisense

ISRN Biochemistry 7

200 ms

1205721C77

1205721C127

(a)

Prepulse interval (s)02 04 06 08 1

0

1

08

06

04

02

0

Frac

tion

of119868 C

a

1205721C77

1205721C127

(b)

0

1

08

06

04

02

119866Ca

119866Ca

max

1205721C77

1205721C127

minus60 minus40 minus20 0 20 40(mV)

(c)

Figure 5 Comparison of electrophysiological properties of 119868Ca through the ldquoatheroscleroticrdquo 1205721C77 and ldquonormalrdquo 120572

1C127 channels expressedin Xenopus oocytes with 120572

2120575-1 subunit and the primary cardiac 1205732a subunit and measured with 25mM Ca2+ as the charge carrier (a)

Representative traces of 119868Ca evoked by 1 s step depolarizations to +20mV from 119881ℎ= minus90mV and normalized to the same amplitude (b)

Fractional recovery of 119868Ca from inactivation (c) Averaged activation (119866119866max-119881) curves fit by the Boltzmann function A 1 s test pulses in therange of minus40 to +50mV (10-mV increments) were applied from 119881

ℎ= minus90mV with 30-s intervals

(a)

AvrII

Serum

1 2 3 4

+minus+minus

minus minus + +

142 pb

459 pb

601 pb

(b)

Figure 6 (a) Trace diagrams of DNA sequencing of the 1205721C PCR amplification products of healthy (top row) and diseased VSM cells (bottom

row) using an antisense primer composed of nucleotides 2923ndash2939 (z34815) A boundary with the sequence of invariant exon 20 is markedby a vertical dotted line (b) Evidence that the AvrII-sensitive exon 22 isoform of 120572

1C is expressed only in proliferating VSM cells Primaryaortic cells were grown to confluent monolayer in 5 serum before serum-deprivation for 5 days (lanes 1 and 2)Then the cells were replatedat low density in 5 serum for 3-4 days (lanes 3 and 4) Total RNA was isolated and exon 2122 isoforms were identified by RT-PCR andAvrII restriction analysis (lanes 2 and 4)

8 ISRN Biochemistry

sequence and supplemented with Sm ribonucleoprotein-binding sequence may be incorporated into the adenovirusvector for high efficiency transfer [62]The cytokine receptors(eg PdGF-120573 receptor) based recognition targeting of viralliposomes or nanoparticles may be especially advantageousin connection with selective gene transfer to VSM cellsaffected by atherosclerosis while reducing the probability ofthe transfection of other cells

6 Cak12 and CREB-DependentTranscriptional Activation

How Cav12 activity is translated into a proliferation-effectedmodality is another important question to be asked Cav12calcium channels generate a transient rise in cytosolic Ca2+-concentration activated by membrane depolarization Cel-lular responses associated with the rise of [Ca2+]i rangefrom sarcomeric contraction to cell growth and proliferationCytoplasmic domains of Cav12 have evolved a fairly intricateCaM-dependent signaling mechanism that provides for thenegative feedback inhibition of the calcium current knownas Ca2+ dependent inactivation (CDI) which is mediated bydifferent determinants of 120572

1C [25 63] Such a mechanism ofCDI resulting in acceleration of 119868Ca inactivation in responseto the rise of intracellular Ca2+ was first identified in cardiacCav12 [64] Similar experiments performed on the recombi-nant Cav12 also showed that the replacement of extracellularCa2+ by Ba2+ eliminates CDI and the channel inactivates by aslower voltage-dependent mechanism [65]The very fact thattwo distantly located determinants one in the pore regionresponsible for slow inactivation and the CaM-binding onein the proximal locus of the C-tail are independently crucialfor CDI indicates that not only their specific molecularstructure but also their mutual folding andor interaction areessential Experimental evidence show that this interactionreacts dynamically to membrane voltage supporting state-dependent transitions of the channel between resting openand inactivated conformations which are essential for CREB-dependent transcriptional activation [66 67]

CREB is a transcription factor of general importance ina large variety of cells CREB phosphorylation promotes theactivation of genes and is regulated by protein kinases undercontrol of the major second messengers cAMP andor Ca2+Indeed CREB functions as a ldquomolecular determinant of VSMcells fate [68]rdquo CREB content depends on proliferation ofVSM cells both in situ and in culture Serum deprivationincreased CREB content in VSM cells while exposure toPdGF decreased it Consistent with this observation anoverexpression of the constitutively active CREB inVSM cellsarrested cell cycle progression

To investigate the association of Cav12 with CREB tran-scriptional activation at the molecular level we combinedpatch clamp with fluorescent resonance energy transfer(FRET) microscopy in the live cell In voltage clampedcells FRET provided optical measurements under state-dependent conditions showing that the shorter N-terminaltail of 120572

1C (eg 1205721C77) does not rearrange vis-a-vis the

plasma membrane in response to voltage gating [25] In

sharp contrast the C-tail shows voltage-dependent confor-mational rearrangements which are much larger in sizethan that for example in the potassium Kv21 channel[69] Measurements of 119868Ca and corrected FRET betweenthe enhanced yellow (EYFP) and cyan fluorescent proteins(ECFP) genetically attached respectively to the N- and C-termini of 120572

1C77 showed no significant effect on voltage-dependence and kinetics of the channel current Howevertherewas a substantial increase in FRET signal accompanyinginactivation of the channel that was fully reversible uponits transition into the resting state in response to hyper-polarization [66] The plasma-membrane anchoring of the1205721C C-tail by the fusion of the pleckstrin homology domain

(PH) eliminated CDI but not 119868Ca Do the voltage-gatedconformational rearrangements of the 120572

1C C-tail and CDIplay a role in Ca2+ signal transduction that is utilized inCa2+-induced activation or CREB-dependent transcriptionTo answer this question we used the test system based onthe measurement of interaction between KID domain ofCREB and KIX domain of coactivator CREB-binding protein(CBP Figure 7(A) inset) under voltage-clamp conditionsby monitoring FRET between EYFP-KID and ECFP-KIX[70] Ca2+-dependent phosphorylation of KID stimulates itsbinding to KIX bringing EYFP and ECFP close enoughto observe the interaction by FRET In perforated wholeclamped cell where the integrity of the cytoplasmic contentis intact (Figure 7(A)) no activation of CREB transcription(Figure 7(B) panel (a)) and rearrangement of the120572

1C77 C-tail(panel (b)) was observed when the C-tail was anchored to theplasma membrane CREB transcriptional activity remainedlow in spite of a large sustained inward 119868Ca (panel (d))and the corresponding increase in [Ca2+]i detected by thefluorescence of Ca2+ indicator Fura4 (panel (c)) Releaseof the 120572

1C77 C-tail by the activation of PIP2hydrolysis

upon activation of M1AchR (Figure 7(C)) at minus90mV causedsignificant elevation of [Ca2+]i that also was not utilizedby the cell for CREB transcriptional activation (Figure 7(C)panel (a)) until a depolarizing pulse to +20-mV was appliedand the released 120572

1C77 C-tail was permitted to rearrange(Figure 7(D))This experiment provides compelling evidencethat neither large inward 119868Ca nor the subsequent rise in[Ca2+]i lead to CREB transcription activation unless theconformational rearrangement of the 120572

1C77 subunit C-tailprovides the precise targeting of the Ca2+ signal transduction(Figure 7(D) scheme) [66]

There is general agreement that CaM binds to LA and IQdomains of the 120572

1C C-tail and acts as a sensor that conveysCDI (for review see [63]) The affinity of CaM for bothdomains depends on [Ca2+]i Our data indicate that CDI andCa2+-signal transduction depend on the voltage-gatedmobil-ity of the120572

1C77 C-tail It is therefore reasonable to suggest thatthe LA-domain is a Ca2+-sensitive apo-CaM-mediated lockfor the mechanism of slow voltage-dependent inactivationof the channel [67] Apo-CaM associated with LA is able tocross-link it to another still unidentified apo-CaM bindingsite in the polypeptide bundle underlying the pore As theresult of this specific localization apo-CaMLA ldquolockrdquo ishidden from the cytoplasmic Ca2+ so that for example the

ISRN Biochemistry 9

10

0

2

10

100 pA125 ms

(a) (a) (a)

(b)(c)

(b)(c)

(b)(c)

(B) (C) (D)

C-tail Anchored ReleasedReleased

0 10No FRET No FRET

(d) (d) (d)

Fluo-4

PH

CYN

C

C

NY

C

Y EYFP

C ECFP

PH Plekstrin homologydomain

PHPHY

C

NC

IQ

LA

LAIQ

(A)

Fluo-4 Fluo-4

FRET FRET FRET

CRE

Gene expressionCREBCREB P

CBP

AC

GPCR

PKA

cAMP

CaM

Nucleus

cAMP

Plasmamembrane

Ca2+ Ca2+

Ca2+

+20mV +20mV

minus90mV minus90mV

1205721C77

1198651198650

Cav 12

Ca2+

minus90mVACh (5 mM)

4 120583

Figure 7 Evidence that Ca2+ signal transduction by Cav12 to activate CREB-dependent transcription is mediated by the voltage-gatedmobility of the 120572

1C C-tail carrying the CaM-caged Ca2+ and is not directly associated with the increase of [Ca2+]i CREB activation wasexamined under perforated patch conditions in COS1 cells expressing recombinant atherosclerotic 120572

1C77 channel (A) Phase-contrast imageof the COS1 cell with a shadow of patch pipette The cell was expressing the 120572

1C77-PHC1205731a1205722120575-1 channel with the membrane-trapped 1205721C77

subunit C-tail type 1muscarinic Ach receptor (to release the anchored C-tail in response to activation by Ach) and EYFP-KID and ECFP-KIXdomains both supplemented with nuclear localization sequences Inset at the bottom Cav12- and cAMP-dependent cell signaling pathwaysmediating CREB-dependent transcription CBP CREB-binding protein CRE cAMP-response element (B) Plasma-membrane anchoring ofthe 1205721C77 C-tail (see schematic diagram) inhibits CREB activation in spite of robust 119868Ca evoked by depolarization (a) A 100-ms images of

FRET between EYFP-KID and ECFP-KIX were recorded at the end of the 12th +20-mV depolarization step applied every 10 s for 1 s from119881ℎ=

minus90mV (b) A corresponding representative image of corrected FRET ratio (in a ldquo3Drdquo MetaMorph transformation) recorded within 50-mswindows at minus90 and at the end of the last test pulse of the applied train of pulses (c) An increase of [Ca2+]i detected by Ca

2+ indicator Fluo4(d) Representative trace of 119868Ca (20mM Ca2+ in bath medium) evoked by depolarization to +20mV from119881

ℎ= minus90mV showing the sustained

component of Ca2+ conductance due to the C-tail anchoring (C) ACh-stimulation of theM1AChR to unbind the 1205721C C-tail caused activation

of IP3-dependent Ca2+ release (panel (c)) but did not show substantial activation of CREB transcription at 119881

ℎ= minus90mV (panel (a)) (D)

CREB-dependent transcription was activated only in response to depolarization applied to membrane channel with the 1205721C77 C-terminal tail

released from the plasma membrane to assume functional voltage-dependent conformation

intracellular Ca2+ released from the intracellular stores orCa2+ caging compounds does not accelerate significantly theinactivation of Cav12 [71] Thus apo-CaM associated withLA binds predominantly Ca2+ ions permeating through thepore A Ca2+-dependent transfer of CaM from LA to the IQ-motif opens the ldquolockrdquo and initiates a large rearrangement ofthe C-terminal tail This in turn facilitates inactivation of thechannel The Ca2+CaM complex with the IQ-motif is thentransferred by themobile C-tail to a downstream target of theCa2+-signaling cascade (such as CaMKII [72]) where Ca2+ isreleased as an activating stimulus while CaM switches back

to LA and returns the C-tail to the resting position availablefor the next cycle of Ca2+-signal transduction

Activation of CREB-dependent transcription by the L-type 119868Ca ismediated throughmultiple cell signaling pathwaysUsing FRET probes of CREB activity and 2D wavelet trans-form analysis we applied principles of quantitative biologyto detail the mechanism of Ca2+-activated CREB-dependenttranscription within localized regions (microdomains) ofthe nucleus We reached this goal by applying continuouswavelet analysis in two dimensions with a 2D wavelet asa deconvolution algorithm for FRET microscopy image

10 ISRN Biochemistry

Control

(A)

05

0

1

05

00 20 40 60 800 20 40 60 800 20 40 60 80

(a) (b) (c) (d)

20 40 60 800

0

20

40

60

80 1

C 3 5 10 mincAMP

C 2 10 mincAMP

C 3 10 min

cAMP

C 1 3 10 min

cAMP

5

cAMP stable (5)

(B)

119868Ca (3 min) cAMP (5 min) cAMP (10 min)

119868Ca cAMP (65) 119868Ca stable (15)

119868Ca119868Ca

119868Ca 119868Ca

119868Ca cAMP transient (15)

Figure 8 Microdomain analysis on the additive effect of 119868Ca and cAMP on CREB signaling in COS1 cells expressing the recombinantldquoatheroscleroticrdquo120572

1C77 channel (A) FRET signal within the nucleus during selected time points during 119868Ca stimulation and cAMP applicationOutlined are four types of signaling microdomains identified using 2D Mexican hat wavelet Red circles represent stable microdomains thatpersist through both 119868Ca and cAMP application White and yellow circles showmicrodomains stable activated by cAMP and 119868Ca respectivelyGreen circles represent transient microdomains of CREB signaling activation Axes show pixel numbers (B) Typical appearances of the fourtypes of CREB signaling microdomains activated by 119868Ca andor cAMP application and recorded in their maximal development in relationto the time of applied stimuli C control recorded before stimulation Color bars in (A) and (B) represent FRET values normalized to themaximum

analysis [73] Continuous wavelet analysis is a mathematicaltechnique that allows us to analyze a signal over severaldifferent frequencies across the entire signal [73 74] It isespecially useful for finding heterogeneity in a signal becauseit can easily find where the pattern (ie frequency) of asignal changes In these experiments we for the first timeobtained evidence of CREB signaling microdomains withinthe nucleus that respond differentially to 119868Ca stimulationand cAMP (Figure 8(A)) Results of the study revealed thatCREB-dependent transcriptional signaling occurs in dis-crete signaling microdomains underlying the architecture ofnuclear signaling Continuous activation ofCREB-dependenttranscriptional signaling by cAMP and Ca2+ resulted in agradual increase of the number of microdomains Four dif-ferent categories of cAMP and Ca2+-induced CREB signalingmicrodomains were characterized in COS1 cells expressingrecombinant Cav12 with the ldquoatheroscleroticrdquo 120572

1C77 splice

variant (Figure 8) In up to 65 of the microdomainstranscription was activated in additive manner by cAMPand Ca2+ Approximately 15 of signaling domains wereactivated only by 119868Ca and 5 of domains were activatedonly by cAMP Finally 15 of the domains were transientand activated by both cAMP and 119868Ca (Figure 8(B)) [75]A similar spatiotemporal organization of CREB-dependentsignaling was observed in spontaneously beating neonatalrat cardiomyocytes Although COS1 cells that were used inour experiments shown in Figures 7 and 8 are naturallydeprived of Cav12 [76] they inherited the ability to replicatethe Cav12-dependent activation of CREB signaling with theexogenous recombinant channel Thus this experimentalapproach fits the task which in my opinion is the mostimportant unresolved issue for the coupling of Cav12 toCREB signaling does the splice variation of 120572

1C affect thespatiotemporal organization of CREB-dependent signaling

ISRN Biochemistry 11

in a way that may affect cell proliferation and other crucialfunction

7 Conclusions

Association of Cav12 with regulation of transcription cellproliferation and its pathophysiology as in the case ofatherosclerosis requires detailed investigation of the roles ofthe naturally occurring 120572

1C splice variants It will limit thetraditionally intuitive approach to Cav12 in physiology andhelp to define new principle approaches to the treatment ofvarious Cav12 channelopathy-related dysfunctions above allcardiovascular diseases Humgenex Inc provides consultingand logistic support on a broad range of issues reported inthis review

Abbreviations

CaM CalmodulinCBD Calmodulin-binding domainCDI Ca2+-dependent inactivationDHP DihydropyridineECFP Enhanced cyan fluorescent proteinEYFP Enhanced yellow fluorescent proteinFALI Fluorophore-assisted light inactivationFRET Fluorescent resonance energy transfer[3H]PMD 26-Dimethyl-3-methoxycarbonyl-5-([23-

3H2]-n-propoxycarbonyl)-4-(21015840-

difluoromethoxyphenyl)-14-dihydropyridine

119868Ca Calcium currentPH Pleckstrin homology domainVSM Vascular smooth muscle

References

[1] Y S Lee M M Sayeed and R D Wurster ldquoInhibition ofcell growth and intracellular Ca2+ mobilization in humanbrain tumor cells by Ca2+ channel antagonistsrdquo Molecular andChemical Neuropathology vol 22 no 2 pp 81ndash95 1994

[2] K Sato J Ishizuka C W Cooper et al ldquoInhibitory effect ofcalcium channel blockers on growth of pancreatic cancer cellsrdquoPancreas vol 9 no 2 pp 193ndash202 1994

[3] V Bertrand M J Bastie N Vaysse and L Pradayrol ldquoInhibi-tion of gastrin-induced proliferation of AR4-2J cells by calciumchannel antagonistsrdquo International Journal of Cancer vol 56no 3 pp 427ndash432 1994

[4] JM Taylor andRU Simpson ldquoInhibition of cancer cell growthby calcium channel antagonists in the athymic mouserdquo CancerResearch vol 52 no 9 pp 2413ndash2418 1992

[5] M G Cattaneo M Gullo and L M Vicentini ldquoC2+ and Ca2+channel antagonists in the control of human small cell lung car-cinoma cell proliferationrdquo European Journal of Pharmacologyvol 247 no 3 pp 325ndash331 1993

[6] C Chen M J Corbley T M Roberts and P Hess ldquoVoltage-sensitive calcium channels in normal and transformed 3T3fibroblastsrdquo Science vol 239 no 4843 pp 1024ndash1026 1988

[7] P C Dartsch M Ritter M Gschwentner H J Lang andF Lang ldquoEffects of calcium channel blockers on NIH 3T3

fibroblasts expressing the Ha-ras oncogenerdquo European Journalof Cell Biology vol 67 no 4 pp 372ndash378 1995

[8] M Biel R Hullin S Freundner et al ldquoTissue-specific expres-sion of high-voltage-activated dihydropyridine-sensitive L-typecalcium channelsrdquo European Journal of Biochemistry vol 200no 1 pp 81ndash88 1991

[9] DM Rodman K Reese J Harral et al ldquoLow-voltage-activated(T-type) calcium channels control proliferation of human pul-monary artery myocytesrdquo Circulation Research vol 96 no 8pp 864ndash872 2005

[10] A Panner L L Cribbs G M Zainelli T C Origitano S Singhand R DWurster ldquoVariation of T-type calcium channel proteinexpression affects cell division of cultured tumor cellsrdquo CellCalcium vol 37 no 2 pp 105ndash119 2005

[11] C Chen and P Hess ldquoCalcium channels in mouse 3T3 andhuman fibroblastsrdquo Biophysical Journal vol 51 p 226a 1987

[12] N M Soldatov ldquoMolecular diversity of L-type Ca2+ channeltranscripts in human fibroblastsrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 89 no10 pp 4628ndash4632 1992

[13] A Fujii H Matsumoto S Nakao H Teshigawara and YAkimoto ldquoEffect of calcium-channel blockers on cell prolifera-tion DNA synthesis and collagen synthesis of cultured gingivalfibroblasts derived fromhumannifedipine responders and non-respondersrdquo Archives of Oral Biology vol 39 no 2 pp 99ndash1041994

[14] N M Soldatov ldquoGenomic structure of human L-type Ca2+channelrdquo Genomics vol 22 no 1 pp 77ndash87 1994

[15] V P Shevchenko N F Myasoedov N M Soldatov G JDuburs V V Kastron and I P Skrastins ldquoSynthesis andevaluation of biological activity of two novel tritium-labeledderivatives of riodipinerdquo Journal of Labelled Compounds andRadiopharmaceuticals vol 27 no 6 pp 721ndash730 1989

[16] S M Dudkin S N Gnedoj N N Chernyuk and N MSoldatov ldquo14-dihydropyridine receptor associated with Ca2+channels in human embryonic fibroblastsrdquo FEBS Letters vol233 no 2 pp 352ndash354 1988

[17] A DePover M A Matlib S W Lee et al ldquoSpecific bindingof [3H]nitrendipine to membranes from coronary arteriesand heart in relation to pharmacological effects Paradoxicalstimulation by diltiazemrdquo Biochemical and Biophysical ResearchCommunications vol 108 no 1 pp 110ndash117 1982

[18] N M Soldatov S N Gnedoj N N Chernyuk I A Britanovaand S M Dudkin ldquoMitogenesis and 14-dihydropyridinereceptor associated with Ca2+-channels in human embryonicfibroblastsrdquo Journal of Membrane Biology vol 5 pp 1161ndash11671988 (Russian)

[19] D Lipscombe D V Madison M Poenie H Reuter R Y Tsienand R W Tsien ldquoSpatial distribution of calcium channels andcytosolic calcium transients in growth cones and cell bodies ofsympathetic neuronsrdquo Proceedings of the National Academy ofSciences of the United States of America vol 85 no 7 pp 2398ndash2402 1988

[20] G S Harms L Cognet P H M Lommerse et al ldquoSingle-molecule imaging of L-type CA2+ channels in live cellsrdquoBiophysical Journal vol 81 no 5 pp 2639ndash2646 2001

[21] V di Biase G J Obermair Z Szabo et al ldquoStable membraneexpression of postsynaptic Cav12 calcium channel clustersis independent of interactions with AKAP79150 and PDZproteinsrdquo Journal of Neuroscience vol 28 no 51 pp 13845ndash13855 2008

12 ISRN Biochemistry

[22] M F Navedo E P Cheng C Yuan et al ldquoIncreased coupledgating of L-type Ca2+ channels during hypertension and timo-thy syndromerdquoCirculationResearch vol 106 no 4 pp 748ndash7562010

[23] E Kobrinsky P Abrahimi S Q Duong et al ldquoEffect of Cav120573subunits on structural organization of Cav12 calcium channelsrdquoPLoS One vol 4 no 5 Article ID e5587 2009

[24] Q Z Lao E Kobrinsky Z Liu and N M Soldatov ldquoOligomer-ization of Cav120573 subunits is an essential correlate of Ca

2+ channelactivityrdquoThe FASEB Journal vol 24 no 12 pp 5013ndash5023 2010

[25] E Kobrinsky S Tiwari V A Maltsev et al ldquoDifferential roleof the 1205721C subunit tails in regulation of the Cav12 channelby membrane potential 120573 subunits and Ca2+ ionsrdquo Journal ofBiological Chemistry vol 280 no 13 pp 12474ndash12485 2005

[26] S Tiwari Y Zhang J Heller D R Abernethy and N MSoldatov ldquoArtherosclerosis-related molecular alteration of thehuman Cav12 calcium channel 1205721C subunitrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 103 no 45 pp 17024ndash17029 2006

[27] I Splawski K W Timothy L M Sharpe et al ldquoCav12 calciumchannel dysfunction causes a multisystem disorder includingarrhythmia and autismrdquo Cell vol 119 no 1 pp 19ndash31 2004

[28] Z Z TangM C Liang S Lu et al ldquoTranscript scanning revealsnovel and extensive splice variations in human L-type voltage-gated calcium channel Cav12 1205721 subunitrdquo Journal of BiologicalChemistry vol 279 no 43 pp 44335ndash44343 2004

[29] P Liao T F Yong M C Liang D T Yue and T W SoongldquoSplicing for alternative structures of Cav12 Ca2+ channels incardiac and smooth musclesrdquo Cardiovascular Research vol 68no 2 pp 197ndash203 2005

[30] Y Blumenstein N Kanevsky G Sahar R Barzilai T Ivaninaand N Dascal ldquoA novel long N-terminal isoform of human L-type Ca2+ channel is up-regulated by protein kinase Crdquo Journalof Biological Chemistry vol 277 no 5 pp 3419ndash3423 2002

[31] N M Soldatov A Bouron and H Reuter ldquoDifferent voltage-dependent inhibition by dihydropyridines of human Ca2+channel splice variantsrdquo Journal of Biological Chemistry vol270 no 18 pp 10540ndash10543 1995

[32] D P Faxon M A Creager S C Smith et al ldquoAtheroscleroticvascular disease conference executive summary atheroscle-rotic vascular disease conference proceeding for healthcareprofessionals from a special writing group of the AmericanHeart Associationrdquo Circulation vol 109 no 21 pp 2595ndash26042004

[33] R Ross ldquoAtherosclerosismdashan inflammatory diseaserdquo The NewEngland Journal of Medicine vol 340 no 2 pp 115ndash126 1999

[34] H Roy S Bhardwaj and S Yla-Herttuala ldquoMolecular geneticsof atherosclerosisrdquo Human Genetics vol 125 no 5-6 pp 467ndash491 2009

[35] M Biel P Ruth E Bosse et al ldquoPrimary structure and func-tional expression of a high voltage activated calcium channelfrom rabbit lungrdquo FEBS Letters vol 269 no 2 pp 409ndash4121990

[36] X Cheng J Liu M Asuncion-Chin et al ldquoA novel Cav12N terminus expressed in smooth muscle cells of resistancesize arteries modifies channel regulation by auxiliary subunitsrdquoJournal of Biological Chemistry vol 282 no 40 pp 29211ndash292212007

[37] W J Koch P T Ellinor and A Schwartz ldquocDNA cloning ofa dihydropyridine-sensitive calcium channel from rat aortaevidence for the existence of alternatively spliced formsrdquo Journalof Biological Chemistry vol 265 no 29 pp 17786ndash17791 1990

[38] S Richard D Neveu G Carnac P Bodin P Travo and J Nar-geot ldquoDifferential expression of voltage-gated Ca2+-currents incultivated aortic myocytesrdquo Biochimica et Biophysica Acta vol1160 no 1 pp 95ndash104 1992

[39] A Schwartz ldquoCalcium antagonists review and perspective onmechanism of actionrdquo American Journal of Cardiology vol 64no 17 pp 3Indash9I 1989

[40] R P Mason ldquoMechanisms of plaque stabilization for thedihydropyridine calcium channel blocker amlodipine review ofthe evidencerdquo Atherosclerosis vol 165 no 2 pp 191ndash199 2002

[41] A Ruiz-Torres R Lozano J Melon and R Carraro ldquoL-calciumchannel blockade induced by diltiazem inhibits proliferationmigration and F-actin membrane rearrangements in humanvascular smooth muscle cells stimulated with insulin and IGF-1rdquo International Journal of Clinical Pharmacology andTherapeu-tics vol 41 no 9 pp 386ndash391 2003

[42] H Koshiyama S Tanaka and J Minamikawa ldquoEffect ofcalcium channel blocker amlodipine on the intimal-medialthickness of carotid arterial wall in type 2 diabetesrdquo Journal ofCardiovascular Pharmacology vol 33 no 6 pp 894ndash896 1999

[43] T Yamashita S KawashimaMOzaki et al ldquoA calcium channelblocker benidipine inhibits intimal thickening in the carotidartery of mice by increasing nitric oxide productionrdquo Journal ofHypertension vol 19 no 3 pp 451ndash458 2001

[44] R P Mason P Marche and T H Hintze ldquoNovel vascularbiology of third-generation L-type calcium channel antagonistsancillary actions of amlodipinerdquo Arteriosclerosis Thrombosisand Vascular Biology vol 23 no 12 pp 2155ndash2163 2003

[45] O Stepien J GogusevD L Zhu et al ldquoAmlodipine inhibition ofserum- thrombin- or fibroblast growth factor- induced vascu-lar smooth-muscle cell proliferationrdquo Journal of CardiovascularPharmacology vol 31 no 5 pp 786ndash793 1998

[46] V Ruiz-Velasco M B Mayer E W Inscho and L J HymelldquoModulation of dihydropyridine receptors in vascular smoothmuscle cells by membrane potential and cell proliferationrdquoEuropean Journal of Pharmacology vol 268 no 3 pp 311ndash3181994

[47] J Nilsson M Sjolund L Palmberg A M von Euler B Jonzonand J Thyberg ldquoThe calcium antagonist nifedipine inhibitsarterial smooth muscle cell proliferationrdquo Atherosclerosis vol58 no 1ndash3 pp 109ndash122 1985

[48] J Thyberg and L Palmberg ldquoThe calcium antagonist nisoldip-ine and the calmodulin antagonist W-7 synergistically inhibitinitiation of DNA synthesis in cultured arterial smooth musclecellsrdquo Biology of the Cell vol 60 no 2 pp 125ndash132 1987

[49] E Munro M Patel P Chan et al ldquoEffect of calcium channelblockers on the growth of human vascular smooth musclecells derived from saphenous vein and vascular graft stenosesrdquoJournal of Cardiovascular Pharmacology vol 23 no 5 pp 779ndash784 1994

[50] T A Kent A Jazayeri and J M Simard ldquoCalcium channelsand nifedipine inhibition of serotonin-induced [3H]thymidineincorporation in cultured cerebral smoothmuscle cellsrdquo Journalof Cerebral Blood Flow and Metabolism vol 12 no 1 pp 139ndash146 1992

[51] A Agrotis P J Little J Saltis and A Bobik ldquoDihydropyridineCa2+ channel antagonists inhibit the salvage pathway for DNAsynthesis in human vascular smooth muscle cellsrdquo EuropeanJournal of Pharmacology vol 244 no 3 pp 269ndash275 1993

[52] I Duque M R Puyol P Ruiz M Gonzalez-Rubio M LD Marques and D R Puyol ldquoCalcium channel blockers

ISRN Biochemistry 13

inhibit hydrogen peroxide-induced proliferation of culturedrat mesangial cellsrdquo Journal of Pharmacology and ExperimentalTherapeutics vol 267 no 2 pp 612ndash616 1993

[53] T Kuga S Kobayashi Y Hirakawa H Kanaide and ATakeshita ldquoCell cycle-dependent expression of L- and T-typeCa2+ currents in rat aortic smooth muscle cells in primaryculturerdquo Circulation Research vol 79 no 1 pp 14ndash19 1996

[54] E M Graf M Bock J F Heubach et al ldquoTissue distribution ofa humanCav12 1205721 subunit splice variant with a 75 bp insertionrdquoCell Calcium vol 38 no 1 pp 11ndash21 2005

[55] P Liao D Yu S Lu et al ldquoSmoothmuscle-selective alternativelyspliced exon generates functional variation in Cav12 calciumchannelsrdquo Journal of Biological Chemistry vol 279 no 48 pp50329ndash50335 2004

[56] O Skalli P Ropraz and A Trzeciak ldquoA monoclonal antibodyagainst 120572-smooth muscle actin a new probe for smooth muscledifferentiationrdquo Journal of Cell Biology vol 103 no 6 pp 2787ndash2796 1986

[57] T M Doherty K Asotra L A Fitzpatrick et al ldquoCalcificationin atherosclerosis bone biology and chronic inflammation atthe arterial crossroadsrdquo Proceedings of the National Academyof Sciences of the United States of America vol 100 no 20 pp11201ndash11206 2003

[58] G K Owens ldquoRegulation of differentiation of vascular smoothmuscle cellsrdquo Physiological Reviews vol 75 no 3 pp 487ndash5171995

[59] J P Bannister A Adebiyi G Zhao et al ldquoSmooth musclecell 1205722120575-1 subunits are essential for vasoregulation by Cav12channelsrdquo Circulation Research vol 105 no 10 pp 948ndash9552009

[60] S SonkusareM Fraer J DMarsh andN J Rusch ldquoDisruptingcalcium channel expression to lower blood pressure newtargeting of awell-known channelrdquoMolecular Interventions vol6 no 6 pp 304ndash310 2006

[61] S Telemaque S Sonkusare TGrain et al ldquoDesign ofmutant1205732subunits as decoy molecules to reduce the expression of func-tional Ca2+ channels in cardiac cellsrdquo Journal of Pharmacologyand Experimental Therapeutics vol 325 no 1 pp 37ndash46 2008

[62] A Goyenvalle A Vulin F Fougerousse et al ldquoRescue of dys-trophic muscle through U7 snRNA-mediated exon skippingrdquoScience vol 306 no 5702 pp 1796ndash1799 2004

[63] D BHalling P Aracena-Parks and S L Hamilton ldquoRegulationof voltage-gated Ca2+ channels by calmodulinrdquo Sciencersquos STKEvol 2005 no 315 p re15 2005

[64] K S Lee E Marban and R W Tsien ldquoInactivation of calciumchannels in mammalian heart cells joint dependence on mem-brane potential and intracellular calciumrdquo Journal of Physiologyvol 364 pp 395ndash411 1985

[65] C Shi and N M Soldatov ldquoMolecular determinants of voltage-dependent slow inactivation of the Ca2+ channelrdquo Journal ofBiological Chemistry vol 277 no 9 pp 6813ndash6821 2002

[66] E Kobrinsky E Schwartz D R Abernethy and NM SoldatovldquoVoltage-gated mobility of the Ca2+ channel cytoplasmic tailsand its regulatory rolerdquo Journal of Biological Chemistry vol 278no 7 pp 5021ndash5028 2003

[67] N M Soldatov ldquoCa2+ channel moving tail link between Ca2+-induced inactivation and Ca2+ signal transductionrdquo Trends inPharmacological Sciences vol 24 no 4 pp 167ndash171 2003

[68] D J Klemm P A Watson M G Frid et al ldquocAMP responseelement-binding protein content is a molecular determinantof smooth muscle cell proliferation and migrationrdquo Journal ofBiological Chemistry vol 276 no 49 pp 46132ndash46141 2001

[69] E Kobrinsky L Stevens Y Kazmi DWray andNM SoldatovldquoMolecular rearrangements of the Kv21 potassium channeltermini associated with voltage gatingrdquo Journal of BiologicalChemistry vol 281 no 28 pp 19233ndash19240 2006

[70] BMMayr G Canettieri andMRMontminy ldquoDistinct effectsof cAMP and mitogenic signals on CREB-binding proteinrecruitment impart specificity to target gene activation viaCREBrdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 98 no 19 pp 10936ndash10941 2001

[71] M Morad and N Soldatov ldquoCalcium channel inactivationpossible role in signal transduction and Ca2+ signalingrdquo CellCalcium vol 38 no 3-4 pp 223ndash231 2005

[72] DGWheeler C F Barrett R DGroth P Safa andRW TsienldquoCaMKII locally encodes L-type channel activity to signal tonuclear CREB in excitation-transcription couplingrdquo Journal ofCell Biology vol 183 no 5 pp 849ndash863 2008

[73] D E Mager E Kobrinsky A Masoudieh A Maltsev D RAbernethy andNM Soldatov ldquoAnalysis of functional signalingdomains from fluorescence imaging and the two-dimensionalcontinuous wavelet transformrdquo Biophysical Journal vol 93 no8 pp 2900ndash2910 2007

[74] E Kobrinsky D E Mager S A Bentil S I Murata DR Abernethy and N M Soldatov ldquoIdentification of plasmamembrane macro- and microdomains from wavelet analysis ofFRET microscopyrdquo Biophysical Journal vol 88 no 5 pp 3625ndash3634 2005

[75] E Kobrinsky S Q Duong A Sheydina and N M Solda-tov ldquoMicrodomain organization and frequency-dependence ofCREB-dependent transcriptional signaling in heart cellsrdquo TheFASEB Journal vol 25 no 5 pp 1544ndash1555 2011

[76] A Ravindran Q Z Lao J B Harry P Abrahimi E Kobrinskyand N M Soldatov ldquoCalmodulin-dependent gating of Cav12calcium channels in the absence of Cav120573 subunitsrdquo Proceedingsof the National Academy of Sciences of the United States ofAmerica vol 105 no 23 pp 8154ndash8159 2008

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Virolog y

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Volume 2014

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Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

2 ISRN Biochemistry

10 serum

Serum-free

0 04 08 12 16

Confluent monolayer

Nonconfluent monolayer10 serum 2 (control)

1

3

4

5

6

lowast

lowast

lowast

lowast

lowast

[3H]PMD binding

+cAMP

+Ca2+

+diltiazem

(a)

10 serum (control)

Serum-free 10 serum

05

1

15

0 1 2 3 4 5 6 7Time (d)

[3H

]PM

D b

indi

ng

(b)

Figure 1 Effect of serum and second messengers on the density of DHP receptors in normal human fibroblasts (a) On day 2 after theplating of fibroblasts at high (bar 1) or low density (bars 2ndash6) the growth medium was supplemented with 1mM 8Br-cAMP (bar 3) 1120583MCa2+ ionophore A23187 (bar 4) 1120583M D-cis-diltiazem (bar 5) or growth medium was replaced by serum-free Eaglersquos medium containing01 BSA (bar 6) After 4 days the density of DHP receptors was measured at 2 nM [3H]-PMD as pmol [3H]PMD specifically bound per 106cells of confluent (bar 1 gt2 times 104 cellscm2) or nonconfluent monolayers (bars 2ndash6 3ndash5 times 103 cellscm2) Mean of 5-6 measurements plusmn SEMlowast

119875 lt 005 (b) Effect of serum deprivation on the density of DHP receptors in fibroblasts On day 2 after plating sparse cultures of fibroblastswere incubated for up to 4 days in serum-free Eaglersquos medium supplemented with 01 BSA The horizontal bars indicate when serum-freemedium was replaced by standard growth medium containing 10 serum The time course of changes in the density of DHP binding siteswas measured as in (a) at different time points (not shown for simplicity) and compared with that in cells not subjected to serum-deprivation(blue line)

estimated from the rate of loss of [3H]PMD binding sitesin response to the net inhibition of protein synthesis bycycloheximide With progression to confluent monolayersthe 119870

119889value for [3H]PMD binding did not change but the

119861max value decreased asymp4 fold (Figure 1(a) compare bars 1and 2) suggesting that expression of Cav12 is responsiveto the arrest of fibroblasts proliferation by cell-cell contactinhibition

The involvement of Cav12 in proliferation of humanfibroblasts was further supported by the finding that con-centration of DHP receptors and respectively of Cav12is strongly affected by mitogens and second messengersDemonstrating a remarkable plasticity of the Cav12 expres-sion in normal human fibroblasts (Figure 1(b)) serum depri-vation induced a 2-fold increase in the density of DHP recep-tors that reached its maximum after 3-4 days of cultivationin the absence of serum (compare also bars 2 and 6 inFigure 1(a)) The elevation of the Cav12 expression wasfully reversible and highly sensitive to serum and othermitogens An addition of 10 serum reduced the densityof DHP receptors to the initial level with almost the sametime course (Figure 1(b)) Thus inhibition of cell growthproliferation and DNA synthesis in fibroblasts by serumdeprivation stimulates expression ofCav12with a time coursecomparable with the DHP receptor turnover rate

The response of fibroblasts to serum deprivation byboosting Cav12 expression is essentially identical to thatcaused by the inhibition of Cav12 by 1 120583M diltiazem

(Figure 1(a) compare bars 5 and 6) the calcium channelblocker that does not compete with DHPs for binding withthe channel In fact diltiazem was present in the DHP bind-ing assay medium throughout all experiments to enhancethe affinity of the DHP probe to the channel receptor [17]Thus it is reasonable to suggest that by boosting the Cav12expression the cell recruits more routes for the Ca2+ entrythrough the plasma membrane to overcome the lack of mito-gens in serum deprivation or lack of conducting channelsin the presence of diltiazem both aimed at supporting cellproliferation until cell-cell contact inhibition terminates itand turns the cells into a quiescent state

If this hypothesis is true then the addition of DHP-insensitive routes for Ca2+ entry through the plasma mem-brane should eliminate the need in higher level of Cav12expression This observation exactly has been made whenthe serum-stimulated cells were supplemented with Ca2+ionophore A23187 (Figure 1(a) bar 4) The A23187-inducedCa2+ entry dramatically reduced the cellular expression ofCav12 A similar effect was observed with 8Br-cAMP theplasma membrane-permeable derivative of cAMP showingthat stimulation of the alternative cAMP-dependent cellsignaling pathway may reduce the needs in Cav12 in prolif-eration of fibroblasts

Stimulation of Cav12 expression in fibroblasts arrested inthe quiescent state by serum deprivation strongly dependson cell proliferation The measurement of [14C]thymidineincorporation as an assay for DNA synthesis in cells showed

ISRN Biochemistry 3

Synthesis of DNA ( of control)

bFGF

EGF

Insulin

Serum-free

10 serum

0 50 100 150 200

0 05 1 15 2

Control

[3H]PMD binding

Figure 2 Effect of mitogens on the density of DHP receptors (openbars) and cell proliferation (dark bars) in normal human fibroblastsOn day 2 after cell plating the growth medium was replaced for4 days with serum-free Eaglersquos medium supplemented with 01BSA Cells were incubated for 4 days in the presence of 10 serum(control) 01 BSA (serum-free) epidermal growth factor (EGF15 ngmL) insulin (10 120583gmL) or basic fibroblast growth factor(bFGF 2 ngmL) The density of DHP receptors was measuredat 2 nM [3H]-PMD as pmol [3H]-PMD specifically bound per106 cells of nonconfluent monolayers (3ndash5 times 103 cellscm2) Cellproliferation was estimated from the synthesis of DNAmeasured by[14C]thymidine incorporation

(Figure 2 dark bars) that individual mitogens stimulated cellproliferation in the order bFGF gt insulin gt EGF In contrastin that very order the same mitogens suppressed expressionof Cav12 in fibroblasts as evidenced by the measurement ofDHP receptor binding (open bars) Consistent with relativelylow cell toxicity of dihydropyridines blockade of Cav12attenuated entry of cells into the S phase of cell cycle [18]Thus the plasticity of Cav12 in fibroblast proliferation is asso-ciated with transient Cav12 expression responsive to bothprimary effectors and secondmessengers of cell proliferation

3 Cak12 Variability

One of themost important features of Cav12 is its remarkablemolecular diversityThe channel consists of the pore-forming1205721C protein which binds calcium channel blockers Three

types of regulatory subunits 120573 1205722120575 and calmodulin (CaM)

are constitutively tethered to 1205721C All these subunits are

products of different genes that are located on differentchromosomes Cav12 shares the accessory subunits withother Cav1 and Cav2 channels Moreover 120572

1C 120573 and 1205722120575 aresubject to individual alternative splicing that generates a largediversity of Cav12 channel complexes The functional signif-icance of the Cav12 splice variation is poorly understood not

only because of the difficulties in identification of individualsplice variants in the naturally occurring oligomeric proteinsThe tendency of Cav12 to form large clusters in the plasmamembrane [19ndash23] as well as homo- and heterooligomeriza-tion of 120573 subunits [24] create additional major challenges forthe investigation of Cav12 in regulation of cell proliferationdifferentiation and other functionsmdashnot to speak of anadequate elucidation of their roles in disease and search fornew therapeutic approaches

Molecular cloning showed that 1205721C transcripts in human

fibroblasts are composed of exons 1ndash50 [14] with alternativesplicing of exons 2122 and 3132 and constitutive splicingof exons 33 and 45 (Figure 3(a)) [12] However variabilityassociated with exons 1 7 8 9 34 41 and 42 that were laterfound in human hippocampus heart and VSM cells [25ndash30]was not observed in fibroblasts

It is not known which Cav12 splice variants are expressedin fibroblasts in response to serum deprivation and whetherthey are structurally different from those present in pro-liferating cells However electrophysiological properties ofthe three of fibroblast 120572

1C splice variants (coexpressed with1205731a and 1205722120575-1) were compared in Xenopus oocyte expression

system [31] Characteristics of the voltage-dependence andkinetics of inactivation of the barium current through the1205721C70 channel encoded by the CACNA1C transcript lacking

exons 22 31 41A and 45 were found to be very similar tothose recorded with 120572

1C77 (lacking exons 21 31 41A and45) and 120572

1C76 (also lacking exon 33) (Figure 3(b)) Howevervoltage dependence of the DHP inhibition of the currentwas significantly different in the 120572

1C70 and 1205721C77 channelsthe IC

50values for the concentration dependence of the

barium current inhibition by (+)isradipine almost identical(62 and 73 nM resp) at minus40mV were significantly differentat minus90mV (680 and 79 nM for 120572

1C70 and 1205721C77 resp) Whilesuch a difference in the pharmacological properties of theexon 21- and exon 22-splice variants is deemed unimportantin the case of fibroblasts it may significantly contribute tothe tissue specificity of this major class of calcium channelblockers in cardiac vascular and other responsive cellsaffected by cardiovascular diseases

4 Cak12 in Atherosclerosis

Atherosclerosis is perhaps the single most deadly diseaseleading to about 600000 deaths annually in the USA mostof these due to the progression of the disease to heartattack or stroke [32] In spite of significant efforts themolecular mechanisms of atherosclerosis are not currentlywell understood and effective molecular targets for preven-tion and treatment are not elaborated Atherosclerosis is aninflammatory process in medium and large size arteries thatcauses endothelial perturbation and local release of cytokinesas well as dedifferentiation proliferation and migration ofVSM cells [33] Arterial VSM cells constitute the media of theartery and play a crucial role in its elasticity and contractilityMigration of VSM cells from the media to the intima of thearterial wall and proliferation of intimal smooth muscle cellsare the major early events in the formation of atheroscleroticlesions Recent advances in molecular genetics studies have

4 ISRN Biochemistry

II III IV

2

3

4

5

6

788A

9

10

1125

12 13 14 15

16

17 18

19

26

23

24 28

27

2934

36

2122

Plasmamembrane

Out

In

33

37+

+

38

39

40

HOOC

4443

454647484950

11A3132 41 4241A

9A

34A30

I

LA IQ

AID

65

++++4

++++

3 51 4

++++

3 51 2 4 26

++++

3 51 2 4

2035

6 6 31 2

H2N

(a)

20 2321 22 30 31 32 33 34

20 2321 22 30 31 32 33 34

20 2321 22 30 31 32 33 34

40 41A 41 44 45 46

40 41A 41 44 45 46

40 41A 41 44 45 46

20 2321 22 30 31 32 33 34

20 2321 22 30 31 32 33 34 40 41A 41 44 45 46

40 41A 41 44 45 46

20 2321 22 30 31 32 33 34 40 41A 41 44 45 46

1205721C69

1205721C70

1205721C71

1205721C73

1205721C76

1205721C77

(b)

Figure 3 Molecular diversity of Cav12 (a) Transmembrane topology of the Cav12 1205721C subunit in schematic diagram Four regions ofhomology (IndashIV) each composed of 6 transmembrane segments (numbered) are folded around the central pore The polypeptide sequenceis segmented by black bars and sequentially numbered (1ndash50) according to the CACNA1C genomic map [14] The segments correspondingto the invariant (blackgray) and alternative (blue) exons are outlined 120572-interaction domain (AID) of a constitutive 120573-binding is shown ingreen In the CaM-binding domain (CBD) apo-CaM and Ca2+-CaM are shared between LA and IQmotifs (red) respectively (b) Alternativesplicing of human fibroblast CACNA1C in the region of exons 20ndash46 The following splice variants were studied 120572

1C69 (GenBank z34809splice variant lacking alternative exons (Δ) 21 32 41A and 45) 120572

1C70 (z34810 Δ 22 31 41A and 45) 1205721C71 (z34811 Δ 22 31 and 45) 120572

1C73(z34812 Δ 22 31 33 and 45) 120572

1C76 (z34814 Δ 21 31 33 41A and 45) and 1205721C77 (z34815 Δ 21 31 41A and 45)

revealed that genetic polymorphisms significantly influencesusceptibility to atherosclerotic vascular diseases [34] How-ever none of the discovered susceptibility genes was directlyimplicated for proliferation and migration of VSM cells oneof the major pathophysiological responses to atherosclerosisat the cellular level

The presence and activity of Cav12 calcium channels inVSM cells has been established both in patch clamp andmolecular cloning experiments [29 35ndash38] Cav12 calciumchannels play a major role in atherosclerosis because they

are essential for Ca2+ signal transduction in VSM cellsContraction of VSM cells is triggered by the Ca2+ current(119868Ca) through Cav12 and thus is affected by Ca2+ channelblockers Since the 1990s it is known that DHPs particularlythe charged 2-aminoethoxymethyl DHP derivative amlodip-ine [39] exert a number of vasoprotective effects includingpotent antiatherogenic action [40] inhibition of migrationof VSM cells [41] and reduction of arterial intimal-medialthickening and plaque formation [42 43] Although it istempting to link this activity in part to pleiotropic effects of

ISRN Biochemistry 5

calcium channel blockers [44] it is the inhibition of prolifer-ation that essentially underlies it [45] The density of DHPreceptors was found to depend on VSM cell proliferation[46] Association of Cav12 with mitogenesis in VSM cells issupported by the findings that DHPs reduced DNA synthesisstimulated by serum and PdGF [47ndash49] serotonin [50] EGF[51] and H

2O2(in mesangial cells) [52] Expression of Cav12

in VSM cells was shown to be cell cycle dependent with thehighest calcium current density in G

1phase [53] Whether

these changes are reflected in the molecular repertoire ofthe Cav12 splice variants is the issue particularly importantfor the elucidation of new therapeutic targets in diseasesleading to pathogenic proliferation of VSM cells such asatherosclerosis

Investigation of the 1205721C alternative splicing in VSM

cells [28 54 55] revealed the involvement of a number ofCACNA1C exons generating impressive diversity of humanvascular 120572

1C that includes possibly the VSM-specific splic-ing of exons 99a [35 55] and exon 34 [28] To establishan association between the disease and CACNA1C splicevariation at the level of cell we have completed [26] thesingle-gene profiling of the 120572

1C molecular remodeling inVSM cells of an artery caused by atherosclerosis The VSMcells were identified in frozen sections of six surgical biopsysamples of femoral and carotid arteries by immunostainingwith an antibody against smoothmuscle 120572-actin [56] used asa marker for VSM cells The 120572-actin staining correlated withimmunostaining by an anti-120572

1C antibody in serial sectionsand was reduced in atherosclerotic regions (Figure 4) consis-tent with dedifferentiation of VSM cells [57 58]The reducedexpression of 120572

1C at the protein level was corroborated bythe quantitative RT-PCR data showing that the relative 120572

1CmRNA level in VSM cells (normalized to 18S RNA) wasreduced 37 plusmn 09 fold (mean plusmn SEM) in the atheroscleroticregion Overall the reduced expression of 120572

1C caused by thelocally elaborated cytokines in the atherosclerotic regions ofarteries resembles the reduced expression of DHP receptorsobserved in fibroblasts exposed to mitogens andor secondmessengers after serum deprivation (Figure 1)

To find out whether the altered expression of 1205721C

in atherosclerotic VSM cells is accompanied by changesin the CACNA1C alternative splicing pattern we isolatedthe immunohistochemically identified VSM cells by laser-capture microdissection from adjacent regions of arteriesaffected and not affected by atherosclerosis and identifiedthe CACNA1C splice variants by RT-PCR Our findingsrevealed an extended repertoire of the exon 21 120572

1C spliceisoforms in nonatherosclerotic VSM cells characterized bya complex splicing pattern of exons 9 9A 31ndash34 and41A including the electrophysiologically characterized 120572

1C71(GenBank z34811) 120572

1C73 (z34812) 1205721C125 (AY830711)

1205721C126 (AY830713) and 1205721C127 (AY830712) splice isoforms

However only the exon 22 isoform of 1205721C (1205721C77) was

identified in atherosclerotic VSM cells Thus the switch ofthe CACNA1C alterative splicing from exon 21 to exon 22(Figure 3(b)) is a molecular signature of the Cav12 remod-eling of VSM cells to a pathophysiological proliferating statein atherosclerosis The age gender ethnicity drug exposureand other co-morbid conditions did not appreciably affect

this common pattern of the 1205721C splice variation in VSM cells

in response to atherosclerosisCareful electrophysiological analysis exhibited a number

of differences in the properties of the ldquoatheroscleroticrdquo 1205721C77

channel as compared to the 1205721C isoforms in healthy VSM

cells The largest differences were found between the 1205721C77

and 1205721C127 channels (Figure 5) In response to step depolar-

ization applied from the holding potential of minus90mV bothchannels generate calcium currents (119868Ca) that inactivate withalmost identical kinetics (Figure 5(a)) However we foundthat 119868Ca through the1205721C77 channel recovers from inactivationsignificantly faster than that in 120572

1C127 (Figure 5(b)) and other1205721C isoforms present in healthy VSM cells This finding

suggests that alternative splicing in atherosclerosis may affectvascular tone as a result of the increase in the 119868Ca density inVSM cells A hyperpolarization shift of the activation curvefor the atherosclerotic 120572

1C77 channel variant as comparedto Cav12 in healthy VSM cells (Figure 5(c)) may also resultin an increase of calcium entry in VSM cells Howeverthe overall 3-4-fold reduction in the expression of Cav12in atherosclerotic VSM cells may scale down some of theobserved electrophysiological changes

5 CACNA1C Exon 22 as a New TherapeuticTarget in Atherosclerosis

Direct DNA sequencing of the crude PCR amplificationproducts indicated that the switch to the exon 22 isoform ofvascular 120572

1C was almost complete in atherosclerosis becauseno distortion of the nucleotide peaks in the region of exon2122 was seen when compared to the exon 20 invariantregion (Figure 6(a)) Thus Cav12 underwent almost quanti-tative exon 2122 remodeling in VSM cells of diseased arteryregions

Although the cellular mechanisms leading to theCACNA1C exon 2122 switch may be very complex theassociation with VSM cell proliferation is obvious Indeeda similar exon switch was observed in primary humanaortic cells in culture after the quiescent nonproliferatingcells containing predominantly exon 21 splice variantswere exposed to serum (Figure 6(b)) Unlike exon 21exon 22 contains the AvrII restriction site that allowsfor the assessment of its presence in PCR amplificationproduct of CACNA1C transcripts isolated from the cellsThe AvrII-sensitive exon 22 isoform of the 120572

1C transcriptwas not detected in the quiescent nonproliferating aorticcells (Figure 6(b) lane 2) However when 5 serum wasadded to the medium with nonconfluent aortic cellsDNA biosynthesis was activated while the level of the1205721C transcript decreased asymp3 fold and the presence of the

AvrII-sensitive exon 22 isoforms of 1205721C was easily detected

(Figure 6(b) lane 4) The isoform remodeling simulated inaortic primary cells in vitro was not complete as comparedto VSM cells in atherosclerotic regions of artery occludedwith heavy plaque burden which were selected for the1205721C molecular profiling However the cell culture results

demonstrate that in a different experimental system there isan obvious association between proliferation of VSM cells

6 ISRN Biochemistry

A Ki-67 NKi-67

50 12058350 120583

(a)

A N120572-actin 120572-actin

(b)

A N1205721C1205721C

(c)

Figure 4 Reduced expression of 1205721C in atherosclerotic (A) region of aorta as compared to the adjacent nonaffected (N) region From (a) to

(c) detection of proliferating cells in an exemplar biopsy of an artery by an antibody against human nuclear protein Ki-67 (a) Red arrowshighlight some proliferating nuclei (black staining) while blue arrows exemplify nonproliferating nuclei (light grey) Staining with antibodiesagainst smooth muscle 120572-actin (b) and against 120572

1C (c) Scale bars 50 120583m

downregulation of the CACNA1C expression and synthesisof the exon 22 120572

1C isoformRecent strategies targeting VSM cells to treat cardiovas-

cular diseases suggest indiscriminate disruption of Cav12[59ndash61] Is it possible to correct the described CACNA1Csplice defects induced by atherosclerosis without affectingthe transcripts of the gene lacking the rdquopathogenicrdquo exon22 Correction of defective genes responsible for diseasedevelopment is achieved by gene therapy Usually it requiresan insertion into the genome of a normal gene in place ofa defective one causing disease Such technique howeveris poorly controlled Currently one of the most promising

cutting-edge therapeutic approaches to correct defects asso-ciated with disease-induced expression of abnormal splicevariants is antisense-mediated exon skipping It is basedon the use of antisense oligonucleotides targeting specificexons to be removed The adenovirus-directed 120572

1C exon 22skipping-induced inhibition of VSM cell proliferation (andrespectively migration) can be used to rescue VSM cells fromremodeling in atherosclerosis The 120572

1C exon 22-skippingwill not alter the open reading frame of the 120572

1C transcriptbecause alternative exons 21 and 22 are of the equal size(60 nt)Themodified nonspliceosomal snRNAU7 gene alongwith its natural promoter and 31015840 elements exon 22-antisense

ISRN Biochemistry 7

200 ms

1205721C77

1205721C127

(a)

Prepulse interval (s)02 04 06 08 1

0

1

08

06

04

02

0

Frac

tion

of119868 C

a

1205721C77

1205721C127

(b)

0

1

08

06

04

02

119866Ca

119866Ca

max

1205721C77

1205721C127

minus60 minus40 minus20 0 20 40(mV)

(c)

Figure 5 Comparison of electrophysiological properties of 119868Ca through the ldquoatheroscleroticrdquo 1205721C77 and ldquonormalrdquo 120572

1C127 channels expressedin Xenopus oocytes with 120572

2120575-1 subunit and the primary cardiac 1205732a subunit and measured with 25mM Ca2+ as the charge carrier (a)

Representative traces of 119868Ca evoked by 1 s step depolarizations to +20mV from 119881ℎ= minus90mV and normalized to the same amplitude (b)

Fractional recovery of 119868Ca from inactivation (c) Averaged activation (119866119866max-119881) curves fit by the Boltzmann function A 1 s test pulses in therange of minus40 to +50mV (10-mV increments) were applied from 119881

ℎ= minus90mV with 30-s intervals

(a)

AvrII

Serum

1 2 3 4

+minus+minus

minus minus + +

142 pb

459 pb

601 pb

(b)

Figure 6 (a) Trace diagrams of DNA sequencing of the 1205721C PCR amplification products of healthy (top row) and diseased VSM cells (bottom

row) using an antisense primer composed of nucleotides 2923ndash2939 (z34815) A boundary with the sequence of invariant exon 20 is markedby a vertical dotted line (b) Evidence that the AvrII-sensitive exon 22 isoform of 120572

1C is expressed only in proliferating VSM cells Primaryaortic cells were grown to confluent monolayer in 5 serum before serum-deprivation for 5 days (lanes 1 and 2)Then the cells were replatedat low density in 5 serum for 3-4 days (lanes 3 and 4) Total RNA was isolated and exon 2122 isoforms were identified by RT-PCR andAvrII restriction analysis (lanes 2 and 4)

8 ISRN Biochemistry

sequence and supplemented with Sm ribonucleoprotein-binding sequence may be incorporated into the adenovirusvector for high efficiency transfer [62]The cytokine receptors(eg PdGF-120573 receptor) based recognition targeting of viralliposomes or nanoparticles may be especially advantageousin connection with selective gene transfer to VSM cellsaffected by atherosclerosis while reducing the probability ofthe transfection of other cells

6 Cak12 and CREB-DependentTranscriptional Activation

How Cav12 activity is translated into a proliferation-effectedmodality is another important question to be asked Cav12calcium channels generate a transient rise in cytosolic Ca2+-concentration activated by membrane depolarization Cel-lular responses associated with the rise of [Ca2+]i rangefrom sarcomeric contraction to cell growth and proliferationCytoplasmic domains of Cav12 have evolved a fairly intricateCaM-dependent signaling mechanism that provides for thenegative feedback inhibition of the calcium current knownas Ca2+ dependent inactivation (CDI) which is mediated bydifferent determinants of 120572

1C [25 63] Such a mechanism ofCDI resulting in acceleration of 119868Ca inactivation in responseto the rise of intracellular Ca2+ was first identified in cardiacCav12 [64] Similar experiments performed on the recombi-nant Cav12 also showed that the replacement of extracellularCa2+ by Ba2+ eliminates CDI and the channel inactivates by aslower voltage-dependent mechanism [65]The very fact thattwo distantly located determinants one in the pore regionresponsible for slow inactivation and the CaM-binding onein the proximal locus of the C-tail are independently crucialfor CDI indicates that not only their specific molecularstructure but also their mutual folding andor interaction areessential Experimental evidence show that this interactionreacts dynamically to membrane voltage supporting state-dependent transitions of the channel between resting openand inactivated conformations which are essential for CREB-dependent transcriptional activation [66 67]

CREB is a transcription factor of general importance ina large variety of cells CREB phosphorylation promotes theactivation of genes and is regulated by protein kinases undercontrol of the major second messengers cAMP andor Ca2+Indeed CREB functions as a ldquomolecular determinant of VSMcells fate [68]rdquo CREB content depends on proliferation ofVSM cells both in situ and in culture Serum deprivationincreased CREB content in VSM cells while exposure toPdGF decreased it Consistent with this observation anoverexpression of the constitutively active CREB inVSM cellsarrested cell cycle progression

To investigate the association of Cav12 with CREB tran-scriptional activation at the molecular level we combinedpatch clamp with fluorescent resonance energy transfer(FRET) microscopy in the live cell In voltage clampedcells FRET provided optical measurements under state-dependent conditions showing that the shorter N-terminaltail of 120572

1C (eg 1205721C77) does not rearrange vis-a-vis the

plasma membrane in response to voltage gating [25] In

sharp contrast the C-tail shows voltage-dependent confor-mational rearrangements which are much larger in sizethan that for example in the potassium Kv21 channel[69] Measurements of 119868Ca and corrected FRET betweenthe enhanced yellow (EYFP) and cyan fluorescent proteins(ECFP) genetically attached respectively to the N- and C-termini of 120572

1C77 showed no significant effect on voltage-dependence and kinetics of the channel current Howevertherewas a substantial increase in FRET signal accompanyinginactivation of the channel that was fully reversible uponits transition into the resting state in response to hyper-polarization [66] The plasma-membrane anchoring of the1205721C C-tail by the fusion of the pleckstrin homology domain

(PH) eliminated CDI but not 119868Ca Do the voltage-gatedconformational rearrangements of the 120572

1C C-tail and CDIplay a role in Ca2+ signal transduction that is utilized inCa2+-induced activation or CREB-dependent transcriptionTo answer this question we used the test system based onthe measurement of interaction between KID domain ofCREB and KIX domain of coactivator CREB-binding protein(CBP Figure 7(A) inset) under voltage-clamp conditionsby monitoring FRET between EYFP-KID and ECFP-KIX[70] Ca2+-dependent phosphorylation of KID stimulates itsbinding to KIX bringing EYFP and ECFP close enoughto observe the interaction by FRET In perforated wholeclamped cell where the integrity of the cytoplasmic contentis intact (Figure 7(A)) no activation of CREB transcription(Figure 7(B) panel (a)) and rearrangement of the120572

1C77 C-tail(panel (b)) was observed when the C-tail was anchored to theplasma membrane CREB transcriptional activity remainedlow in spite of a large sustained inward 119868Ca (panel (d))and the corresponding increase in [Ca2+]i detected by thefluorescence of Ca2+ indicator Fura4 (panel (c)) Releaseof the 120572

1C77 C-tail by the activation of PIP2hydrolysis

upon activation of M1AchR (Figure 7(C)) at minus90mV causedsignificant elevation of [Ca2+]i that also was not utilizedby the cell for CREB transcriptional activation (Figure 7(C)panel (a)) until a depolarizing pulse to +20-mV was appliedand the released 120572

1C77 C-tail was permitted to rearrange(Figure 7(D))This experiment provides compelling evidencethat neither large inward 119868Ca nor the subsequent rise in[Ca2+]i lead to CREB transcription activation unless theconformational rearrangement of the 120572

1C77 subunit C-tailprovides the precise targeting of the Ca2+ signal transduction(Figure 7(D) scheme) [66]

There is general agreement that CaM binds to LA and IQdomains of the 120572

1C C-tail and acts as a sensor that conveysCDI (for review see [63]) The affinity of CaM for bothdomains depends on [Ca2+]i Our data indicate that CDI andCa2+-signal transduction depend on the voltage-gatedmobil-ity of the120572

1C77 C-tail It is therefore reasonable to suggest thatthe LA-domain is a Ca2+-sensitive apo-CaM-mediated lockfor the mechanism of slow voltage-dependent inactivationof the channel [67] Apo-CaM associated with LA is able tocross-link it to another still unidentified apo-CaM bindingsite in the polypeptide bundle underlying the pore As theresult of this specific localization apo-CaMLA ldquolockrdquo ishidden from the cytoplasmic Ca2+ so that for example the

ISRN Biochemistry 9

10

0

2

10

100 pA125 ms

(a) (a) (a)

(b)(c)

(b)(c)

(b)(c)

(B) (C) (D)

C-tail Anchored ReleasedReleased

0 10No FRET No FRET

(d) (d) (d)

Fluo-4

PH

CYN

C

C

NY

C

Y EYFP

C ECFP

PH Plekstrin homologydomain

PHPHY

C

NC

IQ

LA

LAIQ

(A)

Fluo-4 Fluo-4

FRET FRET FRET

CRE

Gene expressionCREBCREB P

CBP

AC

GPCR

PKA

cAMP

CaM

Nucleus

cAMP

Plasmamembrane

Ca2+ Ca2+

Ca2+

+20mV +20mV

minus90mV minus90mV

1205721C77

1198651198650

Cav 12

Ca2+

minus90mVACh (5 mM)

4 120583

Figure 7 Evidence that Ca2+ signal transduction by Cav12 to activate CREB-dependent transcription is mediated by the voltage-gatedmobility of the 120572

1C C-tail carrying the CaM-caged Ca2+ and is not directly associated with the increase of [Ca2+]i CREB activation wasexamined under perforated patch conditions in COS1 cells expressing recombinant atherosclerotic 120572

1C77 channel (A) Phase-contrast imageof the COS1 cell with a shadow of patch pipette The cell was expressing the 120572

1C77-PHC1205731a1205722120575-1 channel with the membrane-trapped 1205721C77

subunit C-tail type 1muscarinic Ach receptor (to release the anchored C-tail in response to activation by Ach) and EYFP-KID and ECFP-KIXdomains both supplemented with nuclear localization sequences Inset at the bottom Cav12- and cAMP-dependent cell signaling pathwaysmediating CREB-dependent transcription CBP CREB-binding protein CRE cAMP-response element (B) Plasma-membrane anchoring ofthe 1205721C77 C-tail (see schematic diagram) inhibits CREB activation in spite of robust 119868Ca evoked by depolarization (a) A 100-ms images of

FRET between EYFP-KID and ECFP-KIX were recorded at the end of the 12th +20-mV depolarization step applied every 10 s for 1 s from119881ℎ=

minus90mV (b) A corresponding representative image of corrected FRET ratio (in a ldquo3Drdquo MetaMorph transformation) recorded within 50-mswindows at minus90 and at the end of the last test pulse of the applied train of pulses (c) An increase of [Ca2+]i detected by Ca

2+ indicator Fluo4(d) Representative trace of 119868Ca (20mM Ca2+ in bath medium) evoked by depolarization to +20mV from119881

ℎ= minus90mV showing the sustained

component of Ca2+ conductance due to the C-tail anchoring (C) ACh-stimulation of theM1AChR to unbind the 1205721C C-tail caused activation

of IP3-dependent Ca2+ release (panel (c)) but did not show substantial activation of CREB transcription at 119881

ℎ= minus90mV (panel (a)) (D)

CREB-dependent transcription was activated only in response to depolarization applied to membrane channel with the 1205721C77 C-terminal tail

released from the plasma membrane to assume functional voltage-dependent conformation

intracellular Ca2+ released from the intracellular stores orCa2+ caging compounds does not accelerate significantly theinactivation of Cav12 [71] Thus apo-CaM associated withLA binds predominantly Ca2+ ions permeating through thepore A Ca2+-dependent transfer of CaM from LA to the IQ-motif opens the ldquolockrdquo and initiates a large rearrangement ofthe C-terminal tail This in turn facilitates inactivation of thechannel The Ca2+CaM complex with the IQ-motif is thentransferred by themobile C-tail to a downstream target of theCa2+-signaling cascade (such as CaMKII [72]) where Ca2+ isreleased as an activating stimulus while CaM switches back

to LA and returns the C-tail to the resting position availablefor the next cycle of Ca2+-signal transduction

Activation of CREB-dependent transcription by the L-type 119868Ca ismediated throughmultiple cell signaling pathwaysUsing FRET probes of CREB activity and 2D wavelet trans-form analysis we applied principles of quantitative biologyto detail the mechanism of Ca2+-activated CREB-dependenttranscription within localized regions (microdomains) ofthe nucleus We reached this goal by applying continuouswavelet analysis in two dimensions with a 2D wavelet asa deconvolution algorithm for FRET microscopy image

10 ISRN Biochemistry

Control

(A)

05

0

1

05

00 20 40 60 800 20 40 60 800 20 40 60 80

(a) (b) (c) (d)

20 40 60 800

0

20

40

60

80 1

C 3 5 10 mincAMP

C 2 10 mincAMP

C 3 10 min

cAMP

C 1 3 10 min

cAMP

5

cAMP stable (5)

(B)

119868Ca (3 min) cAMP (5 min) cAMP (10 min)

119868Ca cAMP (65) 119868Ca stable (15)

119868Ca119868Ca

119868Ca 119868Ca

119868Ca cAMP transient (15)

Figure 8 Microdomain analysis on the additive effect of 119868Ca and cAMP on CREB signaling in COS1 cells expressing the recombinantldquoatheroscleroticrdquo120572

1C77 channel (A) FRET signal within the nucleus during selected time points during 119868Ca stimulation and cAMP applicationOutlined are four types of signaling microdomains identified using 2D Mexican hat wavelet Red circles represent stable microdomains thatpersist through both 119868Ca and cAMP application White and yellow circles showmicrodomains stable activated by cAMP and 119868Ca respectivelyGreen circles represent transient microdomains of CREB signaling activation Axes show pixel numbers (B) Typical appearances of the fourtypes of CREB signaling microdomains activated by 119868Ca andor cAMP application and recorded in their maximal development in relationto the time of applied stimuli C control recorded before stimulation Color bars in (A) and (B) represent FRET values normalized to themaximum

analysis [73] Continuous wavelet analysis is a mathematicaltechnique that allows us to analyze a signal over severaldifferent frequencies across the entire signal [73 74] It isespecially useful for finding heterogeneity in a signal becauseit can easily find where the pattern (ie frequency) of asignal changes In these experiments we for the first timeobtained evidence of CREB signaling microdomains withinthe nucleus that respond differentially to 119868Ca stimulationand cAMP (Figure 8(A)) Results of the study revealed thatCREB-dependent transcriptional signaling occurs in dis-crete signaling microdomains underlying the architecture ofnuclear signaling Continuous activation ofCREB-dependenttranscriptional signaling by cAMP and Ca2+ resulted in agradual increase of the number of microdomains Four dif-ferent categories of cAMP and Ca2+-induced CREB signalingmicrodomains were characterized in COS1 cells expressingrecombinant Cav12 with the ldquoatheroscleroticrdquo 120572

1C77 splice

variant (Figure 8) In up to 65 of the microdomainstranscription was activated in additive manner by cAMPand Ca2+ Approximately 15 of signaling domains wereactivated only by 119868Ca and 5 of domains were activatedonly by cAMP Finally 15 of the domains were transientand activated by both cAMP and 119868Ca (Figure 8(B)) [75]A similar spatiotemporal organization of CREB-dependentsignaling was observed in spontaneously beating neonatalrat cardiomyocytes Although COS1 cells that were used inour experiments shown in Figures 7 and 8 are naturallydeprived of Cav12 [76] they inherited the ability to replicatethe Cav12-dependent activation of CREB signaling with theexogenous recombinant channel Thus this experimentalapproach fits the task which in my opinion is the mostimportant unresolved issue for the coupling of Cav12 toCREB signaling does the splice variation of 120572

1C affect thespatiotemporal organization of CREB-dependent signaling

ISRN Biochemistry 11

in a way that may affect cell proliferation and other crucialfunction

7 Conclusions

Association of Cav12 with regulation of transcription cellproliferation and its pathophysiology as in the case ofatherosclerosis requires detailed investigation of the roles ofthe naturally occurring 120572

1C splice variants It will limit thetraditionally intuitive approach to Cav12 in physiology andhelp to define new principle approaches to the treatment ofvarious Cav12 channelopathy-related dysfunctions above allcardiovascular diseases Humgenex Inc provides consultingand logistic support on a broad range of issues reported inthis review

Abbreviations

CaM CalmodulinCBD Calmodulin-binding domainCDI Ca2+-dependent inactivationDHP DihydropyridineECFP Enhanced cyan fluorescent proteinEYFP Enhanced yellow fluorescent proteinFALI Fluorophore-assisted light inactivationFRET Fluorescent resonance energy transfer[3H]PMD 26-Dimethyl-3-methoxycarbonyl-5-([23-

3H2]-n-propoxycarbonyl)-4-(21015840-

difluoromethoxyphenyl)-14-dihydropyridine

119868Ca Calcium currentPH Pleckstrin homology domainVSM Vascular smooth muscle

References

[1] Y S Lee M M Sayeed and R D Wurster ldquoInhibition ofcell growth and intracellular Ca2+ mobilization in humanbrain tumor cells by Ca2+ channel antagonistsrdquo Molecular andChemical Neuropathology vol 22 no 2 pp 81ndash95 1994

[2] K Sato J Ishizuka C W Cooper et al ldquoInhibitory effect ofcalcium channel blockers on growth of pancreatic cancer cellsrdquoPancreas vol 9 no 2 pp 193ndash202 1994

[3] V Bertrand M J Bastie N Vaysse and L Pradayrol ldquoInhibi-tion of gastrin-induced proliferation of AR4-2J cells by calciumchannel antagonistsrdquo International Journal of Cancer vol 56no 3 pp 427ndash432 1994

[4] JM Taylor andRU Simpson ldquoInhibition of cancer cell growthby calcium channel antagonists in the athymic mouserdquo CancerResearch vol 52 no 9 pp 2413ndash2418 1992

[5] M G Cattaneo M Gullo and L M Vicentini ldquoC2+ and Ca2+channel antagonists in the control of human small cell lung car-cinoma cell proliferationrdquo European Journal of Pharmacologyvol 247 no 3 pp 325ndash331 1993

[6] C Chen M J Corbley T M Roberts and P Hess ldquoVoltage-sensitive calcium channels in normal and transformed 3T3fibroblastsrdquo Science vol 239 no 4843 pp 1024ndash1026 1988

[7] P C Dartsch M Ritter M Gschwentner H J Lang andF Lang ldquoEffects of calcium channel blockers on NIH 3T3

fibroblasts expressing the Ha-ras oncogenerdquo European Journalof Cell Biology vol 67 no 4 pp 372ndash378 1995

[8] M Biel R Hullin S Freundner et al ldquoTissue-specific expres-sion of high-voltage-activated dihydropyridine-sensitive L-typecalcium channelsrdquo European Journal of Biochemistry vol 200no 1 pp 81ndash88 1991

[9] DM Rodman K Reese J Harral et al ldquoLow-voltage-activated(T-type) calcium channels control proliferation of human pul-monary artery myocytesrdquo Circulation Research vol 96 no 8pp 864ndash872 2005

[10] A Panner L L Cribbs G M Zainelli T C Origitano S Singhand R DWurster ldquoVariation of T-type calcium channel proteinexpression affects cell division of cultured tumor cellsrdquo CellCalcium vol 37 no 2 pp 105ndash119 2005

[11] C Chen and P Hess ldquoCalcium channels in mouse 3T3 andhuman fibroblastsrdquo Biophysical Journal vol 51 p 226a 1987

[12] N M Soldatov ldquoMolecular diversity of L-type Ca2+ channeltranscripts in human fibroblastsrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 89 no10 pp 4628ndash4632 1992

[13] A Fujii H Matsumoto S Nakao H Teshigawara and YAkimoto ldquoEffect of calcium-channel blockers on cell prolifera-tion DNA synthesis and collagen synthesis of cultured gingivalfibroblasts derived fromhumannifedipine responders and non-respondersrdquo Archives of Oral Biology vol 39 no 2 pp 99ndash1041994

[14] N M Soldatov ldquoGenomic structure of human L-type Ca2+channelrdquo Genomics vol 22 no 1 pp 77ndash87 1994

[15] V P Shevchenko N F Myasoedov N M Soldatov G JDuburs V V Kastron and I P Skrastins ldquoSynthesis andevaluation of biological activity of two novel tritium-labeledderivatives of riodipinerdquo Journal of Labelled Compounds andRadiopharmaceuticals vol 27 no 6 pp 721ndash730 1989

[16] S M Dudkin S N Gnedoj N N Chernyuk and N MSoldatov ldquo14-dihydropyridine receptor associated with Ca2+channels in human embryonic fibroblastsrdquo FEBS Letters vol233 no 2 pp 352ndash354 1988

[17] A DePover M A Matlib S W Lee et al ldquoSpecific bindingof [3H]nitrendipine to membranes from coronary arteriesand heart in relation to pharmacological effects Paradoxicalstimulation by diltiazemrdquo Biochemical and Biophysical ResearchCommunications vol 108 no 1 pp 110ndash117 1982

[18] N M Soldatov S N Gnedoj N N Chernyuk I A Britanovaand S M Dudkin ldquoMitogenesis and 14-dihydropyridinereceptor associated with Ca2+-channels in human embryonicfibroblastsrdquo Journal of Membrane Biology vol 5 pp 1161ndash11671988 (Russian)

[19] D Lipscombe D V Madison M Poenie H Reuter R Y Tsienand R W Tsien ldquoSpatial distribution of calcium channels andcytosolic calcium transients in growth cones and cell bodies ofsympathetic neuronsrdquo Proceedings of the National Academy ofSciences of the United States of America vol 85 no 7 pp 2398ndash2402 1988

[20] G S Harms L Cognet P H M Lommerse et al ldquoSingle-molecule imaging of L-type CA2+ channels in live cellsrdquoBiophysical Journal vol 81 no 5 pp 2639ndash2646 2001

[21] V di Biase G J Obermair Z Szabo et al ldquoStable membraneexpression of postsynaptic Cav12 calcium channel clustersis independent of interactions with AKAP79150 and PDZproteinsrdquo Journal of Neuroscience vol 28 no 51 pp 13845ndash13855 2008

12 ISRN Biochemistry

[22] M F Navedo E P Cheng C Yuan et al ldquoIncreased coupledgating of L-type Ca2+ channels during hypertension and timo-thy syndromerdquoCirculationResearch vol 106 no 4 pp 748ndash7562010

[23] E Kobrinsky P Abrahimi S Q Duong et al ldquoEffect of Cav120573subunits on structural organization of Cav12 calcium channelsrdquoPLoS One vol 4 no 5 Article ID e5587 2009

[24] Q Z Lao E Kobrinsky Z Liu and N M Soldatov ldquoOligomer-ization of Cav120573 subunits is an essential correlate of Ca

2+ channelactivityrdquoThe FASEB Journal vol 24 no 12 pp 5013ndash5023 2010

[25] E Kobrinsky S Tiwari V A Maltsev et al ldquoDifferential roleof the 1205721C subunit tails in regulation of the Cav12 channelby membrane potential 120573 subunits and Ca2+ ionsrdquo Journal ofBiological Chemistry vol 280 no 13 pp 12474ndash12485 2005

[26] S Tiwari Y Zhang J Heller D R Abernethy and N MSoldatov ldquoArtherosclerosis-related molecular alteration of thehuman Cav12 calcium channel 1205721C subunitrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 103 no 45 pp 17024ndash17029 2006

[27] I Splawski K W Timothy L M Sharpe et al ldquoCav12 calciumchannel dysfunction causes a multisystem disorder includingarrhythmia and autismrdquo Cell vol 119 no 1 pp 19ndash31 2004

[28] Z Z TangM C Liang S Lu et al ldquoTranscript scanning revealsnovel and extensive splice variations in human L-type voltage-gated calcium channel Cav12 1205721 subunitrdquo Journal of BiologicalChemistry vol 279 no 43 pp 44335ndash44343 2004

[29] P Liao T F Yong M C Liang D T Yue and T W SoongldquoSplicing for alternative structures of Cav12 Ca2+ channels incardiac and smooth musclesrdquo Cardiovascular Research vol 68no 2 pp 197ndash203 2005

[30] Y Blumenstein N Kanevsky G Sahar R Barzilai T Ivaninaand N Dascal ldquoA novel long N-terminal isoform of human L-type Ca2+ channel is up-regulated by protein kinase Crdquo Journalof Biological Chemistry vol 277 no 5 pp 3419ndash3423 2002

[31] N M Soldatov A Bouron and H Reuter ldquoDifferent voltage-dependent inhibition by dihydropyridines of human Ca2+channel splice variantsrdquo Journal of Biological Chemistry vol270 no 18 pp 10540ndash10543 1995

[32] D P Faxon M A Creager S C Smith et al ldquoAtheroscleroticvascular disease conference executive summary atheroscle-rotic vascular disease conference proceeding for healthcareprofessionals from a special writing group of the AmericanHeart Associationrdquo Circulation vol 109 no 21 pp 2595ndash26042004

[33] R Ross ldquoAtherosclerosismdashan inflammatory diseaserdquo The NewEngland Journal of Medicine vol 340 no 2 pp 115ndash126 1999

[34] H Roy S Bhardwaj and S Yla-Herttuala ldquoMolecular geneticsof atherosclerosisrdquo Human Genetics vol 125 no 5-6 pp 467ndash491 2009

[35] M Biel P Ruth E Bosse et al ldquoPrimary structure and func-tional expression of a high voltage activated calcium channelfrom rabbit lungrdquo FEBS Letters vol 269 no 2 pp 409ndash4121990

[36] X Cheng J Liu M Asuncion-Chin et al ldquoA novel Cav12N terminus expressed in smooth muscle cells of resistancesize arteries modifies channel regulation by auxiliary subunitsrdquoJournal of Biological Chemistry vol 282 no 40 pp 29211ndash292212007

[37] W J Koch P T Ellinor and A Schwartz ldquocDNA cloning ofa dihydropyridine-sensitive calcium channel from rat aortaevidence for the existence of alternatively spliced formsrdquo Journalof Biological Chemistry vol 265 no 29 pp 17786ndash17791 1990

[38] S Richard D Neveu G Carnac P Bodin P Travo and J Nar-geot ldquoDifferential expression of voltage-gated Ca2+-currents incultivated aortic myocytesrdquo Biochimica et Biophysica Acta vol1160 no 1 pp 95ndash104 1992

[39] A Schwartz ldquoCalcium antagonists review and perspective onmechanism of actionrdquo American Journal of Cardiology vol 64no 17 pp 3Indash9I 1989

[40] R P Mason ldquoMechanisms of plaque stabilization for thedihydropyridine calcium channel blocker amlodipine review ofthe evidencerdquo Atherosclerosis vol 165 no 2 pp 191ndash199 2002

[41] A Ruiz-Torres R Lozano J Melon and R Carraro ldquoL-calciumchannel blockade induced by diltiazem inhibits proliferationmigration and F-actin membrane rearrangements in humanvascular smooth muscle cells stimulated with insulin and IGF-1rdquo International Journal of Clinical Pharmacology andTherapeu-tics vol 41 no 9 pp 386ndash391 2003

[42] H Koshiyama S Tanaka and J Minamikawa ldquoEffect ofcalcium channel blocker amlodipine on the intimal-medialthickness of carotid arterial wall in type 2 diabetesrdquo Journal ofCardiovascular Pharmacology vol 33 no 6 pp 894ndash896 1999

[43] T Yamashita S KawashimaMOzaki et al ldquoA calcium channelblocker benidipine inhibits intimal thickening in the carotidartery of mice by increasing nitric oxide productionrdquo Journal ofHypertension vol 19 no 3 pp 451ndash458 2001

[44] R P Mason P Marche and T H Hintze ldquoNovel vascularbiology of third-generation L-type calcium channel antagonistsancillary actions of amlodipinerdquo Arteriosclerosis Thrombosisand Vascular Biology vol 23 no 12 pp 2155ndash2163 2003

[45] O Stepien J GogusevD L Zhu et al ldquoAmlodipine inhibition ofserum- thrombin- or fibroblast growth factor- induced vascu-lar smooth-muscle cell proliferationrdquo Journal of CardiovascularPharmacology vol 31 no 5 pp 786ndash793 1998

[46] V Ruiz-Velasco M B Mayer E W Inscho and L J HymelldquoModulation of dihydropyridine receptors in vascular smoothmuscle cells by membrane potential and cell proliferationrdquoEuropean Journal of Pharmacology vol 268 no 3 pp 311ndash3181994

[47] J Nilsson M Sjolund L Palmberg A M von Euler B Jonzonand J Thyberg ldquoThe calcium antagonist nifedipine inhibitsarterial smooth muscle cell proliferationrdquo Atherosclerosis vol58 no 1ndash3 pp 109ndash122 1985

[48] J Thyberg and L Palmberg ldquoThe calcium antagonist nisoldip-ine and the calmodulin antagonist W-7 synergistically inhibitinitiation of DNA synthesis in cultured arterial smooth musclecellsrdquo Biology of the Cell vol 60 no 2 pp 125ndash132 1987

[49] E Munro M Patel P Chan et al ldquoEffect of calcium channelblockers on the growth of human vascular smooth musclecells derived from saphenous vein and vascular graft stenosesrdquoJournal of Cardiovascular Pharmacology vol 23 no 5 pp 779ndash784 1994

[50] T A Kent A Jazayeri and J M Simard ldquoCalcium channelsand nifedipine inhibition of serotonin-induced [3H]thymidineincorporation in cultured cerebral smoothmuscle cellsrdquo Journalof Cerebral Blood Flow and Metabolism vol 12 no 1 pp 139ndash146 1992

[51] A Agrotis P J Little J Saltis and A Bobik ldquoDihydropyridineCa2+ channel antagonists inhibit the salvage pathway for DNAsynthesis in human vascular smooth muscle cellsrdquo EuropeanJournal of Pharmacology vol 244 no 3 pp 269ndash275 1993

[52] I Duque M R Puyol P Ruiz M Gonzalez-Rubio M LD Marques and D R Puyol ldquoCalcium channel blockers

ISRN Biochemistry 13

inhibit hydrogen peroxide-induced proliferation of culturedrat mesangial cellsrdquo Journal of Pharmacology and ExperimentalTherapeutics vol 267 no 2 pp 612ndash616 1993

[53] T Kuga S Kobayashi Y Hirakawa H Kanaide and ATakeshita ldquoCell cycle-dependent expression of L- and T-typeCa2+ currents in rat aortic smooth muscle cells in primaryculturerdquo Circulation Research vol 79 no 1 pp 14ndash19 1996

[54] E M Graf M Bock J F Heubach et al ldquoTissue distribution ofa humanCav12 1205721 subunit splice variant with a 75 bp insertionrdquoCell Calcium vol 38 no 1 pp 11ndash21 2005

[55] P Liao D Yu S Lu et al ldquoSmoothmuscle-selective alternativelyspliced exon generates functional variation in Cav12 calciumchannelsrdquo Journal of Biological Chemistry vol 279 no 48 pp50329ndash50335 2004

[56] O Skalli P Ropraz and A Trzeciak ldquoA monoclonal antibodyagainst 120572-smooth muscle actin a new probe for smooth muscledifferentiationrdquo Journal of Cell Biology vol 103 no 6 pp 2787ndash2796 1986

[57] T M Doherty K Asotra L A Fitzpatrick et al ldquoCalcificationin atherosclerosis bone biology and chronic inflammation atthe arterial crossroadsrdquo Proceedings of the National Academyof Sciences of the United States of America vol 100 no 20 pp11201ndash11206 2003

[58] G K Owens ldquoRegulation of differentiation of vascular smoothmuscle cellsrdquo Physiological Reviews vol 75 no 3 pp 487ndash5171995

[59] J P Bannister A Adebiyi G Zhao et al ldquoSmooth musclecell 1205722120575-1 subunits are essential for vasoregulation by Cav12channelsrdquo Circulation Research vol 105 no 10 pp 948ndash9552009

[60] S SonkusareM Fraer J DMarsh andN J Rusch ldquoDisruptingcalcium channel expression to lower blood pressure newtargeting of awell-known channelrdquoMolecular Interventions vol6 no 6 pp 304ndash310 2006

[61] S Telemaque S Sonkusare TGrain et al ldquoDesign ofmutant1205732subunits as decoy molecules to reduce the expression of func-tional Ca2+ channels in cardiac cellsrdquo Journal of Pharmacologyand Experimental Therapeutics vol 325 no 1 pp 37ndash46 2008

[62] A Goyenvalle A Vulin F Fougerousse et al ldquoRescue of dys-trophic muscle through U7 snRNA-mediated exon skippingrdquoScience vol 306 no 5702 pp 1796ndash1799 2004

[63] D BHalling P Aracena-Parks and S L Hamilton ldquoRegulationof voltage-gated Ca2+ channels by calmodulinrdquo Sciencersquos STKEvol 2005 no 315 p re15 2005

[64] K S Lee E Marban and R W Tsien ldquoInactivation of calciumchannels in mammalian heart cells joint dependence on mem-brane potential and intracellular calciumrdquo Journal of Physiologyvol 364 pp 395ndash411 1985

[65] C Shi and N M Soldatov ldquoMolecular determinants of voltage-dependent slow inactivation of the Ca2+ channelrdquo Journal ofBiological Chemistry vol 277 no 9 pp 6813ndash6821 2002

[66] E Kobrinsky E Schwartz D R Abernethy and NM SoldatovldquoVoltage-gated mobility of the Ca2+ channel cytoplasmic tailsand its regulatory rolerdquo Journal of Biological Chemistry vol 278no 7 pp 5021ndash5028 2003

[67] N M Soldatov ldquoCa2+ channel moving tail link between Ca2+-induced inactivation and Ca2+ signal transductionrdquo Trends inPharmacological Sciences vol 24 no 4 pp 167ndash171 2003

[68] D J Klemm P A Watson M G Frid et al ldquocAMP responseelement-binding protein content is a molecular determinantof smooth muscle cell proliferation and migrationrdquo Journal ofBiological Chemistry vol 276 no 49 pp 46132ndash46141 2001

[69] E Kobrinsky L Stevens Y Kazmi DWray andNM SoldatovldquoMolecular rearrangements of the Kv21 potassium channeltermini associated with voltage gatingrdquo Journal of BiologicalChemistry vol 281 no 28 pp 19233ndash19240 2006

[70] BMMayr G Canettieri andMRMontminy ldquoDistinct effectsof cAMP and mitogenic signals on CREB-binding proteinrecruitment impart specificity to target gene activation viaCREBrdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 98 no 19 pp 10936ndash10941 2001

[71] M Morad and N Soldatov ldquoCalcium channel inactivationpossible role in signal transduction and Ca2+ signalingrdquo CellCalcium vol 38 no 3-4 pp 223ndash231 2005

[72] DGWheeler C F Barrett R DGroth P Safa andRW TsienldquoCaMKII locally encodes L-type channel activity to signal tonuclear CREB in excitation-transcription couplingrdquo Journal ofCell Biology vol 183 no 5 pp 849ndash863 2008

[73] D E Mager E Kobrinsky A Masoudieh A Maltsev D RAbernethy andNM Soldatov ldquoAnalysis of functional signalingdomains from fluorescence imaging and the two-dimensionalcontinuous wavelet transformrdquo Biophysical Journal vol 93 no8 pp 2900ndash2910 2007

[74] E Kobrinsky D E Mager S A Bentil S I Murata DR Abernethy and N M Soldatov ldquoIdentification of plasmamembrane macro- and microdomains from wavelet analysis ofFRET microscopyrdquo Biophysical Journal vol 88 no 5 pp 3625ndash3634 2005

[75] E Kobrinsky S Q Duong A Sheydina and N M Solda-tov ldquoMicrodomain organization and frequency-dependence ofCREB-dependent transcriptional signaling in heart cellsrdquo TheFASEB Journal vol 25 no 5 pp 1544ndash1555 2011

[76] A Ravindran Q Z Lao J B Harry P Abrahimi E Kobrinskyand N M Soldatov ldquoCalmodulin-dependent gating of Cav12calcium channels in the absence of Cav120573 subunitsrdquo Proceedingsof the National Academy of Sciences of the United States ofAmerica vol 105 no 23 pp 8154ndash8159 2008

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

ISRN Biochemistry 3

Synthesis of DNA ( of control)

bFGF

EGF

Insulin

Serum-free

10 serum

0 50 100 150 200

0 05 1 15 2

Control

[3H]PMD binding

Figure 2 Effect of mitogens on the density of DHP receptors (openbars) and cell proliferation (dark bars) in normal human fibroblastsOn day 2 after cell plating the growth medium was replaced for4 days with serum-free Eaglersquos medium supplemented with 01BSA Cells were incubated for 4 days in the presence of 10 serum(control) 01 BSA (serum-free) epidermal growth factor (EGF15 ngmL) insulin (10 120583gmL) or basic fibroblast growth factor(bFGF 2 ngmL) The density of DHP receptors was measuredat 2 nM [3H]-PMD as pmol [3H]-PMD specifically bound per106 cells of nonconfluent monolayers (3ndash5 times 103 cellscm2) Cellproliferation was estimated from the synthesis of DNAmeasured by[14C]thymidine incorporation

(Figure 2 dark bars) that individual mitogens stimulated cellproliferation in the order bFGF gt insulin gt EGF In contrastin that very order the same mitogens suppressed expressionof Cav12 in fibroblasts as evidenced by the measurement ofDHP receptor binding (open bars) Consistent with relativelylow cell toxicity of dihydropyridines blockade of Cav12attenuated entry of cells into the S phase of cell cycle [18]Thus the plasticity of Cav12 in fibroblast proliferation is asso-ciated with transient Cav12 expression responsive to bothprimary effectors and secondmessengers of cell proliferation

3 Cak12 Variability

One of themost important features of Cav12 is its remarkablemolecular diversityThe channel consists of the pore-forming1205721C protein which binds calcium channel blockers Three

types of regulatory subunits 120573 1205722120575 and calmodulin (CaM)

are constitutively tethered to 1205721C All these subunits are

products of different genes that are located on differentchromosomes Cav12 shares the accessory subunits withother Cav1 and Cav2 channels Moreover 120572

1C 120573 and 1205722120575 aresubject to individual alternative splicing that generates a largediversity of Cav12 channel complexes The functional signif-icance of the Cav12 splice variation is poorly understood not

only because of the difficulties in identification of individualsplice variants in the naturally occurring oligomeric proteinsThe tendency of Cav12 to form large clusters in the plasmamembrane [19ndash23] as well as homo- and heterooligomeriza-tion of 120573 subunits [24] create additional major challenges forthe investigation of Cav12 in regulation of cell proliferationdifferentiation and other functionsmdashnot to speak of anadequate elucidation of their roles in disease and search fornew therapeutic approaches

Molecular cloning showed that 1205721C transcripts in human

fibroblasts are composed of exons 1ndash50 [14] with alternativesplicing of exons 2122 and 3132 and constitutive splicingof exons 33 and 45 (Figure 3(a)) [12] However variabilityassociated with exons 1 7 8 9 34 41 and 42 that were laterfound in human hippocampus heart and VSM cells [25ndash30]was not observed in fibroblasts

It is not known which Cav12 splice variants are expressedin fibroblasts in response to serum deprivation and whetherthey are structurally different from those present in pro-liferating cells However electrophysiological properties ofthe three of fibroblast 120572

1C splice variants (coexpressed with1205731a and 1205722120575-1) were compared in Xenopus oocyte expression

system [31] Characteristics of the voltage-dependence andkinetics of inactivation of the barium current through the1205721C70 channel encoded by the CACNA1C transcript lacking

exons 22 31 41A and 45 were found to be very similar tothose recorded with 120572

1C77 (lacking exons 21 31 41A and45) and 120572

1C76 (also lacking exon 33) (Figure 3(b)) Howevervoltage dependence of the DHP inhibition of the currentwas significantly different in the 120572

1C70 and 1205721C77 channelsthe IC

50values for the concentration dependence of the

barium current inhibition by (+)isradipine almost identical(62 and 73 nM resp) at minus40mV were significantly differentat minus90mV (680 and 79 nM for 120572

1C70 and 1205721C77 resp) Whilesuch a difference in the pharmacological properties of theexon 21- and exon 22-splice variants is deemed unimportantin the case of fibroblasts it may significantly contribute tothe tissue specificity of this major class of calcium channelblockers in cardiac vascular and other responsive cellsaffected by cardiovascular diseases

4 Cak12 in Atherosclerosis

Atherosclerosis is perhaps the single most deadly diseaseleading to about 600000 deaths annually in the USA mostof these due to the progression of the disease to heartattack or stroke [32] In spite of significant efforts themolecular mechanisms of atherosclerosis are not currentlywell understood and effective molecular targets for preven-tion and treatment are not elaborated Atherosclerosis is aninflammatory process in medium and large size arteries thatcauses endothelial perturbation and local release of cytokinesas well as dedifferentiation proliferation and migration ofVSM cells [33] Arterial VSM cells constitute the media of theartery and play a crucial role in its elasticity and contractilityMigration of VSM cells from the media to the intima of thearterial wall and proliferation of intimal smooth muscle cellsare the major early events in the formation of atheroscleroticlesions Recent advances in molecular genetics studies have

4 ISRN Biochemistry

II III IV

2

3

4

5

6

788A

9

10

1125

12 13 14 15

16

17 18

19

26

23

24 28

27

2934

36

2122

Plasmamembrane

Out

In

33

37+

+

38

39

40

HOOC

4443

454647484950

11A3132 41 4241A

9A

34A30

I

LA IQ

AID

65

++++4

++++

3 51 4

++++

3 51 2 4 26

++++

3 51 2 4

2035

6 6 31 2

H2N

(a)

20 2321 22 30 31 32 33 34

20 2321 22 30 31 32 33 34

20 2321 22 30 31 32 33 34

40 41A 41 44 45 46

40 41A 41 44 45 46

40 41A 41 44 45 46

20 2321 22 30 31 32 33 34

20 2321 22 30 31 32 33 34 40 41A 41 44 45 46

40 41A 41 44 45 46

20 2321 22 30 31 32 33 34 40 41A 41 44 45 46

1205721C69

1205721C70

1205721C71

1205721C73

1205721C76

1205721C77

(b)

Figure 3 Molecular diversity of Cav12 (a) Transmembrane topology of the Cav12 1205721C subunit in schematic diagram Four regions ofhomology (IndashIV) each composed of 6 transmembrane segments (numbered) are folded around the central pore The polypeptide sequenceis segmented by black bars and sequentially numbered (1ndash50) according to the CACNA1C genomic map [14] The segments correspondingto the invariant (blackgray) and alternative (blue) exons are outlined 120572-interaction domain (AID) of a constitutive 120573-binding is shown ingreen In the CaM-binding domain (CBD) apo-CaM and Ca2+-CaM are shared between LA and IQmotifs (red) respectively (b) Alternativesplicing of human fibroblast CACNA1C in the region of exons 20ndash46 The following splice variants were studied 120572

1C69 (GenBank z34809splice variant lacking alternative exons (Δ) 21 32 41A and 45) 120572

1C70 (z34810 Δ 22 31 41A and 45) 1205721C71 (z34811 Δ 22 31 and 45) 120572

1C73(z34812 Δ 22 31 33 and 45) 120572

1C76 (z34814 Δ 21 31 33 41A and 45) and 1205721C77 (z34815 Δ 21 31 41A and 45)

revealed that genetic polymorphisms significantly influencesusceptibility to atherosclerotic vascular diseases [34] How-ever none of the discovered susceptibility genes was directlyimplicated for proliferation and migration of VSM cells oneof the major pathophysiological responses to atherosclerosisat the cellular level

The presence and activity of Cav12 calcium channels inVSM cells has been established both in patch clamp andmolecular cloning experiments [29 35ndash38] Cav12 calciumchannels play a major role in atherosclerosis because they

are essential for Ca2+ signal transduction in VSM cellsContraction of VSM cells is triggered by the Ca2+ current(119868Ca) through Cav12 and thus is affected by Ca2+ channelblockers Since the 1990s it is known that DHPs particularlythe charged 2-aminoethoxymethyl DHP derivative amlodip-ine [39] exert a number of vasoprotective effects includingpotent antiatherogenic action [40] inhibition of migrationof VSM cells [41] and reduction of arterial intimal-medialthickening and plaque formation [42 43] Although it istempting to link this activity in part to pleiotropic effects of

ISRN Biochemistry 5

calcium channel blockers [44] it is the inhibition of prolifer-ation that essentially underlies it [45] The density of DHPreceptors was found to depend on VSM cell proliferation[46] Association of Cav12 with mitogenesis in VSM cells issupported by the findings that DHPs reduced DNA synthesisstimulated by serum and PdGF [47ndash49] serotonin [50] EGF[51] and H

2O2(in mesangial cells) [52] Expression of Cav12

in VSM cells was shown to be cell cycle dependent with thehighest calcium current density in G

1phase [53] Whether

these changes are reflected in the molecular repertoire ofthe Cav12 splice variants is the issue particularly importantfor the elucidation of new therapeutic targets in diseasesleading to pathogenic proliferation of VSM cells such asatherosclerosis

Investigation of the 1205721C alternative splicing in VSM

cells [28 54 55] revealed the involvement of a number ofCACNA1C exons generating impressive diversity of humanvascular 120572

1C that includes possibly the VSM-specific splic-ing of exons 99a [35 55] and exon 34 [28] To establishan association between the disease and CACNA1C splicevariation at the level of cell we have completed [26] thesingle-gene profiling of the 120572

1C molecular remodeling inVSM cells of an artery caused by atherosclerosis The VSMcells were identified in frozen sections of six surgical biopsysamples of femoral and carotid arteries by immunostainingwith an antibody against smoothmuscle 120572-actin [56] used asa marker for VSM cells The 120572-actin staining correlated withimmunostaining by an anti-120572

1C antibody in serial sectionsand was reduced in atherosclerotic regions (Figure 4) consis-tent with dedifferentiation of VSM cells [57 58]The reducedexpression of 120572

1C at the protein level was corroborated bythe quantitative RT-PCR data showing that the relative 120572

1CmRNA level in VSM cells (normalized to 18S RNA) wasreduced 37 plusmn 09 fold (mean plusmn SEM) in the atheroscleroticregion Overall the reduced expression of 120572

1C caused by thelocally elaborated cytokines in the atherosclerotic regions ofarteries resembles the reduced expression of DHP receptorsobserved in fibroblasts exposed to mitogens andor secondmessengers after serum deprivation (Figure 1)

To find out whether the altered expression of 1205721C

in atherosclerotic VSM cells is accompanied by changesin the CACNA1C alternative splicing pattern we isolatedthe immunohistochemically identified VSM cells by laser-capture microdissection from adjacent regions of arteriesaffected and not affected by atherosclerosis and identifiedthe CACNA1C splice variants by RT-PCR Our findingsrevealed an extended repertoire of the exon 21 120572

1C spliceisoforms in nonatherosclerotic VSM cells characterized bya complex splicing pattern of exons 9 9A 31ndash34 and41A including the electrophysiologically characterized 120572

1C71(GenBank z34811) 120572

1C73 (z34812) 1205721C125 (AY830711)

1205721C126 (AY830713) and 1205721C127 (AY830712) splice isoforms

However only the exon 22 isoform of 1205721C (1205721C77) was

identified in atherosclerotic VSM cells Thus the switch ofthe CACNA1C alterative splicing from exon 21 to exon 22(Figure 3(b)) is a molecular signature of the Cav12 remod-eling of VSM cells to a pathophysiological proliferating statein atherosclerosis The age gender ethnicity drug exposureand other co-morbid conditions did not appreciably affect

this common pattern of the 1205721C splice variation in VSM cells

in response to atherosclerosisCareful electrophysiological analysis exhibited a number

of differences in the properties of the ldquoatheroscleroticrdquo 1205721C77

channel as compared to the 1205721C isoforms in healthy VSM

cells The largest differences were found between the 1205721C77

and 1205721C127 channels (Figure 5) In response to step depolar-

ization applied from the holding potential of minus90mV bothchannels generate calcium currents (119868Ca) that inactivate withalmost identical kinetics (Figure 5(a)) However we foundthat 119868Ca through the1205721C77 channel recovers from inactivationsignificantly faster than that in 120572

1C127 (Figure 5(b)) and other1205721C isoforms present in healthy VSM cells This finding

suggests that alternative splicing in atherosclerosis may affectvascular tone as a result of the increase in the 119868Ca density inVSM cells A hyperpolarization shift of the activation curvefor the atherosclerotic 120572

1C77 channel variant as comparedto Cav12 in healthy VSM cells (Figure 5(c)) may also resultin an increase of calcium entry in VSM cells Howeverthe overall 3-4-fold reduction in the expression of Cav12in atherosclerotic VSM cells may scale down some of theobserved electrophysiological changes

5 CACNA1C Exon 22 as a New TherapeuticTarget in Atherosclerosis

Direct DNA sequencing of the crude PCR amplificationproducts indicated that the switch to the exon 22 isoform ofvascular 120572

1C was almost complete in atherosclerosis becauseno distortion of the nucleotide peaks in the region of exon2122 was seen when compared to the exon 20 invariantregion (Figure 6(a)) Thus Cav12 underwent almost quanti-tative exon 2122 remodeling in VSM cells of diseased arteryregions

Although the cellular mechanisms leading to theCACNA1C exon 2122 switch may be very complex theassociation with VSM cell proliferation is obvious Indeeda similar exon switch was observed in primary humanaortic cells in culture after the quiescent nonproliferatingcells containing predominantly exon 21 splice variantswere exposed to serum (Figure 6(b)) Unlike exon 21exon 22 contains the AvrII restriction site that allowsfor the assessment of its presence in PCR amplificationproduct of CACNA1C transcripts isolated from the cellsThe AvrII-sensitive exon 22 isoform of the 120572

1C transcriptwas not detected in the quiescent nonproliferating aorticcells (Figure 6(b) lane 2) However when 5 serum wasadded to the medium with nonconfluent aortic cellsDNA biosynthesis was activated while the level of the1205721C transcript decreased asymp3 fold and the presence of the

AvrII-sensitive exon 22 isoforms of 1205721C was easily detected

(Figure 6(b) lane 4) The isoform remodeling simulated inaortic primary cells in vitro was not complete as comparedto VSM cells in atherosclerotic regions of artery occludedwith heavy plaque burden which were selected for the1205721C molecular profiling However the cell culture results

demonstrate that in a different experimental system there isan obvious association between proliferation of VSM cells

6 ISRN Biochemistry

A Ki-67 NKi-67

50 12058350 120583

(a)

A N120572-actin 120572-actin

(b)

A N1205721C1205721C

(c)

Figure 4 Reduced expression of 1205721C in atherosclerotic (A) region of aorta as compared to the adjacent nonaffected (N) region From (a) to

(c) detection of proliferating cells in an exemplar biopsy of an artery by an antibody against human nuclear protein Ki-67 (a) Red arrowshighlight some proliferating nuclei (black staining) while blue arrows exemplify nonproliferating nuclei (light grey) Staining with antibodiesagainst smooth muscle 120572-actin (b) and against 120572

1C (c) Scale bars 50 120583m

downregulation of the CACNA1C expression and synthesisof the exon 22 120572

1C isoformRecent strategies targeting VSM cells to treat cardiovas-

cular diseases suggest indiscriminate disruption of Cav12[59ndash61] Is it possible to correct the described CACNA1Csplice defects induced by atherosclerosis without affectingthe transcripts of the gene lacking the rdquopathogenicrdquo exon22 Correction of defective genes responsible for diseasedevelopment is achieved by gene therapy Usually it requiresan insertion into the genome of a normal gene in place ofa defective one causing disease Such technique howeveris poorly controlled Currently one of the most promising

cutting-edge therapeutic approaches to correct defects asso-ciated with disease-induced expression of abnormal splicevariants is antisense-mediated exon skipping It is basedon the use of antisense oligonucleotides targeting specificexons to be removed The adenovirus-directed 120572

1C exon 22skipping-induced inhibition of VSM cell proliferation (andrespectively migration) can be used to rescue VSM cells fromremodeling in atherosclerosis The 120572

1C exon 22-skippingwill not alter the open reading frame of the 120572

1C transcriptbecause alternative exons 21 and 22 are of the equal size(60 nt)Themodified nonspliceosomal snRNAU7 gene alongwith its natural promoter and 31015840 elements exon 22-antisense

ISRN Biochemistry 7

200 ms

1205721C77

1205721C127

(a)

Prepulse interval (s)02 04 06 08 1

0

1

08

06

04

02

0

Frac

tion

of119868 C

a

1205721C77

1205721C127

(b)

0

1

08

06

04

02

119866Ca

119866Ca

max

1205721C77

1205721C127

minus60 minus40 minus20 0 20 40(mV)

(c)

Figure 5 Comparison of electrophysiological properties of 119868Ca through the ldquoatheroscleroticrdquo 1205721C77 and ldquonormalrdquo 120572

1C127 channels expressedin Xenopus oocytes with 120572

2120575-1 subunit and the primary cardiac 1205732a subunit and measured with 25mM Ca2+ as the charge carrier (a)

Representative traces of 119868Ca evoked by 1 s step depolarizations to +20mV from 119881ℎ= minus90mV and normalized to the same amplitude (b)

Fractional recovery of 119868Ca from inactivation (c) Averaged activation (119866119866max-119881) curves fit by the Boltzmann function A 1 s test pulses in therange of minus40 to +50mV (10-mV increments) were applied from 119881

ℎ= minus90mV with 30-s intervals

(a)

AvrII

Serum

1 2 3 4

+minus+minus

minus minus + +

142 pb

459 pb

601 pb

(b)

Figure 6 (a) Trace diagrams of DNA sequencing of the 1205721C PCR amplification products of healthy (top row) and diseased VSM cells (bottom

row) using an antisense primer composed of nucleotides 2923ndash2939 (z34815) A boundary with the sequence of invariant exon 20 is markedby a vertical dotted line (b) Evidence that the AvrII-sensitive exon 22 isoform of 120572

1C is expressed only in proliferating VSM cells Primaryaortic cells were grown to confluent monolayer in 5 serum before serum-deprivation for 5 days (lanes 1 and 2)Then the cells were replatedat low density in 5 serum for 3-4 days (lanes 3 and 4) Total RNA was isolated and exon 2122 isoforms were identified by RT-PCR andAvrII restriction analysis (lanes 2 and 4)

8 ISRN Biochemistry

sequence and supplemented with Sm ribonucleoprotein-binding sequence may be incorporated into the adenovirusvector for high efficiency transfer [62]The cytokine receptors(eg PdGF-120573 receptor) based recognition targeting of viralliposomes or nanoparticles may be especially advantageousin connection with selective gene transfer to VSM cellsaffected by atherosclerosis while reducing the probability ofthe transfection of other cells

6 Cak12 and CREB-DependentTranscriptional Activation

How Cav12 activity is translated into a proliferation-effectedmodality is another important question to be asked Cav12calcium channels generate a transient rise in cytosolic Ca2+-concentration activated by membrane depolarization Cel-lular responses associated with the rise of [Ca2+]i rangefrom sarcomeric contraction to cell growth and proliferationCytoplasmic domains of Cav12 have evolved a fairly intricateCaM-dependent signaling mechanism that provides for thenegative feedback inhibition of the calcium current knownas Ca2+ dependent inactivation (CDI) which is mediated bydifferent determinants of 120572

1C [25 63] Such a mechanism ofCDI resulting in acceleration of 119868Ca inactivation in responseto the rise of intracellular Ca2+ was first identified in cardiacCav12 [64] Similar experiments performed on the recombi-nant Cav12 also showed that the replacement of extracellularCa2+ by Ba2+ eliminates CDI and the channel inactivates by aslower voltage-dependent mechanism [65]The very fact thattwo distantly located determinants one in the pore regionresponsible for slow inactivation and the CaM-binding onein the proximal locus of the C-tail are independently crucialfor CDI indicates that not only their specific molecularstructure but also their mutual folding andor interaction areessential Experimental evidence show that this interactionreacts dynamically to membrane voltage supporting state-dependent transitions of the channel between resting openand inactivated conformations which are essential for CREB-dependent transcriptional activation [66 67]

CREB is a transcription factor of general importance ina large variety of cells CREB phosphorylation promotes theactivation of genes and is regulated by protein kinases undercontrol of the major second messengers cAMP andor Ca2+Indeed CREB functions as a ldquomolecular determinant of VSMcells fate [68]rdquo CREB content depends on proliferation ofVSM cells both in situ and in culture Serum deprivationincreased CREB content in VSM cells while exposure toPdGF decreased it Consistent with this observation anoverexpression of the constitutively active CREB inVSM cellsarrested cell cycle progression

To investigate the association of Cav12 with CREB tran-scriptional activation at the molecular level we combinedpatch clamp with fluorescent resonance energy transfer(FRET) microscopy in the live cell In voltage clampedcells FRET provided optical measurements under state-dependent conditions showing that the shorter N-terminaltail of 120572

1C (eg 1205721C77) does not rearrange vis-a-vis the

plasma membrane in response to voltage gating [25] In

sharp contrast the C-tail shows voltage-dependent confor-mational rearrangements which are much larger in sizethan that for example in the potassium Kv21 channel[69] Measurements of 119868Ca and corrected FRET betweenthe enhanced yellow (EYFP) and cyan fluorescent proteins(ECFP) genetically attached respectively to the N- and C-termini of 120572

1C77 showed no significant effect on voltage-dependence and kinetics of the channel current Howevertherewas a substantial increase in FRET signal accompanyinginactivation of the channel that was fully reversible uponits transition into the resting state in response to hyper-polarization [66] The plasma-membrane anchoring of the1205721C C-tail by the fusion of the pleckstrin homology domain

(PH) eliminated CDI but not 119868Ca Do the voltage-gatedconformational rearrangements of the 120572

1C C-tail and CDIplay a role in Ca2+ signal transduction that is utilized inCa2+-induced activation or CREB-dependent transcriptionTo answer this question we used the test system based onthe measurement of interaction between KID domain ofCREB and KIX domain of coactivator CREB-binding protein(CBP Figure 7(A) inset) under voltage-clamp conditionsby monitoring FRET between EYFP-KID and ECFP-KIX[70] Ca2+-dependent phosphorylation of KID stimulates itsbinding to KIX bringing EYFP and ECFP close enoughto observe the interaction by FRET In perforated wholeclamped cell where the integrity of the cytoplasmic contentis intact (Figure 7(A)) no activation of CREB transcription(Figure 7(B) panel (a)) and rearrangement of the120572

1C77 C-tail(panel (b)) was observed when the C-tail was anchored to theplasma membrane CREB transcriptional activity remainedlow in spite of a large sustained inward 119868Ca (panel (d))and the corresponding increase in [Ca2+]i detected by thefluorescence of Ca2+ indicator Fura4 (panel (c)) Releaseof the 120572

1C77 C-tail by the activation of PIP2hydrolysis

upon activation of M1AchR (Figure 7(C)) at minus90mV causedsignificant elevation of [Ca2+]i that also was not utilizedby the cell for CREB transcriptional activation (Figure 7(C)panel (a)) until a depolarizing pulse to +20-mV was appliedand the released 120572

1C77 C-tail was permitted to rearrange(Figure 7(D))This experiment provides compelling evidencethat neither large inward 119868Ca nor the subsequent rise in[Ca2+]i lead to CREB transcription activation unless theconformational rearrangement of the 120572

1C77 subunit C-tailprovides the precise targeting of the Ca2+ signal transduction(Figure 7(D) scheme) [66]

There is general agreement that CaM binds to LA and IQdomains of the 120572

1C C-tail and acts as a sensor that conveysCDI (for review see [63]) The affinity of CaM for bothdomains depends on [Ca2+]i Our data indicate that CDI andCa2+-signal transduction depend on the voltage-gatedmobil-ity of the120572

1C77 C-tail It is therefore reasonable to suggest thatthe LA-domain is a Ca2+-sensitive apo-CaM-mediated lockfor the mechanism of slow voltage-dependent inactivationof the channel [67] Apo-CaM associated with LA is able tocross-link it to another still unidentified apo-CaM bindingsite in the polypeptide bundle underlying the pore As theresult of this specific localization apo-CaMLA ldquolockrdquo ishidden from the cytoplasmic Ca2+ so that for example the

ISRN Biochemistry 9

10

0

2

10

100 pA125 ms

(a) (a) (a)

(b)(c)

(b)(c)

(b)(c)

(B) (C) (D)

C-tail Anchored ReleasedReleased

0 10No FRET No FRET

(d) (d) (d)

Fluo-4

PH

CYN

C

C

NY

C

Y EYFP

C ECFP

PH Plekstrin homologydomain

PHPHY

C

NC

IQ

LA

LAIQ

(A)

Fluo-4 Fluo-4

FRET FRET FRET

CRE

Gene expressionCREBCREB P

CBP

AC

GPCR

PKA

cAMP

CaM

Nucleus

cAMP

Plasmamembrane

Ca2+ Ca2+

Ca2+

+20mV +20mV

minus90mV minus90mV

1205721C77

1198651198650

Cav 12

Ca2+

minus90mVACh (5 mM)

4 120583

Figure 7 Evidence that Ca2+ signal transduction by Cav12 to activate CREB-dependent transcription is mediated by the voltage-gatedmobility of the 120572

1C C-tail carrying the CaM-caged Ca2+ and is not directly associated with the increase of [Ca2+]i CREB activation wasexamined under perforated patch conditions in COS1 cells expressing recombinant atherosclerotic 120572

1C77 channel (A) Phase-contrast imageof the COS1 cell with a shadow of patch pipette The cell was expressing the 120572

1C77-PHC1205731a1205722120575-1 channel with the membrane-trapped 1205721C77

subunit C-tail type 1muscarinic Ach receptor (to release the anchored C-tail in response to activation by Ach) and EYFP-KID and ECFP-KIXdomains both supplemented with nuclear localization sequences Inset at the bottom Cav12- and cAMP-dependent cell signaling pathwaysmediating CREB-dependent transcription CBP CREB-binding protein CRE cAMP-response element (B) Plasma-membrane anchoring ofthe 1205721C77 C-tail (see schematic diagram) inhibits CREB activation in spite of robust 119868Ca evoked by depolarization (a) A 100-ms images of

FRET between EYFP-KID and ECFP-KIX were recorded at the end of the 12th +20-mV depolarization step applied every 10 s for 1 s from119881ℎ=

minus90mV (b) A corresponding representative image of corrected FRET ratio (in a ldquo3Drdquo MetaMorph transformation) recorded within 50-mswindows at minus90 and at the end of the last test pulse of the applied train of pulses (c) An increase of [Ca2+]i detected by Ca

2+ indicator Fluo4(d) Representative trace of 119868Ca (20mM Ca2+ in bath medium) evoked by depolarization to +20mV from119881

ℎ= minus90mV showing the sustained

component of Ca2+ conductance due to the C-tail anchoring (C) ACh-stimulation of theM1AChR to unbind the 1205721C C-tail caused activation

of IP3-dependent Ca2+ release (panel (c)) but did not show substantial activation of CREB transcription at 119881

ℎ= minus90mV (panel (a)) (D)

CREB-dependent transcription was activated only in response to depolarization applied to membrane channel with the 1205721C77 C-terminal tail

released from the plasma membrane to assume functional voltage-dependent conformation

intracellular Ca2+ released from the intracellular stores orCa2+ caging compounds does not accelerate significantly theinactivation of Cav12 [71] Thus apo-CaM associated withLA binds predominantly Ca2+ ions permeating through thepore A Ca2+-dependent transfer of CaM from LA to the IQ-motif opens the ldquolockrdquo and initiates a large rearrangement ofthe C-terminal tail This in turn facilitates inactivation of thechannel The Ca2+CaM complex with the IQ-motif is thentransferred by themobile C-tail to a downstream target of theCa2+-signaling cascade (such as CaMKII [72]) where Ca2+ isreleased as an activating stimulus while CaM switches back

to LA and returns the C-tail to the resting position availablefor the next cycle of Ca2+-signal transduction

Activation of CREB-dependent transcription by the L-type 119868Ca ismediated throughmultiple cell signaling pathwaysUsing FRET probes of CREB activity and 2D wavelet trans-form analysis we applied principles of quantitative biologyto detail the mechanism of Ca2+-activated CREB-dependenttranscription within localized regions (microdomains) ofthe nucleus We reached this goal by applying continuouswavelet analysis in two dimensions with a 2D wavelet asa deconvolution algorithm for FRET microscopy image

10 ISRN Biochemistry

Control

(A)

05

0

1

05

00 20 40 60 800 20 40 60 800 20 40 60 80

(a) (b) (c) (d)

20 40 60 800

0

20

40

60

80 1

C 3 5 10 mincAMP

C 2 10 mincAMP

C 3 10 min

cAMP

C 1 3 10 min

cAMP

5

cAMP stable (5)

(B)

119868Ca (3 min) cAMP (5 min) cAMP (10 min)

119868Ca cAMP (65) 119868Ca stable (15)

119868Ca119868Ca

119868Ca 119868Ca

119868Ca cAMP transient (15)

Figure 8 Microdomain analysis on the additive effect of 119868Ca and cAMP on CREB signaling in COS1 cells expressing the recombinantldquoatheroscleroticrdquo120572

1C77 channel (A) FRET signal within the nucleus during selected time points during 119868Ca stimulation and cAMP applicationOutlined are four types of signaling microdomains identified using 2D Mexican hat wavelet Red circles represent stable microdomains thatpersist through both 119868Ca and cAMP application White and yellow circles showmicrodomains stable activated by cAMP and 119868Ca respectivelyGreen circles represent transient microdomains of CREB signaling activation Axes show pixel numbers (B) Typical appearances of the fourtypes of CREB signaling microdomains activated by 119868Ca andor cAMP application and recorded in their maximal development in relationto the time of applied stimuli C control recorded before stimulation Color bars in (A) and (B) represent FRET values normalized to themaximum

analysis [73] Continuous wavelet analysis is a mathematicaltechnique that allows us to analyze a signal over severaldifferent frequencies across the entire signal [73 74] It isespecially useful for finding heterogeneity in a signal becauseit can easily find where the pattern (ie frequency) of asignal changes In these experiments we for the first timeobtained evidence of CREB signaling microdomains withinthe nucleus that respond differentially to 119868Ca stimulationand cAMP (Figure 8(A)) Results of the study revealed thatCREB-dependent transcriptional signaling occurs in dis-crete signaling microdomains underlying the architecture ofnuclear signaling Continuous activation ofCREB-dependenttranscriptional signaling by cAMP and Ca2+ resulted in agradual increase of the number of microdomains Four dif-ferent categories of cAMP and Ca2+-induced CREB signalingmicrodomains were characterized in COS1 cells expressingrecombinant Cav12 with the ldquoatheroscleroticrdquo 120572

1C77 splice

variant (Figure 8) In up to 65 of the microdomainstranscription was activated in additive manner by cAMPand Ca2+ Approximately 15 of signaling domains wereactivated only by 119868Ca and 5 of domains were activatedonly by cAMP Finally 15 of the domains were transientand activated by both cAMP and 119868Ca (Figure 8(B)) [75]A similar spatiotemporal organization of CREB-dependentsignaling was observed in spontaneously beating neonatalrat cardiomyocytes Although COS1 cells that were used inour experiments shown in Figures 7 and 8 are naturallydeprived of Cav12 [76] they inherited the ability to replicatethe Cav12-dependent activation of CREB signaling with theexogenous recombinant channel Thus this experimentalapproach fits the task which in my opinion is the mostimportant unresolved issue for the coupling of Cav12 toCREB signaling does the splice variation of 120572

1C affect thespatiotemporal organization of CREB-dependent signaling

ISRN Biochemistry 11

in a way that may affect cell proliferation and other crucialfunction

7 Conclusions

Association of Cav12 with regulation of transcription cellproliferation and its pathophysiology as in the case ofatherosclerosis requires detailed investigation of the roles ofthe naturally occurring 120572

1C splice variants It will limit thetraditionally intuitive approach to Cav12 in physiology andhelp to define new principle approaches to the treatment ofvarious Cav12 channelopathy-related dysfunctions above allcardiovascular diseases Humgenex Inc provides consultingand logistic support on a broad range of issues reported inthis review

Abbreviations

CaM CalmodulinCBD Calmodulin-binding domainCDI Ca2+-dependent inactivationDHP DihydropyridineECFP Enhanced cyan fluorescent proteinEYFP Enhanced yellow fluorescent proteinFALI Fluorophore-assisted light inactivationFRET Fluorescent resonance energy transfer[3H]PMD 26-Dimethyl-3-methoxycarbonyl-5-([23-

3H2]-n-propoxycarbonyl)-4-(21015840-

difluoromethoxyphenyl)-14-dihydropyridine

119868Ca Calcium currentPH Pleckstrin homology domainVSM Vascular smooth muscle

References

[1] Y S Lee M M Sayeed and R D Wurster ldquoInhibition ofcell growth and intracellular Ca2+ mobilization in humanbrain tumor cells by Ca2+ channel antagonistsrdquo Molecular andChemical Neuropathology vol 22 no 2 pp 81ndash95 1994

[2] K Sato J Ishizuka C W Cooper et al ldquoInhibitory effect ofcalcium channel blockers on growth of pancreatic cancer cellsrdquoPancreas vol 9 no 2 pp 193ndash202 1994

[3] V Bertrand M J Bastie N Vaysse and L Pradayrol ldquoInhibi-tion of gastrin-induced proliferation of AR4-2J cells by calciumchannel antagonistsrdquo International Journal of Cancer vol 56no 3 pp 427ndash432 1994

[4] JM Taylor andRU Simpson ldquoInhibition of cancer cell growthby calcium channel antagonists in the athymic mouserdquo CancerResearch vol 52 no 9 pp 2413ndash2418 1992

[5] M G Cattaneo M Gullo and L M Vicentini ldquoC2+ and Ca2+channel antagonists in the control of human small cell lung car-cinoma cell proliferationrdquo European Journal of Pharmacologyvol 247 no 3 pp 325ndash331 1993

[6] C Chen M J Corbley T M Roberts and P Hess ldquoVoltage-sensitive calcium channels in normal and transformed 3T3fibroblastsrdquo Science vol 239 no 4843 pp 1024ndash1026 1988

[7] P C Dartsch M Ritter M Gschwentner H J Lang andF Lang ldquoEffects of calcium channel blockers on NIH 3T3

fibroblasts expressing the Ha-ras oncogenerdquo European Journalof Cell Biology vol 67 no 4 pp 372ndash378 1995

[8] M Biel R Hullin S Freundner et al ldquoTissue-specific expres-sion of high-voltage-activated dihydropyridine-sensitive L-typecalcium channelsrdquo European Journal of Biochemistry vol 200no 1 pp 81ndash88 1991

[9] DM Rodman K Reese J Harral et al ldquoLow-voltage-activated(T-type) calcium channels control proliferation of human pul-monary artery myocytesrdquo Circulation Research vol 96 no 8pp 864ndash872 2005

[10] A Panner L L Cribbs G M Zainelli T C Origitano S Singhand R DWurster ldquoVariation of T-type calcium channel proteinexpression affects cell division of cultured tumor cellsrdquo CellCalcium vol 37 no 2 pp 105ndash119 2005

[11] C Chen and P Hess ldquoCalcium channels in mouse 3T3 andhuman fibroblastsrdquo Biophysical Journal vol 51 p 226a 1987

[12] N M Soldatov ldquoMolecular diversity of L-type Ca2+ channeltranscripts in human fibroblastsrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 89 no10 pp 4628ndash4632 1992

[13] A Fujii H Matsumoto S Nakao H Teshigawara and YAkimoto ldquoEffect of calcium-channel blockers on cell prolifera-tion DNA synthesis and collagen synthesis of cultured gingivalfibroblasts derived fromhumannifedipine responders and non-respondersrdquo Archives of Oral Biology vol 39 no 2 pp 99ndash1041994

[14] N M Soldatov ldquoGenomic structure of human L-type Ca2+channelrdquo Genomics vol 22 no 1 pp 77ndash87 1994

[15] V P Shevchenko N F Myasoedov N M Soldatov G JDuburs V V Kastron and I P Skrastins ldquoSynthesis andevaluation of biological activity of two novel tritium-labeledderivatives of riodipinerdquo Journal of Labelled Compounds andRadiopharmaceuticals vol 27 no 6 pp 721ndash730 1989

[16] S M Dudkin S N Gnedoj N N Chernyuk and N MSoldatov ldquo14-dihydropyridine receptor associated with Ca2+channels in human embryonic fibroblastsrdquo FEBS Letters vol233 no 2 pp 352ndash354 1988

[17] A DePover M A Matlib S W Lee et al ldquoSpecific bindingof [3H]nitrendipine to membranes from coronary arteriesand heart in relation to pharmacological effects Paradoxicalstimulation by diltiazemrdquo Biochemical and Biophysical ResearchCommunications vol 108 no 1 pp 110ndash117 1982

[18] N M Soldatov S N Gnedoj N N Chernyuk I A Britanovaand S M Dudkin ldquoMitogenesis and 14-dihydropyridinereceptor associated with Ca2+-channels in human embryonicfibroblastsrdquo Journal of Membrane Biology vol 5 pp 1161ndash11671988 (Russian)

[19] D Lipscombe D V Madison M Poenie H Reuter R Y Tsienand R W Tsien ldquoSpatial distribution of calcium channels andcytosolic calcium transients in growth cones and cell bodies ofsympathetic neuronsrdquo Proceedings of the National Academy ofSciences of the United States of America vol 85 no 7 pp 2398ndash2402 1988

[20] G S Harms L Cognet P H M Lommerse et al ldquoSingle-molecule imaging of L-type CA2+ channels in live cellsrdquoBiophysical Journal vol 81 no 5 pp 2639ndash2646 2001

[21] V di Biase G J Obermair Z Szabo et al ldquoStable membraneexpression of postsynaptic Cav12 calcium channel clustersis independent of interactions with AKAP79150 and PDZproteinsrdquo Journal of Neuroscience vol 28 no 51 pp 13845ndash13855 2008

12 ISRN Biochemistry

[22] M F Navedo E P Cheng C Yuan et al ldquoIncreased coupledgating of L-type Ca2+ channels during hypertension and timo-thy syndromerdquoCirculationResearch vol 106 no 4 pp 748ndash7562010

[23] E Kobrinsky P Abrahimi S Q Duong et al ldquoEffect of Cav120573subunits on structural organization of Cav12 calcium channelsrdquoPLoS One vol 4 no 5 Article ID e5587 2009

[24] Q Z Lao E Kobrinsky Z Liu and N M Soldatov ldquoOligomer-ization of Cav120573 subunits is an essential correlate of Ca

2+ channelactivityrdquoThe FASEB Journal vol 24 no 12 pp 5013ndash5023 2010

[25] E Kobrinsky S Tiwari V A Maltsev et al ldquoDifferential roleof the 1205721C subunit tails in regulation of the Cav12 channelby membrane potential 120573 subunits and Ca2+ ionsrdquo Journal ofBiological Chemistry vol 280 no 13 pp 12474ndash12485 2005

[26] S Tiwari Y Zhang J Heller D R Abernethy and N MSoldatov ldquoArtherosclerosis-related molecular alteration of thehuman Cav12 calcium channel 1205721C subunitrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 103 no 45 pp 17024ndash17029 2006

[27] I Splawski K W Timothy L M Sharpe et al ldquoCav12 calciumchannel dysfunction causes a multisystem disorder includingarrhythmia and autismrdquo Cell vol 119 no 1 pp 19ndash31 2004

[28] Z Z TangM C Liang S Lu et al ldquoTranscript scanning revealsnovel and extensive splice variations in human L-type voltage-gated calcium channel Cav12 1205721 subunitrdquo Journal of BiologicalChemistry vol 279 no 43 pp 44335ndash44343 2004

[29] P Liao T F Yong M C Liang D T Yue and T W SoongldquoSplicing for alternative structures of Cav12 Ca2+ channels incardiac and smooth musclesrdquo Cardiovascular Research vol 68no 2 pp 197ndash203 2005

[30] Y Blumenstein N Kanevsky G Sahar R Barzilai T Ivaninaand N Dascal ldquoA novel long N-terminal isoform of human L-type Ca2+ channel is up-regulated by protein kinase Crdquo Journalof Biological Chemistry vol 277 no 5 pp 3419ndash3423 2002

[31] N M Soldatov A Bouron and H Reuter ldquoDifferent voltage-dependent inhibition by dihydropyridines of human Ca2+channel splice variantsrdquo Journal of Biological Chemistry vol270 no 18 pp 10540ndash10543 1995

[32] D P Faxon M A Creager S C Smith et al ldquoAtheroscleroticvascular disease conference executive summary atheroscle-rotic vascular disease conference proceeding for healthcareprofessionals from a special writing group of the AmericanHeart Associationrdquo Circulation vol 109 no 21 pp 2595ndash26042004

[33] R Ross ldquoAtherosclerosismdashan inflammatory diseaserdquo The NewEngland Journal of Medicine vol 340 no 2 pp 115ndash126 1999

[34] H Roy S Bhardwaj and S Yla-Herttuala ldquoMolecular geneticsof atherosclerosisrdquo Human Genetics vol 125 no 5-6 pp 467ndash491 2009

[35] M Biel P Ruth E Bosse et al ldquoPrimary structure and func-tional expression of a high voltage activated calcium channelfrom rabbit lungrdquo FEBS Letters vol 269 no 2 pp 409ndash4121990

[36] X Cheng J Liu M Asuncion-Chin et al ldquoA novel Cav12N terminus expressed in smooth muscle cells of resistancesize arteries modifies channel regulation by auxiliary subunitsrdquoJournal of Biological Chemistry vol 282 no 40 pp 29211ndash292212007

[37] W J Koch P T Ellinor and A Schwartz ldquocDNA cloning ofa dihydropyridine-sensitive calcium channel from rat aortaevidence for the existence of alternatively spliced formsrdquo Journalof Biological Chemistry vol 265 no 29 pp 17786ndash17791 1990

[38] S Richard D Neveu G Carnac P Bodin P Travo and J Nar-geot ldquoDifferential expression of voltage-gated Ca2+-currents incultivated aortic myocytesrdquo Biochimica et Biophysica Acta vol1160 no 1 pp 95ndash104 1992

[39] A Schwartz ldquoCalcium antagonists review and perspective onmechanism of actionrdquo American Journal of Cardiology vol 64no 17 pp 3Indash9I 1989

[40] R P Mason ldquoMechanisms of plaque stabilization for thedihydropyridine calcium channel blocker amlodipine review ofthe evidencerdquo Atherosclerosis vol 165 no 2 pp 191ndash199 2002

[41] A Ruiz-Torres R Lozano J Melon and R Carraro ldquoL-calciumchannel blockade induced by diltiazem inhibits proliferationmigration and F-actin membrane rearrangements in humanvascular smooth muscle cells stimulated with insulin and IGF-1rdquo International Journal of Clinical Pharmacology andTherapeu-tics vol 41 no 9 pp 386ndash391 2003

[42] H Koshiyama S Tanaka and J Minamikawa ldquoEffect ofcalcium channel blocker amlodipine on the intimal-medialthickness of carotid arterial wall in type 2 diabetesrdquo Journal ofCardiovascular Pharmacology vol 33 no 6 pp 894ndash896 1999

[43] T Yamashita S KawashimaMOzaki et al ldquoA calcium channelblocker benidipine inhibits intimal thickening in the carotidartery of mice by increasing nitric oxide productionrdquo Journal ofHypertension vol 19 no 3 pp 451ndash458 2001

[44] R P Mason P Marche and T H Hintze ldquoNovel vascularbiology of third-generation L-type calcium channel antagonistsancillary actions of amlodipinerdquo Arteriosclerosis Thrombosisand Vascular Biology vol 23 no 12 pp 2155ndash2163 2003

[45] O Stepien J GogusevD L Zhu et al ldquoAmlodipine inhibition ofserum- thrombin- or fibroblast growth factor- induced vascu-lar smooth-muscle cell proliferationrdquo Journal of CardiovascularPharmacology vol 31 no 5 pp 786ndash793 1998

[46] V Ruiz-Velasco M B Mayer E W Inscho and L J HymelldquoModulation of dihydropyridine receptors in vascular smoothmuscle cells by membrane potential and cell proliferationrdquoEuropean Journal of Pharmacology vol 268 no 3 pp 311ndash3181994

[47] J Nilsson M Sjolund L Palmberg A M von Euler B Jonzonand J Thyberg ldquoThe calcium antagonist nifedipine inhibitsarterial smooth muscle cell proliferationrdquo Atherosclerosis vol58 no 1ndash3 pp 109ndash122 1985

[48] J Thyberg and L Palmberg ldquoThe calcium antagonist nisoldip-ine and the calmodulin antagonist W-7 synergistically inhibitinitiation of DNA synthesis in cultured arterial smooth musclecellsrdquo Biology of the Cell vol 60 no 2 pp 125ndash132 1987

[49] E Munro M Patel P Chan et al ldquoEffect of calcium channelblockers on the growth of human vascular smooth musclecells derived from saphenous vein and vascular graft stenosesrdquoJournal of Cardiovascular Pharmacology vol 23 no 5 pp 779ndash784 1994

[50] T A Kent A Jazayeri and J M Simard ldquoCalcium channelsand nifedipine inhibition of serotonin-induced [3H]thymidineincorporation in cultured cerebral smoothmuscle cellsrdquo Journalof Cerebral Blood Flow and Metabolism vol 12 no 1 pp 139ndash146 1992

[51] A Agrotis P J Little J Saltis and A Bobik ldquoDihydropyridineCa2+ channel antagonists inhibit the salvage pathway for DNAsynthesis in human vascular smooth muscle cellsrdquo EuropeanJournal of Pharmacology vol 244 no 3 pp 269ndash275 1993

[52] I Duque M R Puyol P Ruiz M Gonzalez-Rubio M LD Marques and D R Puyol ldquoCalcium channel blockers

ISRN Biochemistry 13

inhibit hydrogen peroxide-induced proliferation of culturedrat mesangial cellsrdquo Journal of Pharmacology and ExperimentalTherapeutics vol 267 no 2 pp 612ndash616 1993

[53] T Kuga S Kobayashi Y Hirakawa H Kanaide and ATakeshita ldquoCell cycle-dependent expression of L- and T-typeCa2+ currents in rat aortic smooth muscle cells in primaryculturerdquo Circulation Research vol 79 no 1 pp 14ndash19 1996

[54] E M Graf M Bock J F Heubach et al ldquoTissue distribution ofa humanCav12 1205721 subunit splice variant with a 75 bp insertionrdquoCell Calcium vol 38 no 1 pp 11ndash21 2005

[55] P Liao D Yu S Lu et al ldquoSmoothmuscle-selective alternativelyspliced exon generates functional variation in Cav12 calciumchannelsrdquo Journal of Biological Chemistry vol 279 no 48 pp50329ndash50335 2004

[56] O Skalli P Ropraz and A Trzeciak ldquoA monoclonal antibodyagainst 120572-smooth muscle actin a new probe for smooth muscledifferentiationrdquo Journal of Cell Biology vol 103 no 6 pp 2787ndash2796 1986

[57] T M Doherty K Asotra L A Fitzpatrick et al ldquoCalcificationin atherosclerosis bone biology and chronic inflammation atthe arterial crossroadsrdquo Proceedings of the National Academyof Sciences of the United States of America vol 100 no 20 pp11201ndash11206 2003

[58] G K Owens ldquoRegulation of differentiation of vascular smoothmuscle cellsrdquo Physiological Reviews vol 75 no 3 pp 487ndash5171995

[59] J P Bannister A Adebiyi G Zhao et al ldquoSmooth musclecell 1205722120575-1 subunits are essential for vasoregulation by Cav12channelsrdquo Circulation Research vol 105 no 10 pp 948ndash9552009

[60] S SonkusareM Fraer J DMarsh andN J Rusch ldquoDisruptingcalcium channel expression to lower blood pressure newtargeting of awell-known channelrdquoMolecular Interventions vol6 no 6 pp 304ndash310 2006

[61] S Telemaque S Sonkusare TGrain et al ldquoDesign ofmutant1205732subunits as decoy molecules to reduce the expression of func-tional Ca2+ channels in cardiac cellsrdquo Journal of Pharmacologyand Experimental Therapeutics vol 325 no 1 pp 37ndash46 2008

[62] A Goyenvalle A Vulin F Fougerousse et al ldquoRescue of dys-trophic muscle through U7 snRNA-mediated exon skippingrdquoScience vol 306 no 5702 pp 1796ndash1799 2004

[63] D BHalling P Aracena-Parks and S L Hamilton ldquoRegulationof voltage-gated Ca2+ channels by calmodulinrdquo Sciencersquos STKEvol 2005 no 315 p re15 2005

[64] K S Lee E Marban and R W Tsien ldquoInactivation of calciumchannels in mammalian heart cells joint dependence on mem-brane potential and intracellular calciumrdquo Journal of Physiologyvol 364 pp 395ndash411 1985

[65] C Shi and N M Soldatov ldquoMolecular determinants of voltage-dependent slow inactivation of the Ca2+ channelrdquo Journal ofBiological Chemistry vol 277 no 9 pp 6813ndash6821 2002

[66] E Kobrinsky E Schwartz D R Abernethy and NM SoldatovldquoVoltage-gated mobility of the Ca2+ channel cytoplasmic tailsand its regulatory rolerdquo Journal of Biological Chemistry vol 278no 7 pp 5021ndash5028 2003

[67] N M Soldatov ldquoCa2+ channel moving tail link between Ca2+-induced inactivation and Ca2+ signal transductionrdquo Trends inPharmacological Sciences vol 24 no 4 pp 167ndash171 2003

[68] D J Klemm P A Watson M G Frid et al ldquocAMP responseelement-binding protein content is a molecular determinantof smooth muscle cell proliferation and migrationrdquo Journal ofBiological Chemistry vol 276 no 49 pp 46132ndash46141 2001

[69] E Kobrinsky L Stevens Y Kazmi DWray andNM SoldatovldquoMolecular rearrangements of the Kv21 potassium channeltermini associated with voltage gatingrdquo Journal of BiologicalChemistry vol 281 no 28 pp 19233ndash19240 2006

[70] BMMayr G Canettieri andMRMontminy ldquoDistinct effectsof cAMP and mitogenic signals on CREB-binding proteinrecruitment impart specificity to target gene activation viaCREBrdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 98 no 19 pp 10936ndash10941 2001

[71] M Morad and N Soldatov ldquoCalcium channel inactivationpossible role in signal transduction and Ca2+ signalingrdquo CellCalcium vol 38 no 3-4 pp 223ndash231 2005

[72] DGWheeler C F Barrett R DGroth P Safa andRW TsienldquoCaMKII locally encodes L-type channel activity to signal tonuclear CREB in excitation-transcription couplingrdquo Journal ofCell Biology vol 183 no 5 pp 849ndash863 2008

[73] D E Mager E Kobrinsky A Masoudieh A Maltsev D RAbernethy andNM Soldatov ldquoAnalysis of functional signalingdomains from fluorescence imaging and the two-dimensionalcontinuous wavelet transformrdquo Biophysical Journal vol 93 no8 pp 2900ndash2910 2007

[74] E Kobrinsky D E Mager S A Bentil S I Murata DR Abernethy and N M Soldatov ldquoIdentification of plasmamembrane macro- and microdomains from wavelet analysis ofFRET microscopyrdquo Biophysical Journal vol 88 no 5 pp 3625ndash3634 2005

[75] E Kobrinsky S Q Duong A Sheydina and N M Solda-tov ldquoMicrodomain organization and frequency-dependence ofCREB-dependent transcriptional signaling in heart cellsrdquo TheFASEB Journal vol 25 no 5 pp 1544ndash1555 2011

[76] A Ravindran Q Z Lao J B Harry P Abrahimi E Kobrinskyand N M Soldatov ldquoCalmodulin-dependent gating of Cav12calcium channels in the absence of Cav120573 subunitsrdquo Proceedingsof the National Academy of Sciences of the United States ofAmerica vol 105 no 23 pp 8154ndash8159 2008

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

4 ISRN Biochemistry

II III IV

2

3

4

5

6

788A

9

10

1125

12 13 14 15

16

17 18

19

26

23

24 28

27

2934

36

2122

Plasmamembrane

Out

In

33

37+

+

38

39

40

HOOC

4443

454647484950

11A3132 41 4241A

9A

34A30

I

LA IQ

AID

65

++++4

++++

3 51 4

++++

3 51 2 4 26

++++

3 51 2 4

2035

6 6 31 2

H2N

(a)

20 2321 22 30 31 32 33 34

20 2321 22 30 31 32 33 34

20 2321 22 30 31 32 33 34

40 41A 41 44 45 46

40 41A 41 44 45 46

40 41A 41 44 45 46

20 2321 22 30 31 32 33 34

20 2321 22 30 31 32 33 34 40 41A 41 44 45 46

40 41A 41 44 45 46

20 2321 22 30 31 32 33 34 40 41A 41 44 45 46

1205721C69

1205721C70

1205721C71

1205721C73

1205721C76

1205721C77

(b)

Figure 3 Molecular diversity of Cav12 (a) Transmembrane topology of the Cav12 1205721C subunit in schematic diagram Four regions ofhomology (IndashIV) each composed of 6 transmembrane segments (numbered) are folded around the central pore The polypeptide sequenceis segmented by black bars and sequentially numbered (1ndash50) according to the CACNA1C genomic map [14] The segments correspondingto the invariant (blackgray) and alternative (blue) exons are outlined 120572-interaction domain (AID) of a constitutive 120573-binding is shown ingreen In the CaM-binding domain (CBD) apo-CaM and Ca2+-CaM are shared between LA and IQmotifs (red) respectively (b) Alternativesplicing of human fibroblast CACNA1C in the region of exons 20ndash46 The following splice variants were studied 120572

1C69 (GenBank z34809splice variant lacking alternative exons (Δ) 21 32 41A and 45) 120572

1C70 (z34810 Δ 22 31 41A and 45) 1205721C71 (z34811 Δ 22 31 and 45) 120572

1C73(z34812 Δ 22 31 33 and 45) 120572

1C76 (z34814 Δ 21 31 33 41A and 45) and 1205721C77 (z34815 Δ 21 31 41A and 45)

revealed that genetic polymorphisms significantly influencesusceptibility to atherosclerotic vascular diseases [34] How-ever none of the discovered susceptibility genes was directlyimplicated for proliferation and migration of VSM cells oneof the major pathophysiological responses to atherosclerosisat the cellular level

The presence and activity of Cav12 calcium channels inVSM cells has been established both in patch clamp andmolecular cloning experiments [29 35ndash38] Cav12 calciumchannels play a major role in atherosclerosis because they

are essential for Ca2+ signal transduction in VSM cellsContraction of VSM cells is triggered by the Ca2+ current(119868Ca) through Cav12 and thus is affected by Ca2+ channelblockers Since the 1990s it is known that DHPs particularlythe charged 2-aminoethoxymethyl DHP derivative amlodip-ine [39] exert a number of vasoprotective effects includingpotent antiatherogenic action [40] inhibition of migrationof VSM cells [41] and reduction of arterial intimal-medialthickening and plaque formation [42 43] Although it istempting to link this activity in part to pleiotropic effects of

ISRN Biochemistry 5

calcium channel blockers [44] it is the inhibition of prolifer-ation that essentially underlies it [45] The density of DHPreceptors was found to depend on VSM cell proliferation[46] Association of Cav12 with mitogenesis in VSM cells issupported by the findings that DHPs reduced DNA synthesisstimulated by serum and PdGF [47ndash49] serotonin [50] EGF[51] and H

2O2(in mesangial cells) [52] Expression of Cav12

in VSM cells was shown to be cell cycle dependent with thehighest calcium current density in G

1phase [53] Whether

these changes are reflected in the molecular repertoire ofthe Cav12 splice variants is the issue particularly importantfor the elucidation of new therapeutic targets in diseasesleading to pathogenic proliferation of VSM cells such asatherosclerosis

Investigation of the 1205721C alternative splicing in VSM

cells [28 54 55] revealed the involvement of a number ofCACNA1C exons generating impressive diversity of humanvascular 120572

1C that includes possibly the VSM-specific splic-ing of exons 99a [35 55] and exon 34 [28] To establishan association between the disease and CACNA1C splicevariation at the level of cell we have completed [26] thesingle-gene profiling of the 120572

1C molecular remodeling inVSM cells of an artery caused by atherosclerosis The VSMcells were identified in frozen sections of six surgical biopsysamples of femoral and carotid arteries by immunostainingwith an antibody against smoothmuscle 120572-actin [56] used asa marker for VSM cells The 120572-actin staining correlated withimmunostaining by an anti-120572

1C antibody in serial sectionsand was reduced in atherosclerotic regions (Figure 4) consis-tent with dedifferentiation of VSM cells [57 58]The reducedexpression of 120572

1C at the protein level was corroborated bythe quantitative RT-PCR data showing that the relative 120572

1CmRNA level in VSM cells (normalized to 18S RNA) wasreduced 37 plusmn 09 fold (mean plusmn SEM) in the atheroscleroticregion Overall the reduced expression of 120572

1C caused by thelocally elaborated cytokines in the atherosclerotic regions ofarteries resembles the reduced expression of DHP receptorsobserved in fibroblasts exposed to mitogens andor secondmessengers after serum deprivation (Figure 1)

To find out whether the altered expression of 1205721C

in atherosclerotic VSM cells is accompanied by changesin the CACNA1C alternative splicing pattern we isolatedthe immunohistochemically identified VSM cells by laser-capture microdissection from adjacent regions of arteriesaffected and not affected by atherosclerosis and identifiedthe CACNA1C splice variants by RT-PCR Our findingsrevealed an extended repertoire of the exon 21 120572

1C spliceisoforms in nonatherosclerotic VSM cells characterized bya complex splicing pattern of exons 9 9A 31ndash34 and41A including the electrophysiologically characterized 120572

1C71(GenBank z34811) 120572

1C73 (z34812) 1205721C125 (AY830711)

1205721C126 (AY830713) and 1205721C127 (AY830712) splice isoforms

However only the exon 22 isoform of 1205721C (1205721C77) was

identified in atherosclerotic VSM cells Thus the switch ofthe CACNA1C alterative splicing from exon 21 to exon 22(Figure 3(b)) is a molecular signature of the Cav12 remod-eling of VSM cells to a pathophysiological proliferating statein atherosclerosis The age gender ethnicity drug exposureand other co-morbid conditions did not appreciably affect

this common pattern of the 1205721C splice variation in VSM cells

in response to atherosclerosisCareful electrophysiological analysis exhibited a number

of differences in the properties of the ldquoatheroscleroticrdquo 1205721C77

channel as compared to the 1205721C isoforms in healthy VSM

cells The largest differences were found between the 1205721C77

and 1205721C127 channels (Figure 5) In response to step depolar-

ization applied from the holding potential of minus90mV bothchannels generate calcium currents (119868Ca) that inactivate withalmost identical kinetics (Figure 5(a)) However we foundthat 119868Ca through the1205721C77 channel recovers from inactivationsignificantly faster than that in 120572

1C127 (Figure 5(b)) and other1205721C isoforms present in healthy VSM cells This finding

suggests that alternative splicing in atherosclerosis may affectvascular tone as a result of the increase in the 119868Ca density inVSM cells A hyperpolarization shift of the activation curvefor the atherosclerotic 120572

1C77 channel variant as comparedto Cav12 in healthy VSM cells (Figure 5(c)) may also resultin an increase of calcium entry in VSM cells Howeverthe overall 3-4-fold reduction in the expression of Cav12in atherosclerotic VSM cells may scale down some of theobserved electrophysiological changes

5 CACNA1C Exon 22 as a New TherapeuticTarget in Atherosclerosis

Direct DNA sequencing of the crude PCR amplificationproducts indicated that the switch to the exon 22 isoform ofvascular 120572

1C was almost complete in atherosclerosis becauseno distortion of the nucleotide peaks in the region of exon2122 was seen when compared to the exon 20 invariantregion (Figure 6(a)) Thus Cav12 underwent almost quanti-tative exon 2122 remodeling in VSM cells of diseased arteryregions

Although the cellular mechanisms leading to theCACNA1C exon 2122 switch may be very complex theassociation with VSM cell proliferation is obvious Indeeda similar exon switch was observed in primary humanaortic cells in culture after the quiescent nonproliferatingcells containing predominantly exon 21 splice variantswere exposed to serum (Figure 6(b)) Unlike exon 21exon 22 contains the AvrII restriction site that allowsfor the assessment of its presence in PCR amplificationproduct of CACNA1C transcripts isolated from the cellsThe AvrII-sensitive exon 22 isoform of the 120572

1C transcriptwas not detected in the quiescent nonproliferating aorticcells (Figure 6(b) lane 2) However when 5 serum wasadded to the medium with nonconfluent aortic cellsDNA biosynthesis was activated while the level of the1205721C transcript decreased asymp3 fold and the presence of the

AvrII-sensitive exon 22 isoforms of 1205721C was easily detected

(Figure 6(b) lane 4) The isoform remodeling simulated inaortic primary cells in vitro was not complete as comparedto VSM cells in atherosclerotic regions of artery occludedwith heavy plaque burden which were selected for the1205721C molecular profiling However the cell culture results

demonstrate that in a different experimental system there isan obvious association between proliferation of VSM cells

6 ISRN Biochemistry

A Ki-67 NKi-67

50 12058350 120583

(a)

A N120572-actin 120572-actin

(b)

A N1205721C1205721C

(c)

Figure 4 Reduced expression of 1205721C in atherosclerotic (A) region of aorta as compared to the adjacent nonaffected (N) region From (a) to

(c) detection of proliferating cells in an exemplar biopsy of an artery by an antibody against human nuclear protein Ki-67 (a) Red arrowshighlight some proliferating nuclei (black staining) while blue arrows exemplify nonproliferating nuclei (light grey) Staining with antibodiesagainst smooth muscle 120572-actin (b) and against 120572

1C (c) Scale bars 50 120583m

downregulation of the CACNA1C expression and synthesisof the exon 22 120572

1C isoformRecent strategies targeting VSM cells to treat cardiovas-

cular diseases suggest indiscriminate disruption of Cav12[59ndash61] Is it possible to correct the described CACNA1Csplice defects induced by atherosclerosis without affectingthe transcripts of the gene lacking the rdquopathogenicrdquo exon22 Correction of defective genes responsible for diseasedevelopment is achieved by gene therapy Usually it requiresan insertion into the genome of a normal gene in place ofa defective one causing disease Such technique howeveris poorly controlled Currently one of the most promising

cutting-edge therapeutic approaches to correct defects asso-ciated with disease-induced expression of abnormal splicevariants is antisense-mediated exon skipping It is basedon the use of antisense oligonucleotides targeting specificexons to be removed The adenovirus-directed 120572

1C exon 22skipping-induced inhibition of VSM cell proliferation (andrespectively migration) can be used to rescue VSM cells fromremodeling in atherosclerosis The 120572

1C exon 22-skippingwill not alter the open reading frame of the 120572

1C transcriptbecause alternative exons 21 and 22 are of the equal size(60 nt)Themodified nonspliceosomal snRNAU7 gene alongwith its natural promoter and 31015840 elements exon 22-antisense

ISRN Biochemistry 7

200 ms

1205721C77

1205721C127

(a)

Prepulse interval (s)02 04 06 08 1

0

1

08

06

04

02

0

Frac

tion

of119868 C

a

1205721C77

1205721C127

(b)

0

1

08

06

04

02

119866Ca

119866Ca

max

1205721C77

1205721C127

minus60 minus40 minus20 0 20 40(mV)

(c)

Figure 5 Comparison of electrophysiological properties of 119868Ca through the ldquoatheroscleroticrdquo 1205721C77 and ldquonormalrdquo 120572

1C127 channels expressedin Xenopus oocytes with 120572

2120575-1 subunit and the primary cardiac 1205732a subunit and measured with 25mM Ca2+ as the charge carrier (a)

Representative traces of 119868Ca evoked by 1 s step depolarizations to +20mV from 119881ℎ= minus90mV and normalized to the same amplitude (b)

Fractional recovery of 119868Ca from inactivation (c) Averaged activation (119866119866max-119881) curves fit by the Boltzmann function A 1 s test pulses in therange of minus40 to +50mV (10-mV increments) were applied from 119881

ℎ= minus90mV with 30-s intervals

(a)

AvrII

Serum

1 2 3 4

+minus+minus

minus minus + +

142 pb

459 pb

601 pb

(b)

Figure 6 (a) Trace diagrams of DNA sequencing of the 1205721C PCR amplification products of healthy (top row) and diseased VSM cells (bottom

row) using an antisense primer composed of nucleotides 2923ndash2939 (z34815) A boundary with the sequence of invariant exon 20 is markedby a vertical dotted line (b) Evidence that the AvrII-sensitive exon 22 isoform of 120572

1C is expressed only in proliferating VSM cells Primaryaortic cells were grown to confluent monolayer in 5 serum before serum-deprivation for 5 days (lanes 1 and 2)Then the cells were replatedat low density in 5 serum for 3-4 days (lanes 3 and 4) Total RNA was isolated and exon 2122 isoforms were identified by RT-PCR andAvrII restriction analysis (lanes 2 and 4)

8 ISRN Biochemistry

sequence and supplemented with Sm ribonucleoprotein-binding sequence may be incorporated into the adenovirusvector for high efficiency transfer [62]The cytokine receptors(eg PdGF-120573 receptor) based recognition targeting of viralliposomes or nanoparticles may be especially advantageousin connection with selective gene transfer to VSM cellsaffected by atherosclerosis while reducing the probability ofthe transfection of other cells

6 Cak12 and CREB-DependentTranscriptional Activation

How Cav12 activity is translated into a proliferation-effectedmodality is another important question to be asked Cav12calcium channels generate a transient rise in cytosolic Ca2+-concentration activated by membrane depolarization Cel-lular responses associated with the rise of [Ca2+]i rangefrom sarcomeric contraction to cell growth and proliferationCytoplasmic domains of Cav12 have evolved a fairly intricateCaM-dependent signaling mechanism that provides for thenegative feedback inhibition of the calcium current knownas Ca2+ dependent inactivation (CDI) which is mediated bydifferent determinants of 120572

1C [25 63] Such a mechanism ofCDI resulting in acceleration of 119868Ca inactivation in responseto the rise of intracellular Ca2+ was first identified in cardiacCav12 [64] Similar experiments performed on the recombi-nant Cav12 also showed that the replacement of extracellularCa2+ by Ba2+ eliminates CDI and the channel inactivates by aslower voltage-dependent mechanism [65]The very fact thattwo distantly located determinants one in the pore regionresponsible for slow inactivation and the CaM-binding onein the proximal locus of the C-tail are independently crucialfor CDI indicates that not only their specific molecularstructure but also their mutual folding andor interaction areessential Experimental evidence show that this interactionreacts dynamically to membrane voltage supporting state-dependent transitions of the channel between resting openand inactivated conformations which are essential for CREB-dependent transcriptional activation [66 67]

CREB is a transcription factor of general importance ina large variety of cells CREB phosphorylation promotes theactivation of genes and is regulated by protein kinases undercontrol of the major second messengers cAMP andor Ca2+Indeed CREB functions as a ldquomolecular determinant of VSMcells fate [68]rdquo CREB content depends on proliferation ofVSM cells both in situ and in culture Serum deprivationincreased CREB content in VSM cells while exposure toPdGF decreased it Consistent with this observation anoverexpression of the constitutively active CREB inVSM cellsarrested cell cycle progression

To investigate the association of Cav12 with CREB tran-scriptional activation at the molecular level we combinedpatch clamp with fluorescent resonance energy transfer(FRET) microscopy in the live cell In voltage clampedcells FRET provided optical measurements under state-dependent conditions showing that the shorter N-terminaltail of 120572

1C (eg 1205721C77) does not rearrange vis-a-vis the

plasma membrane in response to voltage gating [25] In

sharp contrast the C-tail shows voltage-dependent confor-mational rearrangements which are much larger in sizethan that for example in the potassium Kv21 channel[69] Measurements of 119868Ca and corrected FRET betweenthe enhanced yellow (EYFP) and cyan fluorescent proteins(ECFP) genetically attached respectively to the N- and C-termini of 120572

1C77 showed no significant effect on voltage-dependence and kinetics of the channel current Howevertherewas a substantial increase in FRET signal accompanyinginactivation of the channel that was fully reversible uponits transition into the resting state in response to hyper-polarization [66] The plasma-membrane anchoring of the1205721C C-tail by the fusion of the pleckstrin homology domain

(PH) eliminated CDI but not 119868Ca Do the voltage-gatedconformational rearrangements of the 120572

1C C-tail and CDIplay a role in Ca2+ signal transduction that is utilized inCa2+-induced activation or CREB-dependent transcriptionTo answer this question we used the test system based onthe measurement of interaction between KID domain ofCREB and KIX domain of coactivator CREB-binding protein(CBP Figure 7(A) inset) under voltage-clamp conditionsby monitoring FRET between EYFP-KID and ECFP-KIX[70] Ca2+-dependent phosphorylation of KID stimulates itsbinding to KIX bringing EYFP and ECFP close enoughto observe the interaction by FRET In perforated wholeclamped cell where the integrity of the cytoplasmic contentis intact (Figure 7(A)) no activation of CREB transcription(Figure 7(B) panel (a)) and rearrangement of the120572

1C77 C-tail(panel (b)) was observed when the C-tail was anchored to theplasma membrane CREB transcriptional activity remainedlow in spite of a large sustained inward 119868Ca (panel (d))and the corresponding increase in [Ca2+]i detected by thefluorescence of Ca2+ indicator Fura4 (panel (c)) Releaseof the 120572

1C77 C-tail by the activation of PIP2hydrolysis

upon activation of M1AchR (Figure 7(C)) at minus90mV causedsignificant elevation of [Ca2+]i that also was not utilizedby the cell for CREB transcriptional activation (Figure 7(C)panel (a)) until a depolarizing pulse to +20-mV was appliedand the released 120572

1C77 C-tail was permitted to rearrange(Figure 7(D))This experiment provides compelling evidencethat neither large inward 119868Ca nor the subsequent rise in[Ca2+]i lead to CREB transcription activation unless theconformational rearrangement of the 120572

1C77 subunit C-tailprovides the precise targeting of the Ca2+ signal transduction(Figure 7(D) scheme) [66]

There is general agreement that CaM binds to LA and IQdomains of the 120572

1C C-tail and acts as a sensor that conveysCDI (for review see [63]) The affinity of CaM for bothdomains depends on [Ca2+]i Our data indicate that CDI andCa2+-signal transduction depend on the voltage-gatedmobil-ity of the120572

1C77 C-tail It is therefore reasonable to suggest thatthe LA-domain is a Ca2+-sensitive apo-CaM-mediated lockfor the mechanism of slow voltage-dependent inactivationof the channel [67] Apo-CaM associated with LA is able tocross-link it to another still unidentified apo-CaM bindingsite in the polypeptide bundle underlying the pore As theresult of this specific localization apo-CaMLA ldquolockrdquo ishidden from the cytoplasmic Ca2+ so that for example the

ISRN Biochemistry 9

10

0

2

10

100 pA125 ms

(a) (a) (a)

(b)(c)

(b)(c)

(b)(c)

(B) (C) (D)

C-tail Anchored ReleasedReleased

0 10No FRET No FRET

(d) (d) (d)

Fluo-4

PH

CYN

C

C

NY

C

Y EYFP

C ECFP

PH Plekstrin homologydomain

PHPHY

C

NC

IQ

LA

LAIQ

(A)

Fluo-4 Fluo-4

FRET FRET FRET

CRE

Gene expressionCREBCREB P

CBP

AC

GPCR

PKA

cAMP

CaM

Nucleus

cAMP

Plasmamembrane

Ca2+ Ca2+

Ca2+

+20mV +20mV

minus90mV minus90mV

1205721C77

1198651198650

Cav 12

Ca2+

minus90mVACh (5 mM)

4 120583

Figure 7 Evidence that Ca2+ signal transduction by Cav12 to activate CREB-dependent transcription is mediated by the voltage-gatedmobility of the 120572

1C C-tail carrying the CaM-caged Ca2+ and is not directly associated with the increase of [Ca2+]i CREB activation wasexamined under perforated patch conditions in COS1 cells expressing recombinant atherosclerotic 120572

1C77 channel (A) Phase-contrast imageof the COS1 cell with a shadow of patch pipette The cell was expressing the 120572

1C77-PHC1205731a1205722120575-1 channel with the membrane-trapped 1205721C77

subunit C-tail type 1muscarinic Ach receptor (to release the anchored C-tail in response to activation by Ach) and EYFP-KID and ECFP-KIXdomains both supplemented with nuclear localization sequences Inset at the bottom Cav12- and cAMP-dependent cell signaling pathwaysmediating CREB-dependent transcription CBP CREB-binding protein CRE cAMP-response element (B) Plasma-membrane anchoring ofthe 1205721C77 C-tail (see schematic diagram) inhibits CREB activation in spite of robust 119868Ca evoked by depolarization (a) A 100-ms images of

FRET between EYFP-KID and ECFP-KIX were recorded at the end of the 12th +20-mV depolarization step applied every 10 s for 1 s from119881ℎ=

minus90mV (b) A corresponding representative image of corrected FRET ratio (in a ldquo3Drdquo MetaMorph transformation) recorded within 50-mswindows at minus90 and at the end of the last test pulse of the applied train of pulses (c) An increase of [Ca2+]i detected by Ca

2+ indicator Fluo4(d) Representative trace of 119868Ca (20mM Ca2+ in bath medium) evoked by depolarization to +20mV from119881

ℎ= minus90mV showing the sustained

component of Ca2+ conductance due to the C-tail anchoring (C) ACh-stimulation of theM1AChR to unbind the 1205721C C-tail caused activation

of IP3-dependent Ca2+ release (panel (c)) but did not show substantial activation of CREB transcription at 119881

ℎ= minus90mV (panel (a)) (D)

CREB-dependent transcription was activated only in response to depolarization applied to membrane channel with the 1205721C77 C-terminal tail

released from the plasma membrane to assume functional voltage-dependent conformation

intracellular Ca2+ released from the intracellular stores orCa2+ caging compounds does not accelerate significantly theinactivation of Cav12 [71] Thus apo-CaM associated withLA binds predominantly Ca2+ ions permeating through thepore A Ca2+-dependent transfer of CaM from LA to the IQ-motif opens the ldquolockrdquo and initiates a large rearrangement ofthe C-terminal tail This in turn facilitates inactivation of thechannel The Ca2+CaM complex with the IQ-motif is thentransferred by themobile C-tail to a downstream target of theCa2+-signaling cascade (such as CaMKII [72]) where Ca2+ isreleased as an activating stimulus while CaM switches back

to LA and returns the C-tail to the resting position availablefor the next cycle of Ca2+-signal transduction

Activation of CREB-dependent transcription by the L-type 119868Ca ismediated throughmultiple cell signaling pathwaysUsing FRET probes of CREB activity and 2D wavelet trans-form analysis we applied principles of quantitative biologyto detail the mechanism of Ca2+-activated CREB-dependenttranscription within localized regions (microdomains) ofthe nucleus We reached this goal by applying continuouswavelet analysis in two dimensions with a 2D wavelet asa deconvolution algorithm for FRET microscopy image

10 ISRN Biochemistry

Control

(A)

05

0

1

05

00 20 40 60 800 20 40 60 800 20 40 60 80

(a) (b) (c) (d)

20 40 60 800

0

20

40

60

80 1

C 3 5 10 mincAMP

C 2 10 mincAMP

C 3 10 min

cAMP

C 1 3 10 min

cAMP

5

cAMP stable (5)

(B)

119868Ca (3 min) cAMP (5 min) cAMP (10 min)

119868Ca cAMP (65) 119868Ca stable (15)

119868Ca119868Ca

119868Ca 119868Ca

119868Ca cAMP transient (15)

Figure 8 Microdomain analysis on the additive effect of 119868Ca and cAMP on CREB signaling in COS1 cells expressing the recombinantldquoatheroscleroticrdquo120572

1C77 channel (A) FRET signal within the nucleus during selected time points during 119868Ca stimulation and cAMP applicationOutlined are four types of signaling microdomains identified using 2D Mexican hat wavelet Red circles represent stable microdomains thatpersist through both 119868Ca and cAMP application White and yellow circles showmicrodomains stable activated by cAMP and 119868Ca respectivelyGreen circles represent transient microdomains of CREB signaling activation Axes show pixel numbers (B) Typical appearances of the fourtypes of CREB signaling microdomains activated by 119868Ca andor cAMP application and recorded in their maximal development in relationto the time of applied stimuli C control recorded before stimulation Color bars in (A) and (B) represent FRET values normalized to themaximum

analysis [73] Continuous wavelet analysis is a mathematicaltechnique that allows us to analyze a signal over severaldifferent frequencies across the entire signal [73 74] It isespecially useful for finding heterogeneity in a signal becauseit can easily find where the pattern (ie frequency) of asignal changes In these experiments we for the first timeobtained evidence of CREB signaling microdomains withinthe nucleus that respond differentially to 119868Ca stimulationand cAMP (Figure 8(A)) Results of the study revealed thatCREB-dependent transcriptional signaling occurs in dis-crete signaling microdomains underlying the architecture ofnuclear signaling Continuous activation ofCREB-dependenttranscriptional signaling by cAMP and Ca2+ resulted in agradual increase of the number of microdomains Four dif-ferent categories of cAMP and Ca2+-induced CREB signalingmicrodomains were characterized in COS1 cells expressingrecombinant Cav12 with the ldquoatheroscleroticrdquo 120572

1C77 splice

variant (Figure 8) In up to 65 of the microdomainstranscription was activated in additive manner by cAMPand Ca2+ Approximately 15 of signaling domains wereactivated only by 119868Ca and 5 of domains were activatedonly by cAMP Finally 15 of the domains were transientand activated by both cAMP and 119868Ca (Figure 8(B)) [75]A similar spatiotemporal organization of CREB-dependentsignaling was observed in spontaneously beating neonatalrat cardiomyocytes Although COS1 cells that were used inour experiments shown in Figures 7 and 8 are naturallydeprived of Cav12 [76] they inherited the ability to replicatethe Cav12-dependent activation of CREB signaling with theexogenous recombinant channel Thus this experimentalapproach fits the task which in my opinion is the mostimportant unresolved issue for the coupling of Cav12 toCREB signaling does the splice variation of 120572

1C affect thespatiotemporal organization of CREB-dependent signaling

ISRN Biochemistry 11

in a way that may affect cell proliferation and other crucialfunction

7 Conclusions

Association of Cav12 with regulation of transcription cellproliferation and its pathophysiology as in the case ofatherosclerosis requires detailed investigation of the roles ofthe naturally occurring 120572

1C splice variants It will limit thetraditionally intuitive approach to Cav12 in physiology andhelp to define new principle approaches to the treatment ofvarious Cav12 channelopathy-related dysfunctions above allcardiovascular diseases Humgenex Inc provides consultingand logistic support on a broad range of issues reported inthis review

Abbreviations

CaM CalmodulinCBD Calmodulin-binding domainCDI Ca2+-dependent inactivationDHP DihydropyridineECFP Enhanced cyan fluorescent proteinEYFP Enhanced yellow fluorescent proteinFALI Fluorophore-assisted light inactivationFRET Fluorescent resonance energy transfer[3H]PMD 26-Dimethyl-3-methoxycarbonyl-5-([23-

3H2]-n-propoxycarbonyl)-4-(21015840-

difluoromethoxyphenyl)-14-dihydropyridine

119868Ca Calcium currentPH Pleckstrin homology domainVSM Vascular smooth muscle

References

[1] Y S Lee M M Sayeed and R D Wurster ldquoInhibition ofcell growth and intracellular Ca2+ mobilization in humanbrain tumor cells by Ca2+ channel antagonistsrdquo Molecular andChemical Neuropathology vol 22 no 2 pp 81ndash95 1994

[2] K Sato J Ishizuka C W Cooper et al ldquoInhibitory effect ofcalcium channel blockers on growth of pancreatic cancer cellsrdquoPancreas vol 9 no 2 pp 193ndash202 1994

[3] V Bertrand M J Bastie N Vaysse and L Pradayrol ldquoInhibi-tion of gastrin-induced proliferation of AR4-2J cells by calciumchannel antagonistsrdquo International Journal of Cancer vol 56no 3 pp 427ndash432 1994

[4] JM Taylor andRU Simpson ldquoInhibition of cancer cell growthby calcium channel antagonists in the athymic mouserdquo CancerResearch vol 52 no 9 pp 2413ndash2418 1992

[5] M G Cattaneo M Gullo and L M Vicentini ldquoC2+ and Ca2+channel antagonists in the control of human small cell lung car-cinoma cell proliferationrdquo European Journal of Pharmacologyvol 247 no 3 pp 325ndash331 1993

[6] C Chen M J Corbley T M Roberts and P Hess ldquoVoltage-sensitive calcium channels in normal and transformed 3T3fibroblastsrdquo Science vol 239 no 4843 pp 1024ndash1026 1988

[7] P C Dartsch M Ritter M Gschwentner H J Lang andF Lang ldquoEffects of calcium channel blockers on NIH 3T3

fibroblasts expressing the Ha-ras oncogenerdquo European Journalof Cell Biology vol 67 no 4 pp 372ndash378 1995

[8] M Biel R Hullin S Freundner et al ldquoTissue-specific expres-sion of high-voltage-activated dihydropyridine-sensitive L-typecalcium channelsrdquo European Journal of Biochemistry vol 200no 1 pp 81ndash88 1991

[9] DM Rodman K Reese J Harral et al ldquoLow-voltage-activated(T-type) calcium channels control proliferation of human pul-monary artery myocytesrdquo Circulation Research vol 96 no 8pp 864ndash872 2005

[10] A Panner L L Cribbs G M Zainelli T C Origitano S Singhand R DWurster ldquoVariation of T-type calcium channel proteinexpression affects cell division of cultured tumor cellsrdquo CellCalcium vol 37 no 2 pp 105ndash119 2005

[11] C Chen and P Hess ldquoCalcium channels in mouse 3T3 andhuman fibroblastsrdquo Biophysical Journal vol 51 p 226a 1987

[12] N M Soldatov ldquoMolecular diversity of L-type Ca2+ channeltranscripts in human fibroblastsrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 89 no10 pp 4628ndash4632 1992

[13] A Fujii H Matsumoto S Nakao H Teshigawara and YAkimoto ldquoEffect of calcium-channel blockers on cell prolifera-tion DNA synthesis and collagen synthesis of cultured gingivalfibroblasts derived fromhumannifedipine responders and non-respondersrdquo Archives of Oral Biology vol 39 no 2 pp 99ndash1041994

[14] N M Soldatov ldquoGenomic structure of human L-type Ca2+channelrdquo Genomics vol 22 no 1 pp 77ndash87 1994

[15] V P Shevchenko N F Myasoedov N M Soldatov G JDuburs V V Kastron and I P Skrastins ldquoSynthesis andevaluation of biological activity of two novel tritium-labeledderivatives of riodipinerdquo Journal of Labelled Compounds andRadiopharmaceuticals vol 27 no 6 pp 721ndash730 1989

[16] S M Dudkin S N Gnedoj N N Chernyuk and N MSoldatov ldquo14-dihydropyridine receptor associated with Ca2+channels in human embryonic fibroblastsrdquo FEBS Letters vol233 no 2 pp 352ndash354 1988

[17] A DePover M A Matlib S W Lee et al ldquoSpecific bindingof [3H]nitrendipine to membranes from coronary arteriesand heart in relation to pharmacological effects Paradoxicalstimulation by diltiazemrdquo Biochemical and Biophysical ResearchCommunications vol 108 no 1 pp 110ndash117 1982

[18] N M Soldatov S N Gnedoj N N Chernyuk I A Britanovaand S M Dudkin ldquoMitogenesis and 14-dihydropyridinereceptor associated with Ca2+-channels in human embryonicfibroblastsrdquo Journal of Membrane Biology vol 5 pp 1161ndash11671988 (Russian)

[19] D Lipscombe D V Madison M Poenie H Reuter R Y Tsienand R W Tsien ldquoSpatial distribution of calcium channels andcytosolic calcium transients in growth cones and cell bodies ofsympathetic neuronsrdquo Proceedings of the National Academy ofSciences of the United States of America vol 85 no 7 pp 2398ndash2402 1988

[20] G S Harms L Cognet P H M Lommerse et al ldquoSingle-molecule imaging of L-type CA2+ channels in live cellsrdquoBiophysical Journal vol 81 no 5 pp 2639ndash2646 2001

[21] V di Biase G J Obermair Z Szabo et al ldquoStable membraneexpression of postsynaptic Cav12 calcium channel clustersis independent of interactions with AKAP79150 and PDZproteinsrdquo Journal of Neuroscience vol 28 no 51 pp 13845ndash13855 2008

12 ISRN Biochemistry

[22] M F Navedo E P Cheng C Yuan et al ldquoIncreased coupledgating of L-type Ca2+ channels during hypertension and timo-thy syndromerdquoCirculationResearch vol 106 no 4 pp 748ndash7562010

[23] E Kobrinsky P Abrahimi S Q Duong et al ldquoEffect of Cav120573subunits on structural organization of Cav12 calcium channelsrdquoPLoS One vol 4 no 5 Article ID e5587 2009

[24] Q Z Lao E Kobrinsky Z Liu and N M Soldatov ldquoOligomer-ization of Cav120573 subunits is an essential correlate of Ca

2+ channelactivityrdquoThe FASEB Journal vol 24 no 12 pp 5013ndash5023 2010

[25] E Kobrinsky S Tiwari V A Maltsev et al ldquoDifferential roleof the 1205721C subunit tails in regulation of the Cav12 channelby membrane potential 120573 subunits and Ca2+ ionsrdquo Journal ofBiological Chemistry vol 280 no 13 pp 12474ndash12485 2005

[26] S Tiwari Y Zhang J Heller D R Abernethy and N MSoldatov ldquoArtherosclerosis-related molecular alteration of thehuman Cav12 calcium channel 1205721C subunitrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 103 no 45 pp 17024ndash17029 2006

[27] I Splawski K W Timothy L M Sharpe et al ldquoCav12 calciumchannel dysfunction causes a multisystem disorder includingarrhythmia and autismrdquo Cell vol 119 no 1 pp 19ndash31 2004

[28] Z Z TangM C Liang S Lu et al ldquoTranscript scanning revealsnovel and extensive splice variations in human L-type voltage-gated calcium channel Cav12 1205721 subunitrdquo Journal of BiologicalChemistry vol 279 no 43 pp 44335ndash44343 2004

[29] P Liao T F Yong M C Liang D T Yue and T W SoongldquoSplicing for alternative structures of Cav12 Ca2+ channels incardiac and smooth musclesrdquo Cardiovascular Research vol 68no 2 pp 197ndash203 2005

[30] Y Blumenstein N Kanevsky G Sahar R Barzilai T Ivaninaand N Dascal ldquoA novel long N-terminal isoform of human L-type Ca2+ channel is up-regulated by protein kinase Crdquo Journalof Biological Chemistry vol 277 no 5 pp 3419ndash3423 2002

[31] N M Soldatov A Bouron and H Reuter ldquoDifferent voltage-dependent inhibition by dihydropyridines of human Ca2+channel splice variantsrdquo Journal of Biological Chemistry vol270 no 18 pp 10540ndash10543 1995

[32] D P Faxon M A Creager S C Smith et al ldquoAtheroscleroticvascular disease conference executive summary atheroscle-rotic vascular disease conference proceeding for healthcareprofessionals from a special writing group of the AmericanHeart Associationrdquo Circulation vol 109 no 21 pp 2595ndash26042004

[33] R Ross ldquoAtherosclerosismdashan inflammatory diseaserdquo The NewEngland Journal of Medicine vol 340 no 2 pp 115ndash126 1999

[34] H Roy S Bhardwaj and S Yla-Herttuala ldquoMolecular geneticsof atherosclerosisrdquo Human Genetics vol 125 no 5-6 pp 467ndash491 2009

[35] M Biel P Ruth E Bosse et al ldquoPrimary structure and func-tional expression of a high voltage activated calcium channelfrom rabbit lungrdquo FEBS Letters vol 269 no 2 pp 409ndash4121990

[36] X Cheng J Liu M Asuncion-Chin et al ldquoA novel Cav12N terminus expressed in smooth muscle cells of resistancesize arteries modifies channel regulation by auxiliary subunitsrdquoJournal of Biological Chemistry vol 282 no 40 pp 29211ndash292212007

[37] W J Koch P T Ellinor and A Schwartz ldquocDNA cloning ofa dihydropyridine-sensitive calcium channel from rat aortaevidence for the existence of alternatively spliced formsrdquo Journalof Biological Chemistry vol 265 no 29 pp 17786ndash17791 1990

[38] S Richard D Neveu G Carnac P Bodin P Travo and J Nar-geot ldquoDifferential expression of voltage-gated Ca2+-currents incultivated aortic myocytesrdquo Biochimica et Biophysica Acta vol1160 no 1 pp 95ndash104 1992

[39] A Schwartz ldquoCalcium antagonists review and perspective onmechanism of actionrdquo American Journal of Cardiology vol 64no 17 pp 3Indash9I 1989

[40] R P Mason ldquoMechanisms of plaque stabilization for thedihydropyridine calcium channel blocker amlodipine review ofthe evidencerdquo Atherosclerosis vol 165 no 2 pp 191ndash199 2002

[41] A Ruiz-Torres R Lozano J Melon and R Carraro ldquoL-calciumchannel blockade induced by diltiazem inhibits proliferationmigration and F-actin membrane rearrangements in humanvascular smooth muscle cells stimulated with insulin and IGF-1rdquo International Journal of Clinical Pharmacology andTherapeu-tics vol 41 no 9 pp 386ndash391 2003

[42] H Koshiyama S Tanaka and J Minamikawa ldquoEffect ofcalcium channel blocker amlodipine on the intimal-medialthickness of carotid arterial wall in type 2 diabetesrdquo Journal ofCardiovascular Pharmacology vol 33 no 6 pp 894ndash896 1999

[43] T Yamashita S KawashimaMOzaki et al ldquoA calcium channelblocker benidipine inhibits intimal thickening in the carotidartery of mice by increasing nitric oxide productionrdquo Journal ofHypertension vol 19 no 3 pp 451ndash458 2001

[44] R P Mason P Marche and T H Hintze ldquoNovel vascularbiology of third-generation L-type calcium channel antagonistsancillary actions of amlodipinerdquo Arteriosclerosis Thrombosisand Vascular Biology vol 23 no 12 pp 2155ndash2163 2003

[45] O Stepien J GogusevD L Zhu et al ldquoAmlodipine inhibition ofserum- thrombin- or fibroblast growth factor- induced vascu-lar smooth-muscle cell proliferationrdquo Journal of CardiovascularPharmacology vol 31 no 5 pp 786ndash793 1998

[46] V Ruiz-Velasco M B Mayer E W Inscho and L J HymelldquoModulation of dihydropyridine receptors in vascular smoothmuscle cells by membrane potential and cell proliferationrdquoEuropean Journal of Pharmacology vol 268 no 3 pp 311ndash3181994

[47] J Nilsson M Sjolund L Palmberg A M von Euler B Jonzonand J Thyberg ldquoThe calcium antagonist nifedipine inhibitsarterial smooth muscle cell proliferationrdquo Atherosclerosis vol58 no 1ndash3 pp 109ndash122 1985

[48] J Thyberg and L Palmberg ldquoThe calcium antagonist nisoldip-ine and the calmodulin antagonist W-7 synergistically inhibitinitiation of DNA synthesis in cultured arterial smooth musclecellsrdquo Biology of the Cell vol 60 no 2 pp 125ndash132 1987

[49] E Munro M Patel P Chan et al ldquoEffect of calcium channelblockers on the growth of human vascular smooth musclecells derived from saphenous vein and vascular graft stenosesrdquoJournal of Cardiovascular Pharmacology vol 23 no 5 pp 779ndash784 1994

[50] T A Kent A Jazayeri and J M Simard ldquoCalcium channelsand nifedipine inhibition of serotonin-induced [3H]thymidineincorporation in cultured cerebral smoothmuscle cellsrdquo Journalof Cerebral Blood Flow and Metabolism vol 12 no 1 pp 139ndash146 1992

[51] A Agrotis P J Little J Saltis and A Bobik ldquoDihydropyridineCa2+ channel antagonists inhibit the salvage pathway for DNAsynthesis in human vascular smooth muscle cellsrdquo EuropeanJournal of Pharmacology vol 244 no 3 pp 269ndash275 1993

[52] I Duque M R Puyol P Ruiz M Gonzalez-Rubio M LD Marques and D R Puyol ldquoCalcium channel blockers

ISRN Biochemistry 13

inhibit hydrogen peroxide-induced proliferation of culturedrat mesangial cellsrdquo Journal of Pharmacology and ExperimentalTherapeutics vol 267 no 2 pp 612ndash616 1993

[53] T Kuga S Kobayashi Y Hirakawa H Kanaide and ATakeshita ldquoCell cycle-dependent expression of L- and T-typeCa2+ currents in rat aortic smooth muscle cells in primaryculturerdquo Circulation Research vol 79 no 1 pp 14ndash19 1996

[54] E M Graf M Bock J F Heubach et al ldquoTissue distribution ofa humanCav12 1205721 subunit splice variant with a 75 bp insertionrdquoCell Calcium vol 38 no 1 pp 11ndash21 2005

[55] P Liao D Yu S Lu et al ldquoSmoothmuscle-selective alternativelyspliced exon generates functional variation in Cav12 calciumchannelsrdquo Journal of Biological Chemistry vol 279 no 48 pp50329ndash50335 2004

[56] O Skalli P Ropraz and A Trzeciak ldquoA monoclonal antibodyagainst 120572-smooth muscle actin a new probe for smooth muscledifferentiationrdquo Journal of Cell Biology vol 103 no 6 pp 2787ndash2796 1986

[57] T M Doherty K Asotra L A Fitzpatrick et al ldquoCalcificationin atherosclerosis bone biology and chronic inflammation atthe arterial crossroadsrdquo Proceedings of the National Academyof Sciences of the United States of America vol 100 no 20 pp11201ndash11206 2003

[58] G K Owens ldquoRegulation of differentiation of vascular smoothmuscle cellsrdquo Physiological Reviews vol 75 no 3 pp 487ndash5171995

[59] J P Bannister A Adebiyi G Zhao et al ldquoSmooth musclecell 1205722120575-1 subunits are essential for vasoregulation by Cav12channelsrdquo Circulation Research vol 105 no 10 pp 948ndash9552009

[60] S SonkusareM Fraer J DMarsh andN J Rusch ldquoDisruptingcalcium channel expression to lower blood pressure newtargeting of awell-known channelrdquoMolecular Interventions vol6 no 6 pp 304ndash310 2006

[61] S Telemaque S Sonkusare TGrain et al ldquoDesign ofmutant1205732subunits as decoy molecules to reduce the expression of func-tional Ca2+ channels in cardiac cellsrdquo Journal of Pharmacologyand Experimental Therapeutics vol 325 no 1 pp 37ndash46 2008

[62] A Goyenvalle A Vulin F Fougerousse et al ldquoRescue of dys-trophic muscle through U7 snRNA-mediated exon skippingrdquoScience vol 306 no 5702 pp 1796ndash1799 2004

[63] D BHalling P Aracena-Parks and S L Hamilton ldquoRegulationof voltage-gated Ca2+ channels by calmodulinrdquo Sciencersquos STKEvol 2005 no 315 p re15 2005

[64] K S Lee E Marban and R W Tsien ldquoInactivation of calciumchannels in mammalian heart cells joint dependence on mem-brane potential and intracellular calciumrdquo Journal of Physiologyvol 364 pp 395ndash411 1985

[65] C Shi and N M Soldatov ldquoMolecular determinants of voltage-dependent slow inactivation of the Ca2+ channelrdquo Journal ofBiological Chemistry vol 277 no 9 pp 6813ndash6821 2002

[66] E Kobrinsky E Schwartz D R Abernethy and NM SoldatovldquoVoltage-gated mobility of the Ca2+ channel cytoplasmic tailsand its regulatory rolerdquo Journal of Biological Chemistry vol 278no 7 pp 5021ndash5028 2003

[67] N M Soldatov ldquoCa2+ channel moving tail link between Ca2+-induced inactivation and Ca2+ signal transductionrdquo Trends inPharmacological Sciences vol 24 no 4 pp 167ndash171 2003

[68] D J Klemm P A Watson M G Frid et al ldquocAMP responseelement-binding protein content is a molecular determinantof smooth muscle cell proliferation and migrationrdquo Journal ofBiological Chemistry vol 276 no 49 pp 46132ndash46141 2001

[69] E Kobrinsky L Stevens Y Kazmi DWray andNM SoldatovldquoMolecular rearrangements of the Kv21 potassium channeltermini associated with voltage gatingrdquo Journal of BiologicalChemistry vol 281 no 28 pp 19233ndash19240 2006

[70] BMMayr G Canettieri andMRMontminy ldquoDistinct effectsof cAMP and mitogenic signals on CREB-binding proteinrecruitment impart specificity to target gene activation viaCREBrdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 98 no 19 pp 10936ndash10941 2001

[71] M Morad and N Soldatov ldquoCalcium channel inactivationpossible role in signal transduction and Ca2+ signalingrdquo CellCalcium vol 38 no 3-4 pp 223ndash231 2005

[72] DGWheeler C F Barrett R DGroth P Safa andRW TsienldquoCaMKII locally encodes L-type channel activity to signal tonuclear CREB in excitation-transcription couplingrdquo Journal ofCell Biology vol 183 no 5 pp 849ndash863 2008

[73] D E Mager E Kobrinsky A Masoudieh A Maltsev D RAbernethy andNM Soldatov ldquoAnalysis of functional signalingdomains from fluorescence imaging and the two-dimensionalcontinuous wavelet transformrdquo Biophysical Journal vol 93 no8 pp 2900ndash2910 2007

[74] E Kobrinsky D E Mager S A Bentil S I Murata DR Abernethy and N M Soldatov ldquoIdentification of plasmamembrane macro- and microdomains from wavelet analysis ofFRET microscopyrdquo Biophysical Journal vol 88 no 5 pp 3625ndash3634 2005

[75] E Kobrinsky S Q Duong A Sheydina and N M Solda-tov ldquoMicrodomain organization and frequency-dependence ofCREB-dependent transcriptional signaling in heart cellsrdquo TheFASEB Journal vol 25 no 5 pp 1544ndash1555 2011

[76] A Ravindran Q Z Lao J B Harry P Abrahimi E Kobrinskyand N M Soldatov ldquoCalmodulin-dependent gating of Cav12calcium channels in the absence of Cav120573 subunitsrdquo Proceedingsof the National Academy of Sciences of the United States ofAmerica vol 105 no 23 pp 8154ndash8159 2008

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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International Journal of

Microbiology

ISRN Biochemistry 5

calcium channel blockers [44] it is the inhibition of prolifer-ation that essentially underlies it [45] The density of DHPreceptors was found to depend on VSM cell proliferation[46] Association of Cav12 with mitogenesis in VSM cells issupported by the findings that DHPs reduced DNA synthesisstimulated by serum and PdGF [47ndash49] serotonin [50] EGF[51] and H

2O2(in mesangial cells) [52] Expression of Cav12

in VSM cells was shown to be cell cycle dependent with thehighest calcium current density in G

1phase [53] Whether

these changes are reflected in the molecular repertoire ofthe Cav12 splice variants is the issue particularly importantfor the elucidation of new therapeutic targets in diseasesleading to pathogenic proliferation of VSM cells such asatherosclerosis

Investigation of the 1205721C alternative splicing in VSM

cells [28 54 55] revealed the involvement of a number ofCACNA1C exons generating impressive diversity of humanvascular 120572

1C that includes possibly the VSM-specific splic-ing of exons 99a [35 55] and exon 34 [28] To establishan association between the disease and CACNA1C splicevariation at the level of cell we have completed [26] thesingle-gene profiling of the 120572

1C molecular remodeling inVSM cells of an artery caused by atherosclerosis The VSMcells were identified in frozen sections of six surgical biopsysamples of femoral and carotid arteries by immunostainingwith an antibody against smoothmuscle 120572-actin [56] used asa marker for VSM cells The 120572-actin staining correlated withimmunostaining by an anti-120572

1C antibody in serial sectionsand was reduced in atherosclerotic regions (Figure 4) consis-tent with dedifferentiation of VSM cells [57 58]The reducedexpression of 120572

1C at the protein level was corroborated bythe quantitative RT-PCR data showing that the relative 120572

1CmRNA level in VSM cells (normalized to 18S RNA) wasreduced 37 plusmn 09 fold (mean plusmn SEM) in the atheroscleroticregion Overall the reduced expression of 120572

1C caused by thelocally elaborated cytokines in the atherosclerotic regions ofarteries resembles the reduced expression of DHP receptorsobserved in fibroblasts exposed to mitogens andor secondmessengers after serum deprivation (Figure 1)

To find out whether the altered expression of 1205721C

in atherosclerotic VSM cells is accompanied by changesin the CACNA1C alternative splicing pattern we isolatedthe immunohistochemically identified VSM cells by laser-capture microdissection from adjacent regions of arteriesaffected and not affected by atherosclerosis and identifiedthe CACNA1C splice variants by RT-PCR Our findingsrevealed an extended repertoire of the exon 21 120572

1C spliceisoforms in nonatherosclerotic VSM cells characterized bya complex splicing pattern of exons 9 9A 31ndash34 and41A including the electrophysiologically characterized 120572

1C71(GenBank z34811) 120572

1C73 (z34812) 1205721C125 (AY830711)

1205721C126 (AY830713) and 1205721C127 (AY830712) splice isoforms

However only the exon 22 isoform of 1205721C (1205721C77) was

identified in atherosclerotic VSM cells Thus the switch ofthe CACNA1C alterative splicing from exon 21 to exon 22(Figure 3(b)) is a molecular signature of the Cav12 remod-eling of VSM cells to a pathophysiological proliferating statein atherosclerosis The age gender ethnicity drug exposureand other co-morbid conditions did not appreciably affect

this common pattern of the 1205721C splice variation in VSM cells

in response to atherosclerosisCareful electrophysiological analysis exhibited a number

of differences in the properties of the ldquoatheroscleroticrdquo 1205721C77

channel as compared to the 1205721C isoforms in healthy VSM

cells The largest differences were found between the 1205721C77

and 1205721C127 channels (Figure 5) In response to step depolar-

ization applied from the holding potential of minus90mV bothchannels generate calcium currents (119868Ca) that inactivate withalmost identical kinetics (Figure 5(a)) However we foundthat 119868Ca through the1205721C77 channel recovers from inactivationsignificantly faster than that in 120572

1C127 (Figure 5(b)) and other1205721C isoforms present in healthy VSM cells This finding

suggests that alternative splicing in atherosclerosis may affectvascular tone as a result of the increase in the 119868Ca density inVSM cells A hyperpolarization shift of the activation curvefor the atherosclerotic 120572

1C77 channel variant as comparedto Cav12 in healthy VSM cells (Figure 5(c)) may also resultin an increase of calcium entry in VSM cells Howeverthe overall 3-4-fold reduction in the expression of Cav12in atherosclerotic VSM cells may scale down some of theobserved electrophysiological changes

5 CACNA1C Exon 22 as a New TherapeuticTarget in Atherosclerosis

Direct DNA sequencing of the crude PCR amplificationproducts indicated that the switch to the exon 22 isoform ofvascular 120572

1C was almost complete in atherosclerosis becauseno distortion of the nucleotide peaks in the region of exon2122 was seen when compared to the exon 20 invariantregion (Figure 6(a)) Thus Cav12 underwent almost quanti-tative exon 2122 remodeling in VSM cells of diseased arteryregions

Although the cellular mechanisms leading to theCACNA1C exon 2122 switch may be very complex theassociation with VSM cell proliferation is obvious Indeeda similar exon switch was observed in primary humanaortic cells in culture after the quiescent nonproliferatingcells containing predominantly exon 21 splice variantswere exposed to serum (Figure 6(b)) Unlike exon 21exon 22 contains the AvrII restriction site that allowsfor the assessment of its presence in PCR amplificationproduct of CACNA1C transcripts isolated from the cellsThe AvrII-sensitive exon 22 isoform of the 120572

1C transcriptwas not detected in the quiescent nonproliferating aorticcells (Figure 6(b) lane 2) However when 5 serum wasadded to the medium with nonconfluent aortic cellsDNA biosynthesis was activated while the level of the1205721C transcript decreased asymp3 fold and the presence of the

AvrII-sensitive exon 22 isoforms of 1205721C was easily detected

(Figure 6(b) lane 4) The isoform remodeling simulated inaortic primary cells in vitro was not complete as comparedto VSM cells in atherosclerotic regions of artery occludedwith heavy plaque burden which were selected for the1205721C molecular profiling However the cell culture results

demonstrate that in a different experimental system there isan obvious association between proliferation of VSM cells

6 ISRN Biochemistry

A Ki-67 NKi-67

50 12058350 120583

(a)

A N120572-actin 120572-actin

(b)

A N1205721C1205721C

(c)

Figure 4 Reduced expression of 1205721C in atherosclerotic (A) region of aorta as compared to the adjacent nonaffected (N) region From (a) to

(c) detection of proliferating cells in an exemplar biopsy of an artery by an antibody against human nuclear protein Ki-67 (a) Red arrowshighlight some proliferating nuclei (black staining) while blue arrows exemplify nonproliferating nuclei (light grey) Staining with antibodiesagainst smooth muscle 120572-actin (b) and against 120572

1C (c) Scale bars 50 120583m

downregulation of the CACNA1C expression and synthesisof the exon 22 120572

1C isoformRecent strategies targeting VSM cells to treat cardiovas-

cular diseases suggest indiscriminate disruption of Cav12[59ndash61] Is it possible to correct the described CACNA1Csplice defects induced by atherosclerosis without affectingthe transcripts of the gene lacking the rdquopathogenicrdquo exon22 Correction of defective genes responsible for diseasedevelopment is achieved by gene therapy Usually it requiresan insertion into the genome of a normal gene in place ofa defective one causing disease Such technique howeveris poorly controlled Currently one of the most promising

cutting-edge therapeutic approaches to correct defects asso-ciated with disease-induced expression of abnormal splicevariants is antisense-mediated exon skipping It is basedon the use of antisense oligonucleotides targeting specificexons to be removed The adenovirus-directed 120572

1C exon 22skipping-induced inhibition of VSM cell proliferation (andrespectively migration) can be used to rescue VSM cells fromremodeling in atherosclerosis The 120572

1C exon 22-skippingwill not alter the open reading frame of the 120572

1C transcriptbecause alternative exons 21 and 22 are of the equal size(60 nt)Themodified nonspliceosomal snRNAU7 gene alongwith its natural promoter and 31015840 elements exon 22-antisense

ISRN Biochemistry 7

200 ms

1205721C77

1205721C127

(a)

Prepulse interval (s)02 04 06 08 1

0

1

08

06

04

02

0

Frac

tion

of119868 C

a

1205721C77

1205721C127

(b)

0

1

08

06

04

02

119866Ca

119866Ca

max

1205721C77

1205721C127

minus60 minus40 minus20 0 20 40(mV)

(c)

Figure 5 Comparison of electrophysiological properties of 119868Ca through the ldquoatheroscleroticrdquo 1205721C77 and ldquonormalrdquo 120572

1C127 channels expressedin Xenopus oocytes with 120572

2120575-1 subunit and the primary cardiac 1205732a subunit and measured with 25mM Ca2+ as the charge carrier (a)

Representative traces of 119868Ca evoked by 1 s step depolarizations to +20mV from 119881ℎ= minus90mV and normalized to the same amplitude (b)

Fractional recovery of 119868Ca from inactivation (c) Averaged activation (119866119866max-119881) curves fit by the Boltzmann function A 1 s test pulses in therange of minus40 to +50mV (10-mV increments) were applied from 119881

ℎ= minus90mV with 30-s intervals

(a)

AvrII

Serum

1 2 3 4

+minus+minus

minus minus + +

142 pb

459 pb

601 pb

(b)

Figure 6 (a) Trace diagrams of DNA sequencing of the 1205721C PCR amplification products of healthy (top row) and diseased VSM cells (bottom

row) using an antisense primer composed of nucleotides 2923ndash2939 (z34815) A boundary with the sequence of invariant exon 20 is markedby a vertical dotted line (b) Evidence that the AvrII-sensitive exon 22 isoform of 120572

1C is expressed only in proliferating VSM cells Primaryaortic cells were grown to confluent monolayer in 5 serum before serum-deprivation for 5 days (lanes 1 and 2)Then the cells were replatedat low density in 5 serum for 3-4 days (lanes 3 and 4) Total RNA was isolated and exon 2122 isoforms were identified by RT-PCR andAvrII restriction analysis (lanes 2 and 4)

8 ISRN Biochemistry

sequence and supplemented with Sm ribonucleoprotein-binding sequence may be incorporated into the adenovirusvector for high efficiency transfer [62]The cytokine receptors(eg PdGF-120573 receptor) based recognition targeting of viralliposomes or nanoparticles may be especially advantageousin connection with selective gene transfer to VSM cellsaffected by atherosclerosis while reducing the probability ofthe transfection of other cells

6 Cak12 and CREB-DependentTranscriptional Activation

How Cav12 activity is translated into a proliferation-effectedmodality is another important question to be asked Cav12calcium channels generate a transient rise in cytosolic Ca2+-concentration activated by membrane depolarization Cel-lular responses associated with the rise of [Ca2+]i rangefrom sarcomeric contraction to cell growth and proliferationCytoplasmic domains of Cav12 have evolved a fairly intricateCaM-dependent signaling mechanism that provides for thenegative feedback inhibition of the calcium current knownas Ca2+ dependent inactivation (CDI) which is mediated bydifferent determinants of 120572

1C [25 63] Such a mechanism ofCDI resulting in acceleration of 119868Ca inactivation in responseto the rise of intracellular Ca2+ was first identified in cardiacCav12 [64] Similar experiments performed on the recombi-nant Cav12 also showed that the replacement of extracellularCa2+ by Ba2+ eliminates CDI and the channel inactivates by aslower voltage-dependent mechanism [65]The very fact thattwo distantly located determinants one in the pore regionresponsible for slow inactivation and the CaM-binding onein the proximal locus of the C-tail are independently crucialfor CDI indicates that not only their specific molecularstructure but also their mutual folding andor interaction areessential Experimental evidence show that this interactionreacts dynamically to membrane voltage supporting state-dependent transitions of the channel between resting openand inactivated conformations which are essential for CREB-dependent transcriptional activation [66 67]

CREB is a transcription factor of general importance ina large variety of cells CREB phosphorylation promotes theactivation of genes and is regulated by protein kinases undercontrol of the major second messengers cAMP andor Ca2+Indeed CREB functions as a ldquomolecular determinant of VSMcells fate [68]rdquo CREB content depends on proliferation ofVSM cells both in situ and in culture Serum deprivationincreased CREB content in VSM cells while exposure toPdGF decreased it Consistent with this observation anoverexpression of the constitutively active CREB inVSM cellsarrested cell cycle progression

To investigate the association of Cav12 with CREB tran-scriptional activation at the molecular level we combinedpatch clamp with fluorescent resonance energy transfer(FRET) microscopy in the live cell In voltage clampedcells FRET provided optical measurements under state-dependent conditions showing that the shorter N-terminaltail of 120572

1C (eg 1205721C77) does not rearrange vis-a-vis the

plasma membrane in response to voltage gating [25] In

sharp contrast the C-tail shows voltage-dependent confor-mational rearrangements which are much larger in sizethan that for example in the potassium Kv21 channel[69] Measurements of 119868Ca and corrected FRET betweenthe enhanced yellow (EYFP) and cyan fluorescent proteins(ECFP) genetically attached respectively to the N- and C-termini of 120572

1C77 showed no significant effect on voltage-dependence and kinetics of the channel current Howevertherewas a substantial increase in FRET signal accompanyinginactivation of the channel that was fully reversible uponits transition into the resting state in response to hyper-polarization [66] The plasma-membrane anchoring of the1205721C C-tail by the fusion of the pleckstrin homology domain

(PH) eliminated CDI but not 119868Ca Do the voltage-gatedconformational rearrangements of the 120572

1C C-tail and CDIplay a role in Ca2+ signal transduction that is utilized inCa2+-induced activation or CREB-dependent transcriptionTo answer this question we used the test system based onthe measurement of interaction between KID domain ofCREB and KIX domain of coactivator CREB-binding protein(CBP Figure 7(A) inset) under voltage-clamp conditionsby monitoring FRET between EYFP-KID and ECFP-KIX[70] Ca2+-dependent phosphorylation of KID stimulates itsbinding to KIX bringing EYFP and ECFP close enoughto observe the interaction by FRET In perforated wholeclamped cell where the integrity of the cytoplasmic contentis intact (Figure 7(A)) no activation of CREB transcription(Figure 7(B) panel (a)) and rearrangement of the120572

1C77 C-tail(panel (b)) was observed when the C-tail was anchored to theplasma membrane CREB transcriptional activity remainedlow in spite of a large sustained inward 119868Ca (panel (d))and the corresponding increase in [Ca2+]i detected by thefluorescence of Ca2+ indicator Fura4 (panel (c)) Releaseof the 120572

1C77 C-tail by the activation of PIP2hydrolysis

upon activation of M1AchR (Figure 7(C)) at minus90mV causedsignificant elevation of [Ca2+]i that also was not utilizedby the cell for CREB transcriptional activation (Figure 7(C)panel (a)) until a depolarizing pulse to +20-mV was appliedand the released 120572

1C77 C-tail was permitted to rearrange(Figure 7(D))This experiment provides compelling evidencethat neither large inward 119868Ca nor the subsequent rise in[Ca2+]i lead to CREB transcription activation unless theconformational rearrangement of the 120572

1C77 subunit C-tailprovides the precise targeting of the Ca2+ signal transduction(Figure 7(D) scheme) [66]

There is general agreement that CaM binds to LA and IQdomains of the 120572

1C C-tail and acts as a sensor that conveysCDI (for review see [63]) The affinity of CaM for bothdomains depends on [Ca2+]i Our data indicate that CDI andCa2+-signal transduction depend on the voltage-gatedmobil-ity of the120572

1C77 C-tail It is therefore reasonable to suggest thatthe LA-domain is a Ca2+-sensitive apo-CaM-mediated lockfor the mechanism of slow voltage-dependent inactivationof the channel [67] Apo-CaM associated with LA is able tocross-link it to another still unidentified apo-CaM bindingsite in the polypeptide bundle underlying the pore As theresult of this specific localization apo-CaMLA ldquolockrdquo ishidden from the cytoplasmic Ca2+ so that for example the

ISRN Biochemistry 9

10

0

2

10

100 pA125 ms

(a) (a) (a)

(b)(c)

(b)(c)

(b)(c)

(B) (C) (D)

C-tail Anchored ReleasedReleased

0 10No FRET No FRET

(d) (d) (d)

Fluo-4

PH

CYN

C

C

NY

C

Y EYFP

C ECFP

PH Plekstrin homologydomain

PHPHY

C

NC

IQ

LA

LAIQ

(A)

Fluo-4 Fluo-4

FRET FRET FRET

CRE

Gene expressionCREBCREB P

CBP

AC

GPCR

PKA

cAMP

CaM

Nucleus

cAMP

Plasmamembrane

Ca2+ Ca2+

Ca2+

+20mV +20mV

minus90mV minus90mV

1205721C77

1198651198650

Cav 12

Ca2+

minus90mVACh (5 mM)

4 120583

Figure 7 Evidence that Ca2+ signal transduction by Cav12 to activate CREB-dependent transcription is mediated by the voltage-gatedmobility of the 120572

1C C-tail carrying the CaM-caged Ca2+ and is not directly associated with the increase of [Ca2+]i CREB activation wasexamined under perforated patch conditions in COS1 cells expressing recombinant atherosclerotic 120572

1C77 channel (A) Phase-contrast imageof the COS1 cell with a shadow of patch pipette The cell was expressing the 120572

1C77-PHC1205731a1205722120575-1 channel with the membrane-trapped 1205721C77

subunit C-tail type 1muscarinic Ach receptor (to release the anchored C-tail in response to activation by Ach) and EYFP-KID and ECFP-KIXdomains both supplemented with nuclear localization sequences Inset at the bottom Cav12- and cAMP-dependent cell signaling pathwaysmediating CREB-dependent transcription CBP CREB-binding protein CRE cAMP-response element (B) Plasma-membrane anchoring ofthe 1205721C77 C-tail (see schematic diagram) inhibits CREB activation in spite of robust 119868Ca evoked by depolarization (a) A 100-ms images of

FRET between EYFP-KID and ECFP-KIX were recorded at the end of the 12th +20-mV depolarization step applied every 10 s for 1 s from119881ℎ=

minus90mV (b) A corresponding representative image of corrected FRET ratio (in a ldquo3Drdquo MetaMorph transformation) recorded within 50-mswindows at minus90 and at the end of the last test pulse of the applied train of pulses (c) An increase of [Ca2+]i detected by Ca

2+ indicator Fluo4(d) Representative trace of 119868Ca (20mM Ca2+ in bath medium) evoked by depolarization to +20mV from119881

ℎ= minus90mV showing the sustained

component of Ca2+ conductance due to the C-tail anchoring (C) ACh-stimulation of theM1AChR to unbind the 1205721C C-tail caused activation

of IP3-dependent Ca2+ release (panel (c)) but did not show substantial activation of CREB transcription at 119881

ℎ= minus90mV (panel (a)) (D)

CREB-dependent transcription was activated only in response to depolarization applied to membrane channel with the 1205721C77 C-terminal tail

released from the plasma membrane to assume functional voltage-dependent conformation

intracellular Ca2+ released from the intracellular stores orCa2+ caging compounds does not accelerate significantly theinactivation of Cav12 [71] Thus apo-CaM associated withLA binds predominantly Ca2+ ions permeating through thepore A Ca2+-dependent transfer of CaM from LA to the IQ-motif opens the ldquolockrdquo and initiates a large rearrangement ofthe C-terminal tail This in turn facilitates inactivation of thechannel The Ca2+CaM complex with the IQ-motif is thentransferred by themobile C-tail to a downstream target of theCa2+-signaling cascade (such as CaMKII [72]) where Ca2+ isreleased as an activating stimulus while CaM switches back

to LA and returns the C-tail to the resting position availablefor the next cycle of Ca2+-signal transduction

Activation of CREB-dependent transcription by the L-type 119868Ca ismediated throughmultiple cell signaling pathwaysUsing FRET probes of CREB activity and 2D wavelet trans-form analysis we applied principles of quantitative biologyto detail the mechanism of Ca2+-activated CREB-dependenttranscription within localized regions (microdomains) ofthe nucleus We reached this goal by applying continuouswavelet analysis in two dimensions with a 2D wavelet asa deconvolution algorithm for FRET microscopy image

10 ISRN Biochemistry

Control

(A)

05

0

1

05

00 20 40 60 800 20 40 60 800 20 40 60 80

(a) (b) (c) (d)

20 40 60 800

0

20

40

60

80 1

C 3 5 10 mincAMP

C 2 10 mincAMP

C 3 10 min

cAMP

C 1 3 10 min

cAMP

5

cAMP stable (5)

(B)

119868Ca (3 min) cAMP (5 min) cAMP (10 min)

119868Ca cAMP (65) 119868Ca stable (15)

119868Ca119868Ca

119868Ca 119868Ca

119868Ca cAMP transient (15)

Figure 8 Microdomain analysis on the additive effect of 119868Ca and cAMP on CREB signaling in COS1 cells expressing the recombinantldquoatheroscleroticrdquo120572

1C77 channel (A) FRET signal within the nucleus during selected time points during 119868Ca stimulation and cAMP applicationOutlined are four types of signaling microdomains identified using 2D Mexican hat wavelet Red circles represent stable microdomains thatpersist through both 119868Ca and cAMP application White and yellow circles showmicrodomains stable activated by cAMP and 119868Ca respectivelyGreen circles represent transient microdomains of CREB signaling activation Axes show pixel numbers (B) Typical appearances of the fourtypes of CREB signaling microdomains activated by 119868Ca andor cAMP application and recorded in their maximal development in relationto the time of applied stimuli C control recorded before stimulation Color bars in (A) and (B) represent FRET values normalized to themaximum

analysis [73] Continuous wavelet analysis is a mathematicaltechnique that allows us to analyze a signal over severaldifferent frequencies across the entire signal [73 74] It isespecially useful for finding heterogeneity in a signal becauseit can easily find where the pattern (ie frequency) of asignal changes In these experiments we for the first timeobtained evidence of CREB signaling microdomains withinthe nucleus that respond differentially to 119868Ca stimulationand cAMP (Figure 8(A)) Results of the study revealed thatCREB-dependent transcriptional signaling occurs in dis-crete signaling microdomains underlying the architecture ofnuclear signaling Continuous activation ofCREB-dependenttranscriptional signaling by cAMP and Ca2+ resulted in agradual increase of the number of microdomains Four dif-ferent categories of cAMP and Ca2+-induced CREB signalingmicrodomains were characterized in COS1 cells expressingrecombinant Cav12 with the ldquoatheroscleroticrdquo 120572

1C77 splice

variant (Figure 8) In up to 65 of the microdomainstranscription was activated in additive manner by cAMPand Ca2+ Approximately 15 of signaling domains wereactivated only by 119868Ca and 5 of domains were activatedonly by cAMP Finally 15 of the domains were transientand activated by both cAMP and 119868Ca (Figure 8(B)) [75]A similar spatiotemporal organization of CREB-dependentsignaling was observed in spontaneously beating neonatalrat cardiomyocytes Although COS1 cells that were used inour experiments shown in Figures 7 and 8 are naturallydeprived of Cav12 [76] they inherited the ability to replicatethe Cav12-dependent activation of CREB signaling with theexogenous recombinant channel Thus this experimentalapproach fits the task which in my opinion is the mostimportant unresolved issue for the coupling of Cav12 toCREB signaling does the splice variation of 120572

1C affect thespatiotemporal organization of CREB-dependent signaling

ISRN Biochemistry 11

in a way that may affect cell proliferation and other crucialfunction

7 Conclusions

Association of Cav12 with regulation of transcription cellproliferation and its pathophysiology as in the case ofatherosclerosis requires detailed investigation of the roles ofthe naturally occurring 120572

1C splice variants It will limit thetraditionally intuitive approach to Cav12 in physiology andhelp to define new principle approaches to the treatment ofvarious Cav12 channelopathy-related dysfunctions above allcardiovascular diseases Humgenex Inc provides consultingand logistic support on a broad range of issues reported inthis review

Abbreviations

CaM CalmodulinCBD Calmodulin-binding domainCDI Ca2+-dependent inactivationDHP DihydropyridineECFP Enhanced cyan fluorescent proteinEYFP Enhanced yellow fluorescent proteinFALI Fluorophore-assisted light inactivationFRET Fluorescent resonance energy transfer[3H]PMD 26-Dimethyl-3-methoxycarbonyl-5-([23-

3H2]-n-propoxycarbonyl)-4-(21015840-

difluoromethoxyphenyl)-14-dihydropyridine

119868Ca Calcium currentPH Pleckstrin homology domainVSM Vascular smooth muscle

References

[1] Y S Lee M M Sayeed and R D Wurster ldquoInhibition ofcell growth and intracellular Ca2+ mobilization in humanbrain tumor cells by Ca2+ channel antagonistsrdquo Molecular andChemical Neuropathology vol 22 no 2 pp 81ndash95 1994

[2] K Sato J Ishizuka C W Cooper et al ldquoInhibitory effect ofcalcium channel blockers on growth of pancreatic cancer cellsrdquoPancreas vol 9 no 2 pp 193ndash202 1994

[3] V Bertrand M J Bastie N Vaysse and L Pradayrol ldquoInhibi-tion of gastrin-induced proliferation of AR4-2J cells by calciumchannel antagonistsrdquo International Journal of Cancer vol 56no 3 pp 427ndash432 1994

[4] JM Taylor andRU Simpson ldquoInhibition of cancer cell growthby calcium channel antagonists in the athymic mouserdquo CancerResearch vol 52 no 9 pp 2413ndash2418 1992

[5] M G Cattaneo M Gullo and L M Vicentini ldquoC2+ and Ca2+channel antagonists in the control of human small cell lung car-cinoma cell proliferationrdquo European Journal of Pharmacologyvol 247 no 3 pp 325ndash331 1993

[6] C Chen M J Corbley T M Roberts and P Hess ldquoVoltage-sensitive calcium channels in normal and transformed 3T3fibroblastsrdquo Science vol 239 no 4843 pp 1024ndash1026 1988

[7] P C Dartsch M Ritter M Gschwentner H J Lang andF Lang ldquoEffects of calcium channel blockers on NIH 3T3

fibroblasts expressing the Ha-ras oncogenerdquo European Journalof Cell Biology vol 67 no 4 pp 372ndash378 1995

[8] M Biel R Hullin S Freundner et al ldquoTissue-specific expres-sion of high-voltage-activated dihydropyridine-sensitive L-typecalcium channelsrdquo European Journal of Biochemistry vol 200no 1 pp 81ndash88 1991

[9] DM Rodman K Reese J Harral et al ldquoLow-voltage-activated(T-type) calcium channels control proliferation of human pul-monary artery myocytesrdquo Circulation Research vol 96 no 8pp 864ndash872 2005

[10] A Panner L L Cribbs G M Zainelli T C Origitano S Singhand R DWurster ldquoVariation of T-type calcium channel proteinexpression affects cell division of cultured tumor cellsrdquo CellCalcium vol 37 no 2 pp 105ndash119 2005

[11] C Chen and P Hess ldquoCalcium channels in mouse 3T3 andhuman fibroblastsrdquo Biophysical Journal vol 51 p 226a 1987

[12] N M Soldatov ldquoMolecular diversity of L-type Ca2+ channeltranscripts in human fibroblastsrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 89 no10 pp 4628ndash4632 1992

[13] A Fujii H Matsumoto S Nakao H Teshigawara and YAkimoto ldquoEffect of calcium-channel blockers on cell prolifera-tion DNA synthesis and collagen synthesis of cultured gingivalfibroblasts derived fromhumannifedipine responders and non-respondersrdquo Archives of Oral Biology vol 39 no 2 pp 99ndash1041994

[14] N M Soldatov ldquoGenomic structure of human L-type Ca2+channelrdquo Genomics vol 22 no 1 pp 77ndash87 1994

[15] V P Shevchenko N F Myasoedov N M Soldatov G JDuburs V V Kastron and I P Skrastins ldquoSynthesis andevaluation of biological activity of two novel tritium-labeledderivatives of riodipinerdquo Journal of Labelled Compounds andRadiopharmaceuticals vol 27 no 6 pp 721ndash730 1989

[16] S M Dudkin S N Gnedoj N N Chernyuk and N MSoldatov ldquo14-dihydropyridine receptor associated with Ca2+channels in human embryonic fibroblastsrdquo FEBS Letters vol233 no 2 pp 352ndash354 1988

[17] A DePover M A Matlib S W Lee et al ldquoSpecific bindingof [3H]nitrendipine to membranes from coronary arteriesand heart in relation to pharmacological effects Paradoxicalstimulation by diltiazemrdquo Biochemical and Biophysical ResearchCommunications vol 108 no 1 pp 110ndash117 1982

[18] N M Soldatov S N Gnedoj N N Chernyuk I A Britanovaand S M Dudkin ldquoMitogenesis and 14-dihydropyridinereceptor associated with Ca2+-channels in human embryonicfibroblastsrdquo Journal of Membrane Biology vol 5 pp 1161ndash11671988 (Russian)

[19] D Lipscombe D V Madison M Poenie H Reuter R Y Tsienand R W Tsien ldquoSpatial distribution of calcium channels andcytosolic calcium transients in growth cones and cell bodies ofsympathetic neuronsrdquo Proceedings of the National Academy ofSciences of the United States of America vol 85 no 7 pp 2398ndash2402 1988

[20] G S Harms L Cognet P H M Lommerse et al ldquoSingle-molecule imaging of L-type CA2+ channels in live cellsrdquoBiophysical Journal vol 81 no 5 pp 2639ndash2646 2001

[21] V di Biase G J Obermair Z Szabo et al ldquoStable membraneexpression of postsynaptic Cav12 calcium channel clustersis independent of interactions with AKAP79150 and PDZproteinsrdquo Journal of Neuroscience vol 28 no 51 pp 13845ndash13855 2008

12 ISRN Biochemistry

[22] M F Navedo E P Cheng C Yuan et al ldquoIncreased coupledgating of L-type Ca2+ channels during hypertension and timo-thy syndromerdquoCirculationResearch vol 106 no 4 pp 748ndash7562010

[23] E Kobrinsky P Abrahimi S Q Duong et al ldquoEffect of Cav120573subunits on structural organization of Cav12 calcium channelsrdquoPLoS One vol 4 no 5 Article ID e5587 2009

[24] Q Z Lao E Kobrinsky Z Liu and N M Soldatov ldquoOligomer-ization of Cav120573 subunits is an essential correlate of Ca

2+ channelactivityrdquoThe FASEB Journal vol 24 no 12 pp 5013ndash5023 2010

[25] E Kobrinsky S Tiwari V A Maltsev et al ldquoDifferential roleof the 1205721C subunit tails in regulation of the Cav12 channelby membrane potential 120573 subunits and Ca2+ ionsrdquo Journal ofBiological Chemistry vol 280 no 13 pp 12474ndash12485 2005

[26] S Tiwari Y Zhang J Heller D R Abernethy and N MSoldatov ldquoArtherosclerosis-related molecular alteration of thehuman Cav12 calcium channel 1205721C subunitrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 103 no 45 pp 17024ndash17029 2006

[27] I Splawski K W Timothy L M Sharpe et al ldquoCav12 calciumchannel dysfunction causes a multisystem disorder includingarrhythmia and autismrdquo Cell vol 119 no 1 pp 19ndash31 2004

[28] Z Z TangM C Liang S Lu et al ldquoTranscript scanning revealsnovel and extensive splice variations in human L-type voltage-gated calcium channel Cav12 1205721 subunitrdquo Journal of BiologicalChemistry vol 279 no 43 pp 44335ndash44343 2004

[29] P Liao T F Yong M C Liang D T Yue and T W SoongldquoSplicing for alternative structures of Cav12 Ca2+ channels incardiac and smooth musclesrdquo Cardiovascular Research vol 68no 2 pp 197ndash203 2005

[30] Y Blumenstein N Kanevsky G Sahar R Barzilai T Ivaninaand N Dascal ldquoA novel long N-terminal isoform of human L-type Ca2+ channel is up-regulated by protein kinase Crdquo Journalof Biological Chemistry vol 277 no 5 pp 3419ndash3423 2002

[31] N M Soldatov A Bouron and H Reuter ldquoDifferent voltage-dependent inhibition by dihydropyridines of human Ca2+channel splice variantsrdquo Journal of Biological Chemistry vol270 no 18 pp 10540ndash10543 1995

[32] D P Faxon M A Creager S C Smith et al ldquoAtheroscleroticvascular disease conference executive summary atheroscle-rotic vascular disease conference proceeding for healthcareprofessionals from a special writing group of the AmericanHeart Associationrdquo Circulation vol 109 no 21 pp 2595ndash26042004

[33] R Ross ldquoAtherosclerosismdashan inflammatory diseaserdquo The NewEngland Journal of Medicine vol 340 no 2 pp 115ndash126 1999

[34] H Roy S Bhardwaj and S Yla-Herttuala ldquoMolecular geneticsof atherosclerosisrdquo Human Genetics vol 125 no 5-6 pp 467ndash491 2009

[35] M Biel P Ruth E Bosse et al ldquoPrimary structure and func-tional expression of a high voltage activated calcium channelfrom rabbit lungrdquo FEBS Letters vol 269 no 2 pp 409ndash4121990

[36] X Cheng J Liu M Asuncion-Chin et al ldquoA novel Cav12N terminus expressed in smooth muscle cells of resistancesize arteries modifies channel regulation by auxiliary subunitsrdquoJournal of Biological Chemistry vol 282 no 40 pp 29211ndash292212007

[37] W J Koch P T Ellinor and A Schwartz ldquocDNA cloning ofa dihydropyridine-sensitive calcium channel from rat aortaevidence for the existence of alternatively spliced formsrdquo Journalof Biological Chemistry vol 265 no 29 pp 17786ndash17791 1990

[38] S Richard D Neveu G Carnac P Bodin P Travo and J Nar-geot ldquoDifferential expression of voltage-gated Ca2+-currents incultivated aortic myocytesrdquo Biochimica et Biophysica Acta vol1160 no 1 pp 95ndash104 1992

[39] A Schwartz ldquoCalcium antagonists review and perspective onmechanism of actionrdquo American Journal of Cardiology vol 64no 17 pp 3Indash9I 1989

[40] R P Mason ldquoMechanisms of plaque stabilization for thedihydropyridine calcium channel blocker amlodipine review ofthe evidencerdquo Atherosclerosis vol 165 no 2 pp 191ndash199 2002

[41] A Ruiz-Torres R Lozano J Melon and R Carraro ldquoL-calciumchannel blockade induced by diltiazem inhibits proliferationmigration and F-actin membrane rearrangements in humanvascular smooth muscle cells stimulated with insulin and IGF-1rdquo International Journal of Clinical Pharmacology andTherapeu-tics vol 41 no 9 pp 386ndash391 2003

[42] H Koshiyama S Tanaka and J Minamikawa ldquoEffect ofcalcium channel blocker amlodipine on the intimal-medialthickness of carotid arterial wall in type 2 diabetesrdquo Journal ofCardiovascular Pharmacology vol 33 no 6 pp 894ndash896 1999

[43] T Yamashita S KawashimaMOzaki et al ldquoA calcium channelblocker benidipine inhibits intimal thickening in the carotidartery of mice by increasing nitric oxide productionrdquo Journal ofHypertension vol 19 no 3 pp 451ndash458 2001

[44] R P Mason P Marche and T H Hintze ldquoNovel vascularbiology of third-generation L-type calcium channel antagonistsancillary actions of amlodipinerdquo Arteriosclerosis Thrombosisand Vascular Biology vol 23 no 12 pp 2155ndash2163 2003

[45] O Stepien J GogusevD L Zhu et al ldquoAmlodipine inhibition ofserum- thrombin- or fibroblast growth factor- induced vascu-lar smooth-muscle cell proliferationrdquo Journal of CardiovascularPharmacology vol 31 no 5 pp 786ndash793 1998

[46] V Ruiz-Velasco M B Mayer E W Inscho and L J HymelldquoModulation of dihydropyridine receptors in vascular smoothmuscle cells by membrane potential and cell proliferationrdquoEuropean Journal of Pharmacology vol 268 no 3 pp 311ndash3181994

[47] J Nilsson M Sjolund L Palmberg A M von Euler B Jonzonand J Thyberg ldquoThe calcium antagonist nifedipine inhibitsarterial smooth muscle cell proliferationrdquo Atherosclerosis vol58 no 1ndash3 pp 109ndash122 1985

[48] J Thyberg and L Palmberg ldquoThe calcium antagonist nisoldip-ine and the calmodulin antagonist W-7 synergistically inhibitinitiation of DNA synthesis in cultured arterial smooth musclecellsrdquo Biology of the Cell vol 60 no 2 pp 125ndash132 1987

[49] E Munro M Patel P Chan et al ldquoEffect of calcium channelblockers on the growth of human vascular smooth musclecells derived from saphenous vein and vascular graft stenosesrdquoJournal of Cardiovascular Pharmacology vol 23 no 5 pp 779ndash784 1994

[50] T A Kent A Jazayeri and J M Simard ldquoCalcium channelsand nifedipine inhibition of serotonin-induced [3H]thymidineincorporation in cultured cerebral smoothmuscle cellsrdquo Journalof Cerebral Blood Flow and Metabolism vol 12 no 1 pp 139ndash146 1992

[51] A Agrotis P J Little J Saltis and A Bobik ldquoDihydropyridineCa2+ channel antagonists inhibit the salvage pathway for DNAsynthesis in human vascular smooth muscle cellsrdquo EuropeanJournal of Pharmacology vol 244 no 3 pp 269ndash275 1993

[52] I Duque M R Puyol P Ruiz M Gonzalez-Rubio M LD Marques and D R Puyol ldquoCalcium channel blockers

ISRN Biochemistry 13

inhibit hydrogen peroxide-induced proliferation of culturedrat mesangial cellsrdquo Journal of Pharmacology and ExperimentalTherapeutics vol 267 no 2 pp 612ndash616 1993

[53] T Kuga S Kobayashi Y Hirakawa H Kanaide and ATakeshita ldquoCell cycle-dependent expression of L- and T-typeCa2+ currents in rat aortic smooth muscle cells in primaryculturerdquo Circulation Research vol 79 no 1 pp 14ndash19 1996

[54] E M Graf M Bock J F Heubach et al ldquoTissue distribution ofa humanCav12 1205721 subunit splice variant with a 75 bp insertionrdquoCell Calcium vol 38 no 1 pp 11ndash21 2005

[55] P Liao D Yu S Lu et al ldquoSmoothmuscle-selective alternativelyspliced exon generates functional variation in Cav12 calciumchannelsrdquo Journal of Biological Chemistry vol 279 no 48 pp50329ndash50335 2004

[56] O Skalli P Ropraz and A Trzeciak ldquoA monoclonal antibodyagainst 120572-smooth muscle actin a new probe for smooth muscledifferentiationrdquo Journal of Cell Biology vol 103 no 6 pp 2787ndash2796 1986

[57] T M Doherty K Asotra L A Fitzpatrick et al ldquoCalcificationin atherosclerosis bone biology and chronic inflammation atthe arterial crossroadsrdquo Proceedings of the National Academyof Sciences of the United States of America vol 100 no 20 pp11201ndash11206 2003

[58] G K Owens ldquoRegulation of differentiation of vascular smoothmuscle cellsrdquo Physiological Reviews vol 75 no 3 pp 487ndash5171995

[59] J P Bannister A Adebiyi G Zhao et al ldquoSmooth musclecell 1205722120575-1 subunits are essential for vasoregulation by Cav12channelsrdquo Circulation Research vol 105 no 10 pp 948ndash9552009

[60] S SonkusareM Fraer J DMarsh andN J Rusch ldquoDisruptingcalcium channel expression to lower blood pressure newtargeting of awell-known channelrdquoMolecular Interventions vol6 no 6 pp 304ndash310 2006

[61] S Telemaque S Sonkusare TGrain et al ldquoDesign ofmutant1205732subunits as decoy molecules to reduce the expression of func-tional Ca2+ channels in cardiac cellsrdquo Journal of Pharmacologyand Experimental Therapeutics vol 325 no 1 pp 37ndash46 2008

[62] A Goyenvalle A Vulin F Fougerousse et al ldquoRescue of dys-trophic muscle through U7 snRNA-mediated exon skippingrdquoScience vol 306 no 5702 pp 1796ndash1799 2004

[63] D BHalling P Aracena-Parks and S L Hamilton ldquoRegulationof voltage-gated Ca2+ channels by calmodulinrdquo Sciencersquos STKEvol 2005 no 315 p re15 2005

[64] K S Lee E Marban and R W Tsien ldquoInactivation of calciumchannels in mammalian heart cells joint dependence on mem-brane potential and intracellular calciumrdquo Journal of Physiologyvol 364 pp 395ndash411 1985

[65] C Shi and N M Soldatov ldquoMolecular determinants of voltage-dependent slow inactivation of the Ca2+ channelrdquo Journal ofBiological Chemistry vol 277 no 9 pp 6813ndash6821 2002

[66] E Kobrinsky E Schwartz D R Abernethy and NM SoldatovldquoVoltage-gated mobility of the Ca2+ channel cytoplasmic tailsand its regulatory rolerdquo Journal of Biological Chemistry vol 278no 7 pp 5021ndash5028 2003

[67] N M Soldatov ldquoCa2+ channel moving tail link between Ca2+-induced inactivation and Ca2+ signal transductionrdquo Trends inPharmacological Sciences vol 24 no 4 pp 167ndash171 2003

[68] D J Klemm P A Watson M G Frid et al ldquocAMP responseelement-binding protein content is a molecular determinantof smooth muscle cell proliferation and migrationrdquo Journal ofBiological Chemistry vol 276 no 49 pp 46132ndash46141 2001

[69] E Kobrinsky L Stevens Y Kazmi DWray andNM SoldatovldquoMolecular rearrangements of the Kv21 potassium channeltermini associated with voltage gatingrdquo Journal of BiologicalChemistry vol 281 no 28 pp 19233ndash19240 2006

[70] BMMayr G Canettieri andMRMontminy ldquoDistinct effectsof cAMP and mitogenic signals on CREB-binding proteinrecruitment impart specificity to target gene activation viaCREBrdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 98 no 19 pp 10936ndash10941 2001

[71] M Morad and N Soldatov ldquoCalcium channel inactivationpossible role in signal transduction and Ca2+ signalingrdquo CellCalcium vol 38 no 3-4 pp 223ndash231 2005

[72] DGWheeler C F Barrett R DGroth P Safa andRW TsienldquoCaMKII locally encodes L-type channel activity to signal tonuclear CREB in excitation-transcription couplingrdquo Journal ofCell Biology vol 183 no 5 pp 849ndash863 2008

[73] D E Mager E Kobrinsky A Masoudieh A Maltsev D RAbernethy andNM Soldatov ldquoAnalysis of functional signalingdomains from fluorescence imaging and the two-dimensionalcontinuous wavelet transformrdquo Biophysical Journal vol 93 no8 pp 2900ndash2910 2007

[74] E Kobrinsky D E Mager S A Bentil S I Murata DR Abernethy and N M Soldatov ldquoIdentification of plasmamembrane macro- and microdomains from wavelet analysis ofFRET microscopyrdquo Biophysical Journal vol 88 no 5 pp 3625ndash3634 2005

[75] E Kobrinsky S Q Duong A Sheydina and N M Solda-tov ldquoMicrodomain organization and frequency-dependence ofCREB-dependent transcriptional signaling in heart cellsrdquo TheFASEB Journal vol 25 no 5 pp 1544ndash1555 2011

[76] A Ravindran Q Z Lao J B Harry P Abrahimi E Kobrinskyand N M Soldatov ldquoCalmodulin-dependent gating of Cav12calcium channels in the absence of Cav120573 subunitsrdquo Proceedingsof the National Academy of Sciences of the United States ofAmerica vol 105 no 23 pp 8154ndash8159 2008

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

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BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

6 ISRN Biochemistry

A Ki-67 NKi-67

50 12058350 120583

(a)

A N120572-actin 120572-actin

(b)

A N1205721C1205721C

(c)

Figure 4 Reduced expression of 1205721C in atherosclerotic (A) region of aorta as compared to the adjacent nonaffected (N) region From (a) to

(c) detection of proliferating cells in an exemplar biopsy of an artery by an antibody against human nuclear protein Ki-67 (a) Red arrowshighlight some proliferating nuclei (black staining) while blue arrows exemplify nonproliferating nuclei (light grey) Staining with antibodiesagainst smooth muscle 120572-actin (b) and against 120572

1C (c) Scale bars 50 120583m

downregulation of the CACNA1C expression and synthesisof the exon 22 120572

1C isoformRecent strategies targeting VSM cells to treat cardiovas-

cular diseases suggest indiscriminate disruption of Cav12[59ndash61] Is it possible to correct the described CACNA1Csplice defects induced by atherosclerosis without affectingthe transcripts of the gene lacking the rdquopathogenicrdquo exon22 Correction of defective genes responsible for diseasedevelopment is achieved by gene therapy Usually it requiresan insertion into the genome of a normal gene in place ofa defective one causing disease Such technique howeveris poorly controlled Currently one of the most promising

cutting-edge therapeutic approaches to correct defects asso-ciated with disease-induced expression of abnormal splicevariants is antisense-mediated exon skipping It is basedon the use of antisense oligonucleotides targeting specificexons to be removed The adenovirus-directed 120572

1C exon 22skipping-induced inhibition of VSM cell proliferation (andrespectively migration) can be used to rescue VSM cells fromremodeling in atherosclerosis The 120572

1C exon 22-skippingwill not alter the open reading frame of the 120572

1C transcriptbecause alternative exons 21 and 22 are of the equal size(60 nt)Themodified nonspliceosomal snRNAU7 gene alongwith its natural promoter and 31015840 elements exon 22-antisense

ISRN Biochemistry 7

200 ms

1205721C77

1205721C127

(a)

Prepulse interval (s)02 04 06 08 1

0

1

08

06

04

02

0

Frac

tion

of119868 C

a

1205721C77

1205721C127

(b)

0

1

08

06

04

02

119866Ca

119866Ca

max

1205721C77

1205721C127

minus60 minus40 minus20 0 20 40(mV)

(c)

Figure 5 Comparison of electrophysiological properties of 119868Ca through the ldquoatheroscleroticrdquo 1205721C77 and ldquonormalrdquo 120572

1C127 channels expressedin Xenopus oocytes with 120572

2120575-1 subunit and the primary cardiac 1205732a subunit and measured with 25mM Ca2+ as the charge carrier (a)

Representative traces of 119868Ca evoked by 1 s step depolarizations to +20mV from 119881ℎ= minus90mV and normalized to the same amplitude (b)

Fractional recovery of 119868Ca from inactivation (c) Averaged activation (119866119866max-119881) curves fit by the Boltzmann function A 1 s test pulses in therange of minus40 to +50mV (10-mV increments) were applied from 119881

ℎ= minus90mV with 30-s intervals

(a)

AvrII

Serum

1 2 3 4

+minus+minus

minus minus + +

142 pb

459 pb

601 pb

(b)

Figure 6 (a) Trace diagrams of DNA sequencing of the 1205721C PCR amplification products of healthy (top row) and diseased VSM cells (bottom

row) using an antisense primer composed of nucleotides 2923ndash2939 (z34815) A boundary with the sequence of invariant exon 20 is markedby a vertical dotted line (b) Evidence that the AvrII-sensitive exon 22 isoform of 120572

1C is expressed only in proliferating VSM cells Primaryaortic cells were grown to confluent monolayer in 5 serum before serum-deprivation for 5 days (lanes 1 and 2)Then the cells were replatedat low density in 5 serum for 3-4 days (lanes 3 and 4) Total RNA was isolated and exon 2122 isoforms were identified by RT-PCR andAvrII restriction analysis (lanes 2 and 4)

8 ISRN Biochemistry

sequence and supplemented with Sm ribonucleoprotein-binding sequence may be incorporated into the adenovirusvector for high efficiency transfer [62]The cytokine receptors(eg PdGF-120573 receptor) based recognition targeting of viralliposomes or nanoparticles may be especially advantageousin connection with selective gene transfer to VSM cellsaffected by atherosclerosis while reducing the probability ofthe transfection of other cells

6 Cak12 and CREB-DependentTranscriptional Activation

How Cav12 activity is translated into a proliferation-effectedmodality is another important question to be asked Cav12calcium channels generate a transient rise in cytosolic Ca2+-concentration activated by membrane depolarization Cel-lular responses associated with the rise of [Ca2+]i rangefrom sarcomeric contraction to cell growth and proliferationCytoplasmic domains of Cav12 have evolved a fairly intricateCaM-dependent signaling mechanism that provides for thenegative feedback inhibition of the calcium current knownas Ca2+ dependent inactivation (CDI) which is mediated bydifferent determinants of 120572

1C [25 63] Such a mechanism ofCDI resulting in acceleration of 119868Ca inactivation in responseto the rise of intracellular Ca2+ was first identified in cardiacCav12 [64] Similar experiments performed on the recombi-nant Cav12 also showed that the replacement of extracellularCa2+ by Ba2+ eliminates CDI and the channel inactivates by aslower voltage-dependent mechanism [65]The very fact thattwo distantly located determinants one in the pore regionresponsible for slow inactivation and the CaM-binding onein the proximal locus of the C-tail are independently crucialfor CDI indicates that not only their specific molecularstructure but also their mutual folding andor interaction areessential Experimental evidence show that this interactionreacts dynamically to membrane voltage supporting state-dependent transitions of the channel between resting openand inactivated conformations which are essential for CREB-dependent transcriptional activation [66 67]

CREB is a transcription factor of general importance ina large variety of cells CREB phosphorylation promotes theactivation of genes and is regulated by protein kinases undercontrol of the major second messengers cAMP andor Ca2+Indeed CREB functions as a ldquomolecular determinant of VSMcells fate [68]rdquo CREB content depends on proliferation ofVSM cells both in situ and in culture Serum deprivationincreased CREB content in VSM cells while exposure toPdGF decreased it Consistent with this observation anoverexpression of the constitutively active CREB inVSM cellsarrested cell cycle progression

To investigate the association of Cav12 with CREB tran-scriptional activation at the molecular level we combinedpatch clamp with fluorescent resonance energy transfer(FRET) microscopy in the live cell In voltage clampedcells FRET provided optical measurements under state-dependent conditions showing that the shorter N-terminaltail of 120572

1C (eg 1205721C77) does not rearrange vis-a-vis the

plasma membrane in response to voltage gating [25] In

sharp contrast the C-tail shows voltage-dependent confor-mational rearrangements which are much larger in sizethan that for example in the potassium Kv21 channel[69] Measurements of 119868Ca and corrected FRET betweenthe enhanced yellow (EYFP) and cyan fluorescent proteins(ECFP) genetically attached respectively to the N- and C-termini of 120572

1C77 showed no significant effect on voltage-dependence and kinetics of the channel current Howevertherewas a substantial increase in FRET signal accompanyinginactivation of the channel that was fully reversible uponits transition into the resting state in response to hyper-polarization [66] The plasma-membrane anchoring of the1205721C C-tail by the fusion of the pleckstrin homology domain

(PH) eliminated CDI but not 119868Ca Do the voltage-gatedconformational rearrangements of the 120572

1C C-tail and CDIplay a role in Ca2+ signal transduction that is utilized inCa2+-induced activation or CREB-dependent transcriptionTo answer this question we used the test system based onthe measurement of interaction between KID domain ofCREB and KIX domain of coactivator CREB-binding protein(CBP Figure 7(A) inset) under voltage-clamp conditionsby monitoring FRET between EYFP-KID and ECFP-KIX[70] Ca2+-dependent phosphorylation of KID stimulates itsbinding to KIX bringing EYFP and ECFP close enoughto observe the interaction by FRET In perforated wholeclamped cell where the integrity of the cytoplasmic contentis intact (Figure 7(A)) no activation of CREB transcription(Figure 7(B) panel (a)) and rearrangement of the120572

1C77 C-tail(panel (b)) was observed when the C-tail was anchored to theplasma membrane CREB transcriptional activity remainedlow in spite of a large sustained inward 119868Ca (panel (d))and the corresponding increase in [Ca2+]i detected by thefluorescence of Ca2+ indicator Fura4 (panel (c)) Releaseof the 120572

1C77 C-tail by the activation of PIP2hydrolysis

upon activation of M1AchR (Figure 7(C)) at minus90mV causedsignificant elevation of [Ca2+]i that also was not utilizedby the cell for CREB transcriptional activation (Figure 7(C)panel (a)) until a depolarizing pulse to +20-mV was appliedand the released 120572

1C77 C-tail was permitted to rearrange(Figure 7(D))This experiment provides compelling evidencethat neither large inward 119868Ca nor the subsequent rise in[Ca2+]i lead to CREB transcription activation unless theconformational rearrangement of the 120572

1C77 subunit C-tailprovides the precise targeting of the Ca2+ signal transduction(Figure 7(D) scheme) [66]

There is general agreement that CaM binds to LA and IQdomains of the 120572

1C C-tail and acts as a sensor that conveysCDI (for review see [63]) The affinity of CaM for bothdomains depends on [Ca2+]i Our data indicate that CDI andCa2+-signal transduction depend on the voltage-gatedmobil-ity of the120572

1C77 C-tail It is therefore reasonable to suggest thatthe LA-domain is a Ca2+-sensitive apo-CaM-mediated lockfor the mechanism of slow voltage-dependent inactivationof the channel [67] Apo-CaM associated with LA is able tocross-link it to another still unidentified apo-CaM bindingsite in the polypeptide bundle underlying the pore As theresult of this specific localization apo-CaMLA ldquolockrdquo ishidden from the cytoplasmic Ca2+ so that for example the

ISRN Biochemistry 9

10

0

2

10

100 pA125 ms

(a) (a) (a)

(b)(c)

(b)(c)

(b)(c)

(B) (C) (D)

C-tail Anchored ReleasedReleased

0 10No FRET No FRET

(d) (d) (d)

Fluo-4

PH

CYN

C

C

NY

C

Y EYFP

C ECFP

PH Plekstrin homologydomain

PHPHY

C

NC

IQ

LA

LAIQ

(A)

Fluo-4 Fluo-4

FRET FRET FRET

CRE

Gene expressionCREBCREB P

CBP

AC

GPCR

PKA

cAMP

CaM

Nucleus

cAMP

Plasmamembrane

Ca2+ Ca2+

Ca2+

+20mV +20mV

minus90mV minus90mV

1205721C77

1198651198650

Cav 12

Ca2+

minus90mVACh (5 mM)

4 120583

Figure 7 Evidence that Ca2+ signal transduction by Cav12 to activate CREB-dependent transcription is mediated by the voltage-gatedmobility of the 120572

1C C-tail carrying the CaM-caged Ca2+ and is not directly associated with the increase of [Ca2+]i CREB activation wasexamined under perforated patch conditions in COS1 cells expressing recombinant atherosclerotic 120572

1C77 channel (A) Phase-contrast imageof the COS1 cell with a shadow of patch pipette The cell was expressing the 120572

1C77-PHC1205731a1205722120575-1 channel with the membrane-trapped 1205721C77

subunit C-tail type 1muscarinic Ach receptor (to release the anchored C-tail in response to activation by Ach) and EYFP-KID and ECFP-KIXdomains both supplemented with nuclear localization sequences Inset at the bottom Cav12- and cAMP-dependent cell signaling pathwaysmediating CREB-dependent transcription CBP CREB-binding protein CRE cAMP-response element (B) Plasma-membrane anchoring ofthe 1205721C77 C-tail (see schematic diagram) inhibits CREB activation in spite of robust 119868Ca evoked by depolarization (a) A 100-ms images of

FRET between EYFP-KID and ECFP-KIX were recorded at the end of the 12th +20-mV depolarization step applied every 10 s for 1 s from119881ℎ=

minus90mV (b) A corresponding representative image of corrected FRET ratio (in a ldquo3Drdquo MetaMorph transformation) recorded within 50-mswindows at minus90 and at the end of the last test pulse of the applied train of pulses (c) An increase of [Ca2+]i detected by Ca

2+ indicator Fluo4(d) Representative trace of 119868Ca (20mM Ca2+ in bath medium) evoked by depolarization to +20mV from119881

ℎ= minus90mV showing the sustained

component of Ca2+ conductance due to the C-tail anchoring (C) ACh-stimulation of theM1AChR to unbind the 1205721C C-tail caused activation

of IP3-dependent Ca2+ release (panel (c)) but did not show substantial activation of CREB transcription at 119881

ℎ= minus90mV (panel (a)) (D)

CREB-dependent transcription was activated only in response to depolarization applied to membrane channel with the 1205721C77 C-terminal tail

released from the plasma membrane to assume functional voltage-dependent conformation

intracellular Ca2+ released from the intracellular stores orCa2+ caging compounds does not accelerate significantly theinactivation of Cav12 [71] Thus apo-CaM associated withLA binds predominantly Ca2+ ions permeating through thepore A Ca2+-dependent transfer of CaM from LA to the IQ-motif opens the ldquolockrdquo and initiates a large rearrangement ofthe C-terminal tail This in turn facilitates inactivation of thechannel The Ca2+CaM complex with the IQ-motif is thentransferred by themobile C-tail to a downstream target of theCa2+-signaling cascade (such as CaMKII [72]) where Ca2+ isreleased as an activating stimulus while CaM switches back

to LA and returns the C-tail to the resting position availablefor the next cycle of Ca2+-signal transduction

Activation of CREB-dependent transcription by the L-type 119868Ca ismediated throughmultiple cell signaling pathwaysUsing FRET probes of CREB activity and 2D wavelet trans-form analysis we applied principles of quantitative biologyto detail the mechanism of Ca2+-activated CREB-dependenttranscription within localized regions (microdomains) ofthe nucleus We reached this goal by applying continuouswavelet analysis in two dimensions with a 2D wavelet asa deconvolution algorithm for FRET microscopy image

10 ISRN Biochemistry

Control

(A)

05

0

1

05

00 20 40 60 800 20 40 60 800 20 40 60 80

(a) (b) (c) (d)

20 40 60 800

0

20

40

60

80 1

C 3 5 10 mincAMP

C 2 10 mincAMP

C 3 10 min

cAMP

C 1 3 10 min

cAMP

5

cAMP stable (5)

(B)

119868Ca (3 min) cAMP (5 min) cAMP (10 min)

119868Ca cAMP (65) 119868Ca stable (15)

119868Ca119868Ca

119868Ca 119868Ca

119868Ca cAMP transient (15)

Figure 8 Microdomain analysis on the additive effect of 119868Ca and cAMP on CREB signaling in COS1 cells expressing the recombinantldquoatheroscleroticrdquo120572

1C77 channel (A) FRET signal within the nucleus during selected time points during 119868Ca stimulation and cAMP applicationOutlined are four types of signaling microdomains identified using 2D Mexican hat wavelet Red circles represent stable microdomains thatpersist through both 119868Ca and cAMP application White and yellow circles showmicrodomains stable activated by cAMP and 119868Ca respectivelyGreen circles represent transient microdomains of CREB signaling activation Axes show pixel numbers (B) Typical appearances of the fourtypes of CREB signaling microdomains activated by 119868Ca andor cAMP application and recorded in their maximal development in relationto the time of applied stimuli C control recorded before stimulation Color bars in (A) and (B) represent FRET values normalized to themaximum

analysis [73] Continuous wavelet analysis is a mathematicaltechnique that allows us to analyze a signal over severaldifferent frequencies across the entire signal [73 74] It isespecially useful for finding heterogeneity in a signal becauseit can easily find where the pattern (ie frequency) of asignal changes In these experiments we for the first timeobtained evidence of CREB signaling microdomains withinthe nucleus that respond differentially to 119868Ca stimulationand cAMP (Figure 8(A)) Results of the study revealed thatCREB-dependent transcriptional signaling occurs in dis-crete signaling microdomains underlying the architecture ofnuclear signaling Continuous activation ofCREB-dependenttranscriptional signaling by cAMP and Ca2+ resulted in agradual increase of the number of microdomains Four dif-ferent categories of cAMP and Ca2+-induced CREB signalingmicrodomains were characterized in COS1 cells expressingrecombinant Cav12 with the ldquoatheroscleroticrdquo 120572

1C77 splice

variant (Figure 8) In up to 65 of the microdomainstranscription was activated in additive manner by cAMPand Ca2+ Approximately 15 of signaling domains wereactivated only by 119868Ca and 5 of domains were activatedonly by cAMP Finally 15 of the domains were transientand activated by both cAMP and 119868Ca (Figure 8(B)) [75]A similar spatiotemporal organization of CREB-dependentsignaling was observed in spontaneously beating neonatalrat cardiomyocytes Although COS1 cells that were used inour experiments shown in Figures 7 and 8 are naturallydeprived of Cav12 [76] they inherited the ability to replicatethe Cav12-dependent activation of CREB signaling with theexogenous recombinant channel Thus this experimentalapproach fits the task which in my opinion is the mostimportant unresolved issue for the coupling of Cav12 toCREB signaling does the splice variation of 120572

1C affect thespatiotemporal organization of CREB-dependent signaling

ISRN Biochemistry 11

in a way that may affect cell proliferation and other crucialfunction

7 Conclusions

Association of Cav12 with regulation of transcription cellproliferation and its pathophysiology as in the case ofatherosclerosis requires detailed investigation of the roles ofthe naturally occurring 120572

1C splice variants It will limit thetraditionally intuitive approach to Cav12 in physiology andhelp to define new principle approaches to the treatment ofvarious Cav12 channelopathy-related dysfunctions above allcardiovascular diseases Humgenex Inc provides consultingand logistic support on a broad range of issues reported inthis review

Abbreviations

CaM CalmodulinCBD Calmodulin-binding domainCDI Ca2+-dependent inactivationDHP DihydropyridineECFP Enhanced cyan fluorescent proteinEYFP Enhanced yellow fluorescent proteinFALI Fluorophore-assisted light inactivationFRET Fluorescent resonance energy transfer[3H]PMD 26-Dimethyl-3-methoxycarbonyl-5-([23-

3H2]-n-propoxycarbonyl)-4-(21015840-

difluoromethoxyphenyl)-14-dihydropyridine

119868Ca Calcium currentPH Pleckstrin homology domainVSM Vascular smooth muscle

References

[1] Y S Lee M M Sayeed and R D Wurster ldquoInhibition ofcell growth and intracellular Ca2+ mobilization in humanbrain tumor cells by Ca2+ channel antagonistsrdquo Molecular andChemical Neuropathology vol 22 no 2 pp 81ndash95 1994

[2] K Sato J Ishizuka C W Cooper et al ldquoInhibitory effect ofcalcium channel blockers on growth of pancreatic cancer cellsrdquoPancreas vol 9 no 2 pp 193ndash202 1994

[3] V Bertrand M J Bastie N Vaysse and L Pradayrol ldquoInhibi-tion of gastrin-induced proliferation of AR4-2J cells by calciumchannel antagonistsrdquo International Journal of Cancer vol 56no 3 pp 427ndash432 1994

[4] JM Taylor andRU Simpson ldquoInhibition of cancer cell growthby calcium channel antagonists in the athymic mouserdquo CancerResearch vol 52 no 9 pp 2413ndash2418 1992

[5] M G Cattaneo M Gullo and L M Vicentini ldquoC2+ and Ca2+channel antagonists in the control of human small cell lung car-cinoma cell proliferationrdquo European Journal of Pharmacologyvol 247 no 3 pp 325ndash331 1993

[6] C Chen M J Corbley T M Roberts and P Hess ldquoVoltage-sensitive calcium channels in normal and transformed 3T3fibroblastsrdquo Science vol 239 no 4843 pp 1024ndash1026 1988

[7] P C Dartsch M Ritter M Gschwentner H J Lang andF Lang ldquoEffects of calcium channel blockers on NIH 3T3

fibroblasts expressing the Ha-ras oncogenerdquo European Journalof Cell Biology vol 67 no 4 pp 372ndash378 1995

[8] M Biel R Hullin S Freundner et al ldquoTissue-specific expres-sion of high-voltage-activated dihydropyridine-sensitive L-typecalcium channelsrdquo European Journal of Biochemistry vol 200no 1 pp 81ndash88 1991

[9] DM Rodman K Reese J Harral et al ldquoLow-voltage-activated(T-type) calcium channels control proliferation of human pul-monary artery myocytesrdquo Circulation Research vol 96 no 8pp 864ndash872 2005

[10] A Panner L L Cribbs G M Zainelli T C Origitano S Singhand R DWurster ldquoVariation of T-type calcium channel proteinexpression affects cell division of cultured tumor cellsrdquo CellCalcium vol 37 no 2 pp 105ndash119 2005

[11] C Chen and P Hess ldquoCalcium channels in mouse 3T3 andhuman fibroblastsrdquo Biophysical Journal vol 51 p 226a 1987

[12] N M Soldatov ldquoMolecular diversity of L-type Ca2+ channeltranscripts in human fibroblastsrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 89 no10 pp 4628ndash4632 1992

[13] A Fujii H Matsumoto S Nakao H Teshigawara and YAkimoto ldquoEffect of calcium-channel blockers on cell prolifera-tion DNA synthesis and collagen synthesis of cultured gingivalfibroblasts derived fromhumannifedipine responders and non-respondersrdquo Archives of Oral Biology vol 39 no 2 pp 99ndash1041994

[14] N M Soldatov ldquoGenomic structure of human L-type Ca2+channelrdquo Genomics vol 22 no 1 pp 77ndash87 1994

[15] V P Shevchenko N F Myasoedov N M Soldatov G JDuburs V V Kastron and I P Skrastins ldquoSynthesis andevaluation of biological activity of two novel tritium-labeledderivatives of riodipinerdquo Journal of Labelled Compounds andRadiopharmaceuticals vol 27 no 6 pp 721ndash730 1989

[16] S M Dudkin S N Gnedoj N N Chernyuk and N MSoldatov ldquo14-dihydropyridine receptor associated with Ca2+channels in human embryonic fibroblastsrdquo FEBS Letters vol233 no 2 pp 352ndash354 1988

[17] A DePover M A Matlib S W Lee et al ldquoSpecific bindingof [3H]nitrendipine to membranes from coronary arteriesand heart in relation to pharmacological effects Paradoxicalstimulation by diltiazemrdquo Biochemical and Biophysical ResearchCommunications vol 108 no 1 pp 110ndash117 1982

[18] N M Soldatov S N Gnedoj N N Chernyuk I A Britanovaand S M Dudkin ldquoMitogenesis and 14-dihydropyridinereceptor associated with Ca2+-channels in human embryonicfibroblastsrdquo Journal of Membrane Biology vol 5 pp 1161ndash11671988 (Russian)

[19] D Lipscombe D V Madison M Poenie H Reuter R Y Tsienand R W Tsien ldquoSpatial distribution of calcium channels andcytosolic calcium transients in growth cones and cell bodies ofsympathetic neuronsrdquo Proceedings of the National Academy ofSciences of the United States of America vol 85 no 7 pp 2398ndash2402 1988

[20] G S Harms L Cognet P H M Lommerse et al ldquoSingle-molecule imaging of L-type CA2+ channels in live cellsrdquoBiophysical Journal vol 81 no 5 pp 2639ndash2646 2001

[21] V di Biase G J Obermair Z Szabo et al ldquoStable membraneexpression of postsynaptic Cav12 calcium channel clustersis independent of interactions with AKAP79150 and PDZproteinsrdquo Journal of Neuroscience vol 28 no 51 pp 13845ndash13855 2008

12 ISRN Biochemistry

[22] M F Navedo E P Cheng C Yuan et al ldquoIncreased coupledgating of L-type Ca2+ channels during hypertension and timo-thy syndromerdquoCirculationResearch vol 106 no 4 pp 748ndash7562010

[23] E Kobrinsky P Abrahimi S Q Duong et al ldquoEffect of Cav120573subunits on structural organization of Cav12 calcium channelsrdquoPLoS One vol 4 no 5 Article ID e5587 2009

[24] Q Z Lao E Kobrinsky Z Liu and N M Soldatov ldquoOligomer-ization of Cav120573 subunits is an essential correlate of Ca

2+ channelactivityrdquoThe FASEB Journal vol 24 no 12 pp 5013ndash5023 2010

[25] E Kobrinsky S Tiwari V A Maltsev et al ldquoDifferential roleof the 1205721C subunit tails in regulation of the Cav12 channelby membrane potential 120573 subunits and Ca2+ ionsrdquo Journal ofBiological Chemistry vol 280 no 13 pp 12474ndash12485 2005

[26] S Tiwari Y Zhang J Heller D R Abernethy and N MSoldatov ldquoArtherosclerosis-related molecular alteration of thehuman Cav12 calcium channel 1205721C subunitrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 103 no 45 pp 17024ndash17029 2006

[27] I Splawski K W Timothy L M Sharpe et al ldquoCav12 calciumchannel dysfunction causes a multisystem disorder includingarrhythmia and autismrdquo Cell vol 119 no 1 pp 19ndash31 2004

[28] Z Z TangM C Liang S Lu et al ldquoTranscript scanning revealsnovel and extensive splice variations in human L-type voltage-gated calcium channel Cav12 1205721 subunitrdquo Journal of BiologicalChemistry vol 279 no 43 pp 44335ndash44343 2004

[29] P Liao T F Yong M C Liang D T Yue and T W SoongldquoSplicing for alternative structures of Cav12 Ca2+ channels incardiac and smooth musclesrdquo Cardiovascular Research vol 68no 2 pp 197ndash203 2005

[30] Y Blumenstein N Kanevsky G Sahar R Barzilai T Ivaninaand N Dascal ldquoA novel long N-terminal isoform of human L-type Ca2+ channel is up-regulated by protein kinase Crdquo Journalof Biological Chemistry vol 277 no 5 pp 3419ndash3423 2002

[31] N M Soldatov A Bouron and H Reuter ldquoDifferent voltage-dependent inhibition by dihydropyridines of human Ca2+channel splice variantsrdquo Journal of Biological Chemistry vol270 no 18 pp 10540ndash10543 1995

[32] D P Faxon M A Creager S C Smith et al ldquoAtheroscleroticvascular disease conference executive summary atheroscle-rotic vascular disease conference proceeding for healthcareprofessionals from a special writing group of the AmericanHeart Associationrdquo Circulation vol 109 no 21 pp 2595ndash26042004

[33] R Ross ldquoAtherosclerosismdashan inflammatory diseaserdquo The NewEngland Journal of Medicine vol 340 no 2 pp 115ndash126 1999

[34] H Roy S Bhardwaj and S Yla-Herttuala ldquoMolecular geneticsof atherosclerosisrdquo Human Genetics vol 125 no 5-6 pp 467ndash491 2009

[35] M Biel P Ruth E Bosse et al ldquoPrimary structure and func-tional expression of a high voltage activated calcium channelfrom rabbit lungrdquo FEBS Letters vol 269 no 2 pp 409ndash4121990

[36] X Cheng J Liu M Asuncion-Chin et al ldquoA novel Cav12N terminus expressed in smooth muscle cells of resistancesize arteries modifies channel regulation by auxiliary subunitsrdquoJournal of Biological Chemistry vol 282 no 40 pp 29211ndash292212007

[37] W J Koch P T Ellinor and A Schwartz ldquocDNA cloning ofa dihydropyridine-sensitive calcium channel from rat aortaevidence for the existence of alternatively spliced formsrdquo Journalof Biological Chemistry vol 265 no 29 pp 17786ndash17791 1990

[38] S Richard D Neveu G Carnac P Bodin P Travo and J Nar-geot ldquoDifferential expression of voltage-gated Ca2+-currents incultivated aortic myocytesrdquo Biochimica et Biophysica Acta vol1160 no 1 pp 95ndash104 1992

[39] A Schwartz ldquoCalcium antagonists review and perspective onmechanism of actionrdquo American Journal of Cardiology vol 64no 17 pp 3Indash9I 1989

[40] R P Mason ldquoMechanisms of plaque stabilization for thedihydropyridine calcium channel blocker amlodipine review ofthe evidencerdquo Atherosclerosis vol 165 no 2 pp 191ndash199 2002

[41] A Ruiz-Torres R Lozano J Melon and R Carraro ldquoL-calciumchannel blockade induced by diltiazem inhibits proliferationmigration and F-actin membrane rearrangements in humanvascular smooth muscle cells stimulated with insulin and IGF-1rdquo International Journal of Clinical Pharmacology andTherapeu-tics vol 41 no 9 pp 386ndash391 2003

[42] H Koshiyama S Tanaka and J Minamikawa ldquoEffect ofcalcium channel blocker amlodipine on the intimal-medialthickness of carotid arterial wall in type 2 diabetesrdquo Journal ofCardiovascular Pharmacology vol 33 no 6 pp 894ndash896 1999

[43] T Yamashita S KawashimaMOzaki et al ldquoA calcium channelblocker benidipine inhibits intimal thickening in the carotidartery of mice by increasing nitric oxide productionrdquo Journal ofHypertension vol 19 no 3 pp 451ndash458 2001

[44] R P Mason P Marche and T H Hintze ldquoNovel vascularbiology of third-generation L-type calcium channel antagonistsancillary actions of amlodipinerdquo Arteriosclerosis Thrombosisand Vascular Biology vol 23 no 12 pp 2155ndash2163 2003

[45] O Stepien J GogusevD L Zhu et al ldquoAmlodipine inhibition ofserum- thrombin- or fibroblast growth factor- induced vascu-lar smooth-muscle cell proliferationrdquo Journal of CardiovascularPharmacology vol 31 no 5 pp 786ndash793 1998

[46] V Ruiz-Velasco M B Mayer E W Inscho and L J HymelldquoModulation of dihydropyridine receptors in vascular smoothmuscle cells by membrane potential and cell proliferationrdquoEuropean Journal of Pharmacology vol 268 no 3 pp 311ndash3181994

[47] J Nilsson M Sjolund L Palmberg A M von Euler B Jonzonand J Thyberg ldquoThe calcium antagonist nifedipine inhibitsarterial smooth muscle cell proliferationrdquo Atherosclerosis vol58 no 1ndash3 pp 109ndash122 1985

[48] J Thyberg and L Palmberg ldquoThe calcium antagonist nisoldip-ine and the calmodulin antagonist W-7 synergistically inhibitinitiation of DNA synthesis in cultured arterial smooth musclecellsrdquo Biology of the Cell vol 60 no 2 pp 125ndash132 1987

[49] E Munro M Patel P Chan et al ldquoEffect of calcium channelblockers on the growth of human vascular smooth musclecells derived from saphenous vein and vascular graft stenosesrdquoJournal of Cardiovascular Pharmacology vol 23 no 5 pp 779ndash784 1994

[50] T A Kent A Jazayeri and J M Simard ldquoCalcium channelsand nifedipine inhibition of serotonin-induced [3H]thymidineincorporation in cultured cerebral smoothmuscle cellsrdquo Journalof Cerebral Blood Flow and Metabolism vol 12 no 1 pp 139ndash146 1992

[51] A Agrotis P J Little J Saltis and A Bobik ldquoDihydropyridineCa2+ channel antagonists inhibit the salvage pathway for DNAsynthesis in human vascular smooth muscle cellsrdquo EuropeanJournal of Pharmacology vol 244 no 3 pp 269ndash275 1993

[52] I Duque M R Puyol P Ruiz M Gonzalez-Rubio M LD Marques and D R Puyol ldquoCalcium channel blockers

ISRN Biochemistry 13

inhibit hydrogen peroxide-induced proliferation of culturedrat mesangial cellsrdquo Journal of Pharmacology and ExperimentalTherapeutics vol 267 no 2 pp 612ndash616 1993

[53] T Kuga S Kobayashi Y Hirakawa H Kanaide and ATakeshita ldquoCell cycle-dependent expression of L- and T-typeCa2+ currents in rat aortic smooth muscle cells in primaryculturerdquo Circulation Research vol 79 no 1 pp 14ndash19 1996

[54] E M Graf M Bock J F Heubach et al ldquoTissue distribution ofa humanCav12 1205721 subunit splice variant with a 75 bp insertionrdquoCell Calcium vol 38 no 1 pp 11ndash21 2005

[55] P Liao D Yu S Lu et al ldquoSmoothmuscle-selective alternativelyspliced exon generates functional variation in Cav12 calciumchannelsrdquo Journal of Biological Chemistry vol 279 no 48 pp50329ndash50335 2004

[56] O Skalli P Ropraz and A Trzeciak ldquoA monoclonal antibodyagainst 120572-smooth muscle actin a new probe for smooth muscledifferentiationrdquo Journal of Cell Biology vol 103 no 6 pp 2787ndash2796 1986

[57] T M Doherty K Asotra L A Fitzpatrick et al ldquoCalcificationin atherosclerosis bone biology and chronic inflammation atthe arterial crossroadsrdquo Proceedings of the National Academyof Sciences of the United States of America vol 100 no 20 pp11201ndash11206 2003

[58] G K Owens ldquoRegulation of differentiation of vascular smoothmuscle cellsrdquo Physiological Reviews vol 75 no 3 pp 487ndash5171995

[59] J P Bannister A Adebiyi G Zhao et al ldquoSmooth musclecell 1205722120575-1 subunits are essential for vasoregulation by Cav12channelsrdquo Circulation Research vol 105 no 10 pp 948ndash9552009

[60] S SonkusareM Fraer J DMarsh andN J Rusch ldquoDisruptingcalcium channel expression to lower blood pressure newtargeting of awell-known channelrdquoMolecular Interventions vol6 no 6 pp 304ndash310 2006

[61] S Telemaque S Sonkusare TGrain et al ldquoDesign ofmutant1205732subunits as decoy molecules to reduce the expression of func-tional Ca2+ channels in cardiac cellsrdquo Journal of Pharmacologyand Experimental Therapeutics vol 325 no 1 pp 37ndash46 2008

[62] A Goyenvalle A Vulin F Fougerousse et al ldquoRescue of dys-trophic muscle through U7 snRNA-mediated exon skippingrdquoScience vol 306 no 5702 pp 1796ndash1799 2004

[63] D BHalling P Aracena-Parks and S L Hamilton ldquoRegulationof voltage-gated Ca2+ channels by calmodulinrdquo Sciencersquos STKEvol 2005 no 315 p re15 2005

[64] K S Lee E Marban and R W Tsien ldquoInactivation of calciumchannels in mammalian heart cells joint dependence on mem-brane potential and intracellular calciumrdquo Journal of Physiologyvol 364 pp 395ndash411 1985

[65] C Shi and N M Soldatov ldquoMolecular determinants of voltage-dependent slow inactivation of the Ca2+ channelrdquo Journal ofBiological Chemistry vol 277 no 9 pp 6813ndash6821 2002

[66] E Kobrinsky E Schwartz D R Abernethy and NM SoldatovldquoVoltage-gated mobility of the Ca2+ channel cytoplasmic tailsand its regulatory rolerdquo Journal of Biological Chemistry vol 278no 7 pp 5021ndash5028 2003

[67] N M Soldatov ldquoCa2+ channel moving tail link between Ca2+-induced inactivation and Ca2+ signal transductionrdquo Trends inPharmacological Sciences vol 24 no 4 pp 167ndash171 2003

[68] D J Klemm P A Watson M G Frid et al ldquocAMP responseelement-binding protein content is a molecular determinantof smooth muscle cell proliferation and migrationrdquo Journal ofBiological Chemistry vol 276 no 49 pp 46132ndash46141 2001

[69] E Kobrinsky L Stevens Y Kazmi DWray andNM SoldatovldquoMolecular rearrangements of the Kv21 potassium channeltermini associated with voltage gatingrdquo Journal of BiologicalChemistry vol 281 no 28 pp 19233ndash19240 2006

[70] BMMayr G Canettieri andMRMontminy ldquoDistinct effectsof cAMP and mitogenic signals on CREB-binding proteinrecruitment impart specificity to target gene activation viaCREBrdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 98 no 19 pp 10936ndash10941 2001

[71] M Morad and N Soldatov ldquoCalcium channel inactivationpossible role in signal transduction and Ca2+ signalingrdquo CellCalcium vol 38 no 3-4 pp 223ndash231 2005

[72] DGWheeler C F Barrett R DGroth P Safa andRW TsienldquoCaMKII locally encodes L-type channel activity to signal tonuclear CREB in excitation-transcription couplingrdquo Journal ofCell Biology vol 183 no 5 pp 849ndash863 2008

[73] D E Mager E Kobrinsky A Masoudieh A Maltsev D RAbernethy andNM Soldatov ldquoAnalysis of functional signalingdomains from fluorescence imaging and the two-dimensionalcontinuous wavelet transformrdquo Biophysical Journal vol 93 no8 pp 2900ndash2910 2007

[74] E Kobrinsky D E Mager S A Bentil S I Murata DR Abernethy and N M Soldatov ldquoIdentification of plasmamembrane macro- and microdomains from wavelet analysis ofFRET microscopyrdquo Biophysical Journal vol 88 no 5 pp 3625ndash3634 2005

[75] E Kobrinsky S Q Duong A Sheydina and N M Solda-tov ldquoMicrodomain organization and frequency-dependence ofCREB-dependent transcriptional signaling in heart cellsrdquo TheFASEB Journal vol 25 no 5 pp 1544ndash1555 2011

[76] A Ravindran Q Z Lao J B Harry P Abrahimi E Kobrinskyand N M Soldatov ldquoCalmodulin-dependent gating of Cav12calcium channels in the absence of Cav120573 subunitsrdquo Proceedingsof the National Academy of Sciences of the United States ofAmerica vol 105 no 23 pp 8154ndash8159 2008

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

ISRN Biochemistry 7

200 ms

1205721C77

1205721C127

(a)

Prepulse interval (s)02 04 06 08 1

0

1

08

06

04

02

0

Frac

tion

of119868 C

a

1205721C77

1205721C127

(b)

0

1

08

06

04

02

119866Ca

119866Ca

max

1205721C77

1205721C127

minus60 minus40 minus20 0 20 40(mV)

(c)

Figure 5 Comparison of electrophysiological properties of 119868Ca through the ldquoatheroscleroticrdquo 1205721C77 and ldquonormalrdquo 120572

1C127 channels expressedin Xenopus oocytes with 120572

2120575-1 subunit and the primary cardiac 1205732a subunit and measured with 25mM Ca2+ as the charge carrier (a)

Representative traces of 119868Ca evoked by 1 s step depolarizations to +20mV from 119881ℎ= minus90mV and normalized to the same amplitude (b)

Fractional recovery of 119868Ca from inactivation (c) Averaged activation (119866119866max-119881) curves fit by the Boltzmann function A 1 s test pulses in therange of minus40 to +50mV (10-mV increments) were applied from 119881

ℎ= minus90mV with 30-s intervals

(a)

AvrII

Serum

1 2 3 4

+minus+minus

minus minus + +

142 pb

459 pb

601 pb

(b)

Figure 6 (a) Trace diagrams of DNA sequencing of the 1205721C PCR amplification products of healthy (top row) and diseased VSM cells (bottom

row) using an antisense primer composed of nucleotides 2923ndash2939 (z34815) A boundary with the sequence of invariant exon 20 is markedby a vertical dotted line (b) Evidence that the AvrII-sensitive exon 22 isoform of 120572

1C is expressed only in proliferating VSM cells Primaryaortic cells were grown to confluent monolayer in 5 serum before serum-deprivation for 5 days (lanes 1 and 2)Then the cells were replatedat low density in 5 serum for 3-4 days (lanes 3 and 4) Total RNA was isolated and exon 2122 isoforms were identified by RT-PCR andAvrII restriction analysis (lanes 2 and 4)

8 ISRN Biochemistry

sequence and supplemented with Sm ribonucleoprotein-binding sequence may be incorporated into the adenovirusvector for high efficiency transfer [62]The cytokine receptors(eg PdGF-120573 receptor) based recognition targeting of viralliposomes or nanoparticles may be especially advantageousin connection with selective gene transfer to VSM cellsaffected by atherosclerosis while reducing the probability ofthe transfection of other cells

6 Cak12 and CREB-DependentTranscriptional Activation

How Cav12 activity is translated into a proliferation-effectedmodality is another important question to be asked Cav12calcium channels generate a transient rise in cytosolic Ca2+-concentration activated by membrane depolarization Cel-lular responses associated with the rise of [Ca2+]i rangefrom sarcomeric contraction to cell growth and proliferationCytoplasmic domains of Cav12 have evolved a fairly intricateCaM-dependent signaling mechanism that provides for thenegative feedback inhibition of the calcium current knownas Ca2+ dependent inactivation (CDI) which is mediated bydifferent determinants of 120572

1C [25 63] Such a mechanism ofCDI resulting in acceleration of 119868Ca inactivation in responseto the rise of intracellular Ca2+ was first identified in cardiacCav12 [64] Similar experiments performed on the recombi-nant Cav12 also showed that the replacement of extracellularCa2+ by Ba2+ eliminates CDI and the channel inactivates by aslower voltage-dependent mechanism [65]The very fact thattwo distantly located determinants one in the pore regionresponsible for slow inactivation and the CaM-binding onein the proximal locus of the C-tail are independently crucialfor CDI indicates that not only their specific molecularstructure but also their mutual folding andor interaction areessential Experimental evidence show that this interactionreacts dynamically to membrane voltage supporting state-dependent transitions of the channel between resting openand inactivated conformations which are essential for CREB-dependent transcriptional activation [66 67]

CREB is a transcription factor of general importance ina large variety of cells CREB phosphorylation promotes theactivation of genes and is regulated by protein kinases undercontrol of the major second messengers cAMP andor Ca2+Indeed CREB functions as a ldquomolecular determinant of VSMcells fate [68]rdquo CREB content depends on proliferation ofVSM cells both in situ and in culture Serum deprivationincreased CREB content in VSM cells while exposure toPdGF decreased it Consistent with this observation anoverexpression of the constitutively active CREB inVSM cellsarrested cell cycle progression

To investigate the association of Cav12 with CREB tran-scriptional activation at the molecular level we combinedpatch clamp with fluorescent resonance energy transfer(FRET) microscopy in the live cell In voltage clampedcells FRET provided optical measurements under state-dependent conditions showing that the shorter N-terminaltail of 120572

1C (eg 1205721C77) does not rearrange vis-a-vis the

plasma membrane in response to voltage gating [25] In

sharp contrast the C-tail shows voltage-dependent confor-mational rearrangements which are much larger in sizethan that for example in the potassium Kv21 channel[69] Measurements of 119868Ca and corrected FRET betweenthe enhanced yellow (EYFP) and cyan fluorescent proteins(ECFP) genetically attached respectively to the N- and C-termini of 120572

1C77 showed no significant effect on voltage-dependence and kinetics of the channel current Howevertherewas a substantial increase in FRET signal accompanyinginactivation of the channel that was fully reversible uponits transition into the resting state in response to hyper-polarization [66] The plasma-membrane anchoring of the1205721C C-tail by the fusion of the pleckstrin homology domain

(PH) eliminated CDI but not 119868Ca Do the voltage-gatedconformational rearrangements of the 120572

1C C-tail and CDIplay a role in Ca2+ signal transduction that is utilized inCa2+-induced activation or CREB-dependent transcriptionTo answer this question we used the test system based onthe measurement of interaction between KID domain ofCREB and KIX domain of coactivator CREB-binding protein(CBP Figure 7(A) inset) under voltage-clamp conditionsby monitoring FRET between EYFP-KID and ECFP-KIX[70] Ca2+-dependent phosphorylation of KID stimulates itsbinding to KIX bringing EYFP and ECFP close enoughto observe the interaction by FRET In perforated wholeclamped cell where the integrity of the cytoplasmic contentis intact (Figure 7(A)) no activation of CREB transcription(Figure 7(B) panel (a)) and rearrangement of the120572

1C77 C-tail(panel (b)) was observed when the C-tail was anchored to theplasma membrane CREB transcriptional activity remainedlow in spite of a large sustained inward 119868Ca (panel (d))and the corresponding increase in [Ca2+]i detected by thefluorescence of Ca2+ indicator Fura4 (panel (c)) Releaseof the 120572

1C77 C-tail by the activation of PIP2hydrolysis

upon activation of M1AchR (Figure 7(C)) at minus90mV causedsignificant elevation of [Ca2+]i that also was not utilizedby the cell for CREB transcriptional activation (Figure 7(C)panel (a)) until a depolarizing pulse to +20-mV was appliedand the released 120572

1C77 C-tail was permitted to rearrange(Figure 7(D))This experiment provides compelling evidencethat neither large inward 119868Ca nor the subsequent rise in[Ca2+]i lead to CREB transcription activation unless theconformational rearrangement of the 120572

1C77 subunit C-tailprovides the precise targeting of the Ca2+ signal transduction(Figure 7(D) scheme) [66]

There is general agreement that CaM binds to LA and IQdomains of the 120572

1C C-tail and acts as a sensor that conveysCDI (for review see [63]) The affinity of CaM for bothdomains depends on [Ca2+]i Our data indicate that CDI andCa2+-signal transduction depend on the voltage-gatedmobil-ity of the120572

1C77 C-tail It is therefore reasonable to suggest thatthe LA-domain is a Ca2+-sensitive apo-CaM-mediated lockfor the mechanism of slow voltage-dependent inactivationof the channel [67] Apo-CaM associated with LA is able tocross-link it to another still unidentified apo-CaM bindingsite in the polypeptide bundle underlying the pore As theresult of this specific localization apo-CaMLA ldquolockrdquo ishidden from the cytoplasmic Ca2+ so that for example the

ISRN Biochemistry 9

10

0

2

10

100 pA125 ms

(a) (a) (a)

(b)(c)

(b)(c)

(b)(c)

(B) (C) (D)

C-tail Anchored ReleasedReleased

0 10No FRET No FRET

(d) (d) (d)

Fluo-4

PH

CYN

C

C

NY

C

Y EYFP

C ECFP

PH Plekstrin homologydomain

PHPHY

C

NC

IQ

LA

LAIQ

(A)

Fluo-4 Fluo-4

FRET FRET FRET

CRE

Gene expressionCREBCREB P

CBP

AC

GPCR

PKA

cAMP

CaM

Nucleus

cAMP

Plasmamembrane

Ca2+ Ca2+

Ca2+

+20mV +20mV

minus90mV minus90mV

1205721C77

1198651198650

Cav 12

Ca2+

minus90mVACh (5 mM)

4 120583

Figure 7 Evidence that Ca2+ signal transduction by Cav12 to activate CREB-dependent transcription is mediated by the voltage-gatedmobility of the 120572

1C C-tail carrying the CaM-caged Ca2+ and is not directly associated with the increase of [Ca2+]i CREB activation wasexamined under perforated patch conditions in COS1 cells expressing recombinant atherosclerotic 120572

1C77 channel (A) Phase-contrast imageof the COS1 cell with a shadow of patch pipette The cell was expressing the 120572

1C77-PHC1205731a1205722120575-1 channel with the membrane-trapped 1205721C77

subunit C-tail type 1muscarinic Ach receptor (to release the anchored C-tail in response to activation by Ach) and EYFP-KID and ECFP-KIXdomains both supplemented with nuclear localization sequences Inset at the bottom Cav12- and cAMP-dependent cell signaling pathwaysmediating CREB-dependent transcription CBP CREB-binding protein CRE cAMP-response element (B) Plasma-membrane anchoring ofthe 1205721C77 C-tail (see schematic diagram) inhibits CREB activation in spite of robust 119868Ca evoked by depolarization (a) A 100-ms images of

FRET between EYFP-KID and ECFP-KIX were recorded at the end of the 12th +20-mV depolarization step applied every 10 s for 1 s from119881ℎ=

minus90mV (b) A corresponding representative image of corrected FRET ratio (in a ldquo3Drdquo MetaMorph transformation) recorded within 50-mswindows at minus90 and at the end of the last test pulse of the applied train of pulses (c) An increase of [Ca2+]i detected by Ca

2+ indicator Fluo4(d) Representative trace of 119868Ca (20mM Ca2+ in bath medium) evoked by depolarization to +20mV from119881

ℎ= minus90mV showing the sustained

component of Ca2+ conductance due to the C-tail anchoring (C) ACh-stimulation of theM1AChR to unbind the 1205721C C-tail caused activation

of IP3-dependent Ca2+ release (panel (c)) but did not show substantial activation of CREB transcription at 119881

ℎ= minus90mV (panel (a)) (D)

CREB-dependent transcription was activated only in response to depolarization applied to membrane channel with the 1205721C77 C-terminal tail

released from the plasma membrane to assume functional voltage-dependent conformation

intracellular Ca2+ released from the intracellular stores orCa2+ caging compounds does not accelerate significantly theinactivation of Cav12 [71] Thus apo-CaM associated withLA binds predominantly Ca2+ ions permeating through thepore A Ca2+-dependent transfer of CaM from LA to the IQ-motif opens the ldquolockrdquo and initiates a large rearrangement ofthe C-terminal tail This in turn facilitates inactivation of thechannel The Ca2+CaM complex with the IQ-motif is thentransferred by themobile C-tail to a downstream target of theCa2+-signaling cascade (such as CaMKII [72]) where Ca2+ isreleased as an activating stimulus while CaM switches back

to LA and returns the C-tail to the resting position availablefor the next cycle of Ca2+-signal transduction

Activation of CREB-dependent transcription by the L-type 119868Ca ismediated throughmultiple cell signaling pathwaysUsing FRET probes of CREB activity and 2D wavelet trans-form analysis we applied principles of quantitative biologyto detail the mechanism of Ca2+-activated CREB-dependenttranscription within localized regions (microdomains) ofthe nucleus We reached this goal by applying continuouswavelet analysis in two dimensions with a 2D wavelet asa deconvolution algorithm for FRET microscopy image

10 ISRN Biochemistry

Control

(A)

05

0

1

05

00 20 40 60 800 20 40 60 800 20 40 60 80

(a) (b) (c) (d)

20 40 60 800

0

20

40

60

80 1

C 3 5 10 mincAMP

C 2 10 mincAMP

C 3 10 min

cAMP

C 1 3 10 min

cAMP

5

cAMP stable (5)

(B)

119868Ca (3 min) cAMP (5 min) cAMP (10 min)

119868Ca cAMP (65) 119868Ca stable (15)

119868Ca119868Ca

119868Ca 119868Ca

119868Ca cAMP transient (15)

Figure 8 Microdomain analysis on the additive effect of 119868Ca and cAMP on CREB signaling in COS1 cells expressing the recombinantldquoatheroscleroticrdquo120572

1C77 channel (A) FRET signal within the nucleus during selected time points during 119868Ca stimulation and cAMP applicationOutlined are four types of signaling microdomains identified using 2D Mexican hat wavelet Red circles represent stable microdomains thatpersist through both 119868Ca and cAMP application White and yellow circles showmicrodomains stable activated by cAMP and 119868Ca respectivelyGreen circles represent transient microdomains of CREB signaling activation Axes show pixel numbers (B) Typical appearances of the fourtypes of CREB signaling microdomains activated by 119868Ca andor cAMP application and recorded in their maximal development in relationto the time of applied stimuli C control recorded before stimulation Color bars in (A) and (B) represent FRET values normalized to themaximum

analysis [73] Continuous wavelet analysis is a mathematicaltechnique that allows us to analyze a signal over severaldifferent frequencies across the entire signal [73 74] It isespecially useful for finding heterogeneity in a signal becauseit can easily find where the pattern (ie frequency) of asignal changes In these experiments we for the first timeobtained evidence of CREB signaling microdomains withinthe nucleus that respond differentially to 119868Ca stimulationand cAMP (Figure 8(A)) Results of the study revealed thatCREB-dependent transcriptional signaling occurs in dis-crete signaling microdomains underlying the architecture ofnuclear signaling Continuous activation ofCREB-dependenttranscriptional signaling by cAMP and Ca2+ resulted in agradual increase of the number of microdomains Four dif-ferent categories of cAMP and Ca2+-induced CREB signalingmicrodomains were characterized in COS1 cells expressingrecombinant Cav12 with the ldquoatheroscleroticrdquo 120572

1C77 splice

variant (Figure 8) In up to 65 of the microdomainstranscription was activated in additive manner by cAMPand Ca2+ Approximately 15 of signaling domains wereactivated only by 119868Ca and 5 of domains were activatedonly by cAMP Finally 15 of the domains were transientand activated by both cAMP and 119868Ca (Figure 8(B)) [75]A similar spatiotemporal organization of CREB-dependentsignaling was observed in spontaneously beating neonatalrat cardiomyocytes Although COS1 cells that were used inour experiments shown in Figures 7 and 8 are naturallydeprived of Cav12 [76] they inherited the ability to replicatethe Cav12-dependent activation of CREB signaling with theexogenous recombinant channel Thus this experimentalapproach fits the task which in my opinion is the mostimportant unresolved issue for the coupling of Cav12 toCREB signaling does the splice variation of 120572

1C affect thespatiotemporal organization of CREB-dependent signaling

ISRN Biochemistry 11

in a way that may affect cell proliferation and other crucialfunction

7 Conclusions

Association of Cav12 with regulation of transcription cellproliferation and its pathophysiology as in the case ofatherosclerosis requires detailed investigation of the roles ofthe naturally occurring 120572

1C splice variants It will limit thetraditionally intuitive approach to Cav12 in physiology andhelp to define new principle approaches to the treatment ofvarious Cav12 channelopathy-related dysfunctions above allcardiovascular diseases Humgenex Inc provides consultingand logistic support on a broad range of issues reported inthis review

Abbreviations

CaM CalmodulinCBD Calmodulin-binding domainCDI Ca2+-dependent inactivationDHP DihydropyridineECFP Enhanced cyan fluorescent proteinEYFP Enhanced yellow fluorescent proteinFALI Fluorophore-assisted light inactivationFRET Fluorescent resonance energy transfer[3H]PMD 26-Dimethyl-3-methoxycarbonyl-5-([23-

3H2]-n-propoxycarbonyl)-4-(21015840-

difluoromethoxyphenyl)-14-dihydropyridine

119868Ca Calcium currentPH Pleckstrin homology domainVSM Vascular smooth muscle

References

[1] Y S Lee M M Sayeed and R D Wurster ldquoInhibition ofcell growth and intracellular Ca2+ mobilization in humanbrain tumor cells by Ca2+ channel antagonistsrdquo Molecular andChemical Neuropathology vol 22 no 2 pp 81ndash95 1994

[2] K Sato J Ishizuka C W Cooper et al ldquoInhibitory effect ofcalcium channel blockers on growth of pancreatic cancer cellsrdquoPancreas vol 9 no 2 pp 193ndash202 1994

[3] V Bertrand M J Bastie N Vaysse and L Pradayrol ldquoInhibi-tion of gastrin-induced proliferation of AR4-2J cells by calciumchannel antagonistsrdquo International Journal of Cancer vol 56no 3 pp 427ndash432 1994

[4] JM Taylor andRU Simpson ldquoInhibition of cancer cell growthby calcium channel antagonists in the athymic mouserdquo CancerResearch vol 52 no 9 pp 2413ndash2418 1992

[5] M G Cattaneo M Gullo and L M Vicentini ldquoC2+ and Ca2+channel antagonists in the control of human small cell lung car-cinoma cell proliferationrdquo European Journal of Pharmacologyvol 247 no 3 pp 325ndash331 1993

[6] C Chen M J Corbley T M Roberts and P Hess ldquoVoltage-sensitive calcium channels in normal and transformed 3T3fibroblastsrdquo Science vol 239 no 4843 pp 1024ndash1026 1988

[7] P C Dartsch M Ritter M Gschwentner H J Lang andF Lang ldquoEffects of calcium channel blockers on NIH 3T3

fibroblasts expressing the Ha-ras oncogenerdquo European Journalof Cell Biology vol 67 no 4 pp 372ndash378 1995

[8] M Biel R Hullin S Freundner et al ldquoTissue-specific expres-sion of high-voltage-activated dihydropyridine-sensitive L-typecalcium channelsrdquo European Journal of Biochemistry vol 200no 1 pp 81ndash88 1991

[9] DM Rodman K Reese J Harral et al ldquoLow-voltage-activated(T-type) calcium channels control proliferation of human pul-monary artery myocytesrdquo Circulation Research vol 96 no 8pp 864ndash872 2005

[10] A Panner L L Cribbs G M Zainelli T C Origitano S Singhand R DWurster ldquoVariation of T-type calcium channel proteinexpression affects cell division of cultured tumor cellsrdquo CellCalcium vol 37 no 2 pp 105ndash119 2005

[11] C Chen and P Hess ldquoCalcium channels in mouse 3T3 andhuman fibroblastsrdquo Biophysical Journal vol 51 p 226a 1987

[12] N M Soldatov ldquoMolecular diversity of L-type Ca2+ channeltranscripts in human fibroblastsrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 89 no10 pp 4628ndash4632 1992

[13] A Fujii H Matsumoto S Nakao H Teshigawara and YAkimoto ldquoEffect of calcium-channel blockers on cell prolifera-tion DNA synthesis and collagen synthesis of cultured gingivalfibroblasts derived fromhumannifedipine responders and non-respondersrdquo Archives of Oral Biology vol 39 no 2 pp 99ndash1041994

[14] N M Soldatov ldquoGenomic structure of human L-type Ca2+channelrdquo Genomics vol 22 no 1 pp 77ndash87 1994

[15] V P Shevchenko N F Myasoedov N M Soldatov G JDuburs V V Kastron and I P Skrastins ldquoSynthesis andevaluation of biological activity of two novel tritium-labeledderivatives of riodipinerdquo Journal of Labelled Compounds andRadiopharmaceuticals vol 27 no 6 pp 721ndash730 1989

[16] S M Dudkin S N Gnedoj N N Chernyuk and N MSoldatov ldquo14-dihydropyridine receptor associated with Ca2+channels in human embryonic fibroblastsrdquo FEBS Letters vol233 no 2 pp 352ndash354 1988

[17] A DePover M A Matlib S W Lee et al ldquoSpecific bindingof [3H]nitrendipine to membranes from coronary arteriesand heart in relation to pharmacological effects Paradoxicalstimulation by diltiazemrdquo Biochemical and Biophysical ResearchCommunications vol 108 no 1 pp 110ndash117 1982

[18] N M Soldatov S N Gnedoj N N Chernyuk I A Britanovaand S M Dudkin ldquoMitogenesis and 14-dihydropyridinereceptor associated with Ca2+-channels in human embryonicfibroblastsrdquo Journal of Membrane Biology vol 5 pp 1161ndash11671988 (Russian)

[19] D Lipscombe D V Madison M Poenie H Reuter R Y Tsienand R W Tsien ldquoSpatial distribution of calcium channels andcytosolic calcium transients in growth cones and cell bodies ofsympathetic neuronsrdquo Proceedings of the National Academy ofSciences of the United States of America vol 85 no 7 pp 2398ndash2402 1988

[20] G S Harms L Cognet P H M Lommerse et al ldquoSingle-molecule imaging of L-type CA2+ channels in live cellsrdquoBiophysical Journal vol 81 no 5 pp 2639ndash2646 2001

[21] V di Biase G J Obermair Z Szabo et al ldquoStable membraneexpression of postsynaptic Cav12 calcium channel clustersis independent of interactions with AKAP79150 and PDZproteinsrdquo Journal of Neuroscience vol 28 no 51 pp 13845ndash13855 2008

12 ISRN Biochemistry

[22] M F Navedo E P Cheng C Yuan et al ldquoIncreased coupledgating of L-type Ca2+ channels during hypertension and timo-thy syndromerdquoCirculationResearch vol 106 no 4 pp 748ndash7562010

[23] E Kobrinsky P Abrahimi S Q Duong et al ldquoEffect of Cav120573subunits on structural organization of Cav12 calcium channelsrdquoPLoS One vol 4 no 5 Article ID e5587 2009

[24] Q Z Lao E Kobrinsky Z Liu and N M Soldatov ldquoOligomer-ization of Cav120573 subunits is an essential correlate of Ca

2+ channelactivityrdquoThe FASEB Journal vol 24 no 12 pp 5013ndash5023 2010

[25] E Kobrinsky S Tiwari V A Maltsev et al ldquoDifferential roleof the 1205721C subunit tails in regulation of the Cav12 channelby membrane potential 120573 subunits and Ca2+ ionsrdquo Journal ofBiological Chemistry vol 280 no 13 pp 12474ndash12485 2005

[26] S Tiwari Y Zhang J Heller D R Abernethy and N MSoldatov ldquoArtherosclerosis-related molecular alteration of thehuman Cav12 calcium channel 1205721C subunitrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 103 no 45 pp 17024ndash17029 2006

[27] I Splawski K W Timothy L M Sharpe et al ldquoCav12 calciumchannel dysfunction causes a multisystem disorder includingarrhythmia and autismrdquo Cell vol 119 no 1 pp 19ndash31 2004

[28] Z Z TangM C Liang S Lu et al ldquoTranscript scanning revealsnovel and extensive splice variations in human L-type voltage-gated calcium channel Cav12 1205721 subunitrdquo Journal of BiologicalChemistry vol 279 no 43 pp 44335ndash44343 2004

[29] P Liao T F Yong M C Liang D T Yue and T W SoongldquoSplicing for alternative structures of Cav12 Ca2+ channels incardiac and smooth musclesrdquo Cardiovascular Research vol 68no 2 pp 197ndash203 2005

[30] Y Blumenstein N Kanevsky G Sahar R Barzilai T Ivaninaand N Dascal ldquoA novel long N-terminal isoform of human L-type Ca2+ channel is up-regulated by protein kinase Crdquo Journalof Biological Chemistry vol 277 no 5 pp 3419ndash3423 2002

[31] N M Soldatov A Bouron and H Reuter ldquoDifferent voltage-dependent inhibition by dihydropyridines of human Ca2+channel splice variantsrdquo Journal of Biological Chemistry vol270 no 18 pp 10540ndash10543 1995

[32] D P Faxon M A Creager S C Smith et al ldquoAtheroscleroticvascular disease conference executive summary atheroscle-rotic vascular disease conference proceeding for healthcareprofessionals from a special writing group of the AmericanHeart Associationrdquo Circulation vol 109 no 21 pp 2595ndash26042004

[33] R Ross ldquoAtherosclerosismdashan inflammatory diseaserdquo The NewEngland Journal of Medicine vol 340 no 2 pp 115ndash126 1999

[34] H Roy S Bhardwaj and S Yla-Herttuala ldquoMolecular geneticsof atherosclerosisrdquo Human Genetics vol 125 no 5-6 pp 467ndash491 2009

[35] M Biel P Ruth E Bosse et al ldquoPrimary structure and func-tional expression of a high voltage activated calcium channelfrom rabbit lungrdquo FEBS Letters vol 269 no 2 pp 409ndash4121990

[36] X Cheng J Liu M Asuncion-Chin et al ldquoA novel Cav12N terminus expressed in smooth muscle cells of resistancesize arteries modifies channel regulation by auxiliary subunitsrdquoJournal of Biological Chemistry vol 282 no 40 pp 29211ndash292212007

[37] W J Koch P T Ellinor and A Schwartz ldquocDNA cloning ofa dihydropyridine-sensitive calcium channel from rat aortaevidence for the existence of alternatively spliced formsrdquo Journalof Biological Chemistry vol 265 no 29 pp 17786ndash17791 1990

[38] S Richard D Neveu G Carnac P Bodin P Travo and J Nar-geot ldquoDifferential expression of voltage-gated Ca2+-currents incultivated aortic myocytesrdquo Biochimica et Biophysica Acta vol1160 no 1 pp 95ndash104 1992

[39] A Schwartz ldquoCalcium antagonists review and perspective onmechanism of actionrdquo American Journal of Cardiology vol 64no 17 pp 3Indash9I 1989

[40] R P Mason ldquoMechanisms of plaque stabilization for thedihydropyridine calcium channel blocker amlodipine review ofthe evidencerdquo Atherosclerosis vol 165 no 2 pp 191ndash199 2002

[41] A Ruiz-Torres R Lozano J Melon and R Carraro ldquoL-calciumchannel blockade induced by diltiazem inhibits proliferationmigration and F-actin membrane rearrangements in humanvascular smooth muscle cells stimulated with insulin and IGF-1rdquo International Journal of Clinical Pharmacology andTherapeu-tics vol 41 no 9 pp 386ndash391 2003

[42] H Koshiyama S Tanaka and J Minamikawa ldquoEffect ofcalcium channel blocker amlodipine on the intimal-medialthickness of carotid arterial wall in type 2 diabetesrdquo Journal ofCardiovascular Pharmacology vol 33 no 6 pp 894ndash896 1999

[43] T Yamashita S KawashimaMOzaki et al ldquoA calcium channelblocker benidipine inhibits intimal thickening in the carotidartery of mice by increasing nitric oxide productionrdquo Journal ofHypertension vol 19 no 3 pp 451ndash458 2001

[44] R P Mason P Marche and T H Hintze ldquoNovel vascularbiology of third-generation L-type calcium channel antagonistsancillary actions of amlodipinerdquo Arteriosclerosis Thrombosisand Vascular Biology vol 23 no 12 pp 2155ndash2163 2003

[45] O Stepien J GogusevD L Zhu et al ldquoAmlodipine inhibition ofserum- thrombin- or fibroblast growth factor- induced vascu-lar smooth-muscle cell proliferationrdquo Journal of CardiovascularPharmacology vol 31 no 5 pp 786ndash793 1998

[46] V Ruiz-Velasco M B Mayer E W Inscho and L J HymelldquoModulation of dihydropyridine receptors in vascular smoothmuscle cells by membrane potential and cell proliferationrdquoEuropean Journal of Pharmacology vol 268 no 3 pp 311ndash3181994

[47] J Nilsson M Sjolund L Palmberg A M von Euler B Jonzonand J Thyberg ldquoThe calcium antagonist nifedipine inhibitsarterial smooth muscle cell proliferationrdquo Atherosclerosis vol58 no 1ndash3 pp 109ndash122 1985

[48] J Thyberg and L Palmberg ldquoThe calcium antagonist nisoldip-ine and the calmodulin antagonist W-7 synergistically inhibitinitiation of DNA synthesis in cultured arterial smooth musclecellsrdquo Biology of the Cell vol 60 no 2 pp 125ndash132 1987

[49] E Munro M Patel P Chan et al ldquoEffect of calcium channelblockers on the growth of human vascular smooth musclecells derived from saphenous vein and vascular graft stenosesrdquoJournal of Cardiovascular Pharmacology vol 23 no 5 pp 779ndash784 1994

[50] T A Kent A Jazayeri and J M Simard ldquoCalcium channelsand nifedipine inhibition of serotonin-induced [3H]thymidineincorporation in cultured cerebral smoothmuscle cellsrdquo Journalof Cerebral Blood Flow and Metabolism vol 12 no 1 pp 139ndash146 1992

[51] A Agrotis P J Little J Saltis and A Bobik ldquoDihydropyridineCa2+ channel antagonists inhibit the salvage pathway for DNAsynthesis in human vascular smooth muscle cellsrdquo EuropeanJournal of Pharmacology vol 244 no 3 pp 269ndash275 1993

[52] I Duque M R Puyol P Ruiz M Gonzalez-Rubio M LD Marques and D R Puyol ldquoCalcium channel blockers

ISRN Biochemistry 13

inhibit hydrogen peroxide-induced proliferation of culturedrat mesangial cellsrdquo Journal of Pharmacology and ExperimentalTherapeutics vol 267 no 2 pp 612ndash616 1993

[53] T Kuga S Kobayashi Y Hirakawa H Kanaide and ATakeshita ldquoCell cycle-dependent expression of L- and T-typeCa2+ currents in rat aortic smooth muscle cells in primaryculturerdquo Circulation Research vol 79 no 1 pp 14ndash19 1996

[54] E M Graf M Bock J F Heubach et al ldquoTissue distribution ofa humanCav12 1205721 subunit splice variant with a 75 bp insertionrdquoCell Calcium vol 38 no 1 pp 11ndash21 2005

[55] P Liao D Yu S Lu et al ldquoSmoothmuscle-selective alternativelyspliced exon generates functional variation in Cav12 calciumchannelsrdquo Journal of Biological Chemistry vol 279 no 48 pp50329ndash50335 2004

[56] O Skalli P Ropraz and A Trzeciak ldquoA monoclonal antibodyagainst 120572-smooth muscle actin a new probe for smooth muscledifferentiationrdquo Journal of Cell Biology vol 103 no 6 pp 2787ndash2796 1986

[57] T M Doherty K Asotra L A Fitzpatrick et al ldquoCalcificationin atherosclerosis bone biology and chronic inflammation atthe arterial crossroadsrdquo Proceedings of the National Academyof Sciences of the United States of America vol 100 no 20 pp11201ndash11206 2003

[58] G K Owens ldquoRegulation of differentiation of vascular smoothmuscle cellsrdquo Physiological Reviews vol 75 no 3 pp 487ndash5171995

[59] J P Bannister A Adebiyi G Zhao et al ldquoSmooth musclecell 1205722120575-1 subunits are essential for vasoregulation by Cav12channelsrdquo Circulation Research vol 105 no 10 pp 948ndash9552009

[60] S SonkusareM Fraer J DMarsh andN J Rusch ldquoDisruptingcalcium channel expression to lower blood pressure newtargeting of awell-known channelrdquoMolecular Interventions vol6 no 6 pp 304ndash310 2006

[61] S Telemaque S Sonkusare TGrain et al ldquoDesign ofmutant1205732subunits as decoy molecules to reduce the expression of func-tional Ca2+ channels in cardiac cellsrdquo Journal of Pharmacologyand Experimental Therapeutics vol 325 no 1 pp 37ndash46 2008

[62] A Goyenvalle A Vulin F Fougerousse et al ldquoRescue of dys-trophic muscle through U7 snRNA-mediated exon skippingrdquoScience vol 306 no 5702 pp 1796ndash1799 2004

[63] D BHalling P Aracena-Parks and S L Hamilton ldquoRegulationof voltage-gated Ca2+ channels by calmodulinrdquo Sciencersquos STKEvol 2005 no 315 p re15 2005

[64] K S Lee E Marban and R W Tsien ldquoInactivation of calciumchannels in mammalian heart cells joint dependence on mem-brane potential and intracellular calciumrdquo Journal of Physiologyvol 364 pp 395ndash411 1985

[65] C Shi and N M Soldatov ldquoMolecular determinants of voltage-dependent slow inactivation of the Ca2+ channelrdquo Journal ofBiological Chemistry vol 277 no 9 pp 6813ndash6821 2002

[66] E Kobrinsky E Schwartz D R Abernethy and NM SoldatovldquoVoltage-gated mobility of the Ca2+ channel cytoplasmic tailsand its regulatory rolerdquo Journal of Biological Chemistry vol 278no 7 pp 5021ndash5028 2003

[67] N M Soldatov ldquoCa2+ channel moving tail link between Ca2+-induced inactivation and Ca2+ signal transductionrdquo Trends inPharmacological Sciences vol 24 no 4 pp 167ndash171 2003

[68] D J Klemm P A Watson M G Frid et al ldquocAMP responseelement-binding protein content is a molecular determinantof smooth muscle cell proliferation and migrationrdquo Journal ofBiological Chemistry vol 276 no 49 pp 46132ndash46141 2001

[69] E Kobrinsky L Stevens Y Kazmi DWray andNM SoldatovldquoMolecular rearrangements of the Kv21 potassium channeltermini associated with voltage gatingrdquo Journal of BiologicalChemistry vol 281 no 28 pp 19233ndash19240 2006

[70] BMMayr G Canettieri andMRMontminy ldquoDistinct effectsof cAMP and mitogenic signals on CREB-binding proteinrecruitment impart specificity to target gene activation viaCREBrdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 98 no 19 pp 10936ndash10941 2001

[71] M Morad and N Soldatov ldquoCalcium channel inactivationpossible role in signal transduction and Ca2+ signalingrdquo CellCalcium vol 38 no 3-4 pp 223ndash231 2005

[72] DGWheeler C F Barrett R DGroth P Safa andRW TsienldquoCaMKII locally encodes L-type channel activity to signal tonuclear CREB in excitation-transcription couplingrdquo Journal ofCell Biology vol 183 no 5 pp 849ndash863 2008

[73] D E Mager E Kobrinsky A Masoudieh A Maltsev D RAbernethy andNM Soldatov ldquoAnalysis of functional signalingdomains from fluorescence imaging and the two-dimensionalcontinuous wavelet transformrdquo Biophysical Journal vol 93 no8 pp 2900ndash2910 2007

[74] E Kobrinsky D E Mager S A Bentil S I Murata DR Abernethy and N M Soldatov ldquoIdentification of plasmamembrane macro- and microdomains from wavelet analysis ofFRET microscopyrdquo Biophysical Journal vol 88 no 5 pp 3625ndash3634 2005

[75] E Kobrinsky S Q Duong A Sheydina and N M Solda-tov ldquoMicrodomain organization and frequency-dependence ofCREB-dependent transcriptional signaling in heart cellsrdquo TheFASEB Journal vol 25 no 5 pp 1544ndash1555 2011

[76] A Ravindran Q Z Lao J B Harry P Abrahimi E Kobrinskyand N M Soldatov ldquoCalmodulin-dependent gating of Cav12calcium channels in the absence of Cav120573 subunitsrdquo Proceedingsof the National Academy of Sciences of the United States ofAmerica vol 105 no 23 pp 8154ndash8159 2008

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

8 ISRN Biochemistry

sequence and supplemented with Sm ribonucleoprotein-binding sequence may be incorporated into the adenovirusvector for high efficiency transfer [62]The cytokine receptors(eg PdGF-120573 receptor) based recognition targeting of viralliposomes or nanoparticles may be especially advantageousin connection with selective gene transfer to VSM cellsaffected by atherosclerosis while reducing the probability ofthe transfection of other cells

6 Cak12 and CREB-DependentTranscriptional Activation

How Cav12 activity is translated into a proliferation-effectedmodality is another important question to be asked Cav12calcium channels generate a transient rise in cytosolic Ca2+-concentration activated by membrane depolarization Cel-lular responses associated with the rise of [Ca2+]i rangefrom sarcomeric contraction to cell growth and proliferationCytoplasmic domains of Cav12 have evolved a fairly intricateCaM-dependent signaling mechanism that provides for thenegative feedback inhibition of the calcium current knownas Ca2+ dependent inactivation (CDI) which is mediated bydifferent determinants of 120572

1C [25 63] Such a mechanism ofCDI resulting in acceleration of 119868Ca inactivation in responseto the rise of intracellular Ca2+ was first identified in cardiacCav12 [64] Similar experiments performed on the recombi-nant Cav12 also showed that the replacement of extracellularCa2+ by Ba2+ eliminates CDI and the channel inactivates by aslower voltage-dependent mechanism [65]The very fact thattwo distantly located determinants one in the pore regionresponsible for slow inactivation and the CaM-binding onein the proximal locus of the C-tail are independently crucialfor CDI indicates that not only their specific molecularstructure but also their mutual folding andor interaction areessential Experimental evidence show that this interactionreacts dynamically to membrane voltage supporting state-dependent transitions of the channel between resting openand inactivated conformations which are essential for CREB-dependent transcriptional activation [66 67]

CREB is a transcription factor of general importance ina large variety of cells CREB phosphorylation promotes theactivation of genes and is regulated by protein kinases undercontrol of the major second messengers cAMP andor Ca2+Indeed CREB functions as a ldquomolecular determinant of VSMcells fate [68]rdquo CREB content depends on proliferation ofVSM cells both in situ and in culture Serum deprivationincreased CREB content in VSM cells while exposure toPdGF decreased it Consistent with this observation anoverexpression of the constitutively active CREB inVSM cellsarrested cell cycle progression

To investigate the association of Cav12 with CREB tran-scriptional activation at the molecular level we combinedpatch clamp with fluorescent resonance energy transfer(FRET) microscopy in the live cell In voltage clampedcells FRET provided optical measurements under state-dependent conditions showing that the shorter N-terminaltail of 120572

1C (eg 1205721C77) does not rearrange vis-a-vis the

plasma membrane in response to voltage gating [25] In

sharp contrast the C-tail shows voltage-dependent confor-mational rearrangements which are much larger in sizethan that for example in the potassium Kv21 channel[69] Measurements of 119868Ca and corrected FRET betweenthe enhanced yellow (EYFP) and cyan fluorescent proteins(ECFP) genetically attached respectively to the N- and C-termini of 120572

1C77 showed no significant effect on voltage-dependence and kinetics of the channel current Howevertherewas a substantial increase in FRET signal accompanyinginactivation of the channel that was fully reversible uponits transition into the resting state in response to hyper-polarization [66] The plasma-membrane anchoring of the1205721C C-tail by the fusion of the pleckstrin homology domain

(PH) eliminated CDI but not 119868Ca Do the voltage-gatedconformational rearrangements of the 120572

1C C-tail and CDIplay a role in Ca2+ signal transduction that is utilized inCa2+-induced activation or CREB-dependent transcriptionTo answer this question we used the test system based onthe measurement of interaction between KID domain ofCREB and KIX domain of coactivator CREB-binding protein(CBP Figure 7(A) inset) under voltage-clamp conditionsby monitoring FRET between EYFP-KID and ECFP-KIX[70] Ca2+-dependent phosphorylation of KID stimulates itsbinding to KIX bringing EYFP and ECFP close enoughto observe the interaction by FRET In perforated wholeclamped cell where the integrity of the cytoplasmic contentis intact (Figure 7(A)) no activation of CREB transcription(Figure 7(B) panel (a)) and rearrangement of the120572

1C77 C-tail(panel (b)) was observed when the C-tail was anchored to theplasma membrane CREB transcriptional activity remainedlow in spite of a large sustained inward 119868Ca (panel (d))and the corresponding increase in [Ca2+]i detected by thefluorescence of Ca2+ indicator Fura4 (panel (c)) Releaseof the 120572

1C77 C-tail by the activation of PIP2hydrolysis

upon activation of M1AchR (Figure 7(C)) at minus90mV causedsignificant elevation of [Ca2+]i that also was not utilizedby the cell for CREB transcriptional activation (Figure 7(C)panel (a)) until a depolarizing pulse to +20-mV was appliedand the released 120572

1C77 C-tail was permitted to rearrange(Figure 7(D))This experiment provides compelling evidencethat neither large inward 119868Ca nor the subsequent rise in[Ca2+]i lead to CREB transcription activation unless theconformational rearrangement of the 120572

1C77 subunit C-tailprovides the precise targeting of the Ca2+ signal transduction(Figure 7(D) scheme) [66]

There is general agreement that CaM binds to LA and IQdomains of the 120572

1C C-tail and acts as a sensor that conveysCDI (for review see [63]) The affinity of CaM for bothdomains depends on [Ca2+]i Our data indicate that CDI andCa2+-signal transduction depend on the voltage-gatedmobil-ity of the120572

1C77 C-tail It is therefore reasonable to suggest thatthe LA-domain is a Ca2+-sensitive apo-CaM-mediated lockfor the mechanism of slow voltage-dependent inactivationof the channel [67] Apo-CaM associated with LA is able tocross-link it to another still unidentified apo-CaM bindingsite in the polypeptide bundle underlying the pore As theresult of this specific localization apo-CaMLA ldquolockrdquo ishidden from the cytoplasmic Ca2+ so that for example the

ISRN Biochemistry 9

10

0

2

10

100 pA125 ms

(a) (a) (a)

(b)(c)

(b)(c)

(b)(c)

(B) (C) (D)

C-tail Anchored ReleasedReleased

0 10No FRET No FRET

(d) (d) (d)

Fluo-4

PH

CYN

C

C

NY

C

Y EYFP

C ECFP

PH Plekstrin homologydomain

PHPHY

C

NC

IQ

LA

LAIQ

(A)

Fluo-4 Fluo-4

FRET FRET FRET

CRE

Gene expressionCREBCREB P

CBP

AC

GPCR

PKA

cAMP

CaM

Nucleus

cAMP

Plasmamembrane

Ca2+ Ca2+

Ca2+

+20mV +20mV

minus90mV minus90mV

1205721C77

1198651198650

Cav 12

Ca2+

minus90mVACh (5 mM)

4 120583

Figure 7 Evidence that Ca2+ signal transduction by Cav12 to activate CREB-dependent transcription is mediated by the voltage-gatedmobility of the 120572

1C C-tail carrying the CaM-caged Ca2+ and is not directly associated with the increase of [Ca2+]i CREB activation wasexamined under perforated patch conditions in COS1 cells expressing recombinant atherosclerotic 120572

1C77 channel (A) Phase-contrast imageof the COS1 cell with a shadow of patch pipette The cell was expressing the 120572

1C77-PHC1205731a1205722120575-1 channel with the membrane-trapped 1205721C77

subunit C-tail type 1muscarinic Ach receptor (to release the anchored C-tail in response to activation by Ach) and EYFP-KID and ECFP-KIXdomains both supplemented with nuclear localization sequences Inset at the bottom Cav12- and cAMP-dependent cell signaling pathwaysmediating CREB-dependent transcription CBP CREB-binding protein CRE cAMP-response element (B) Plasma-membrane anchoring ofthe 1205721C77 C-tail (see schematic diagram) inhibits CREB activation in spite of robust 119868Ca evoked by depolarization (a) A 100-ms images of

FRET between EYFP-KID and ECFP-KIX were recorded at the end of the 12th +20-mV depolarization step applied every 10 s for 1 s from119881ℎ=

minus90mV (b) A corresponding representative image of corrected FRET ratio (in a ldquo3Drdquo MetaMorph transformation) recorded within 50-mswindows at minus90 and at the end of the last test pulse of the applied train of pulses (c) An increase of [Ca2+]i detected by Ca

2+ indicator Fluo4(d) Representative trace of 119868Ca (20mM Ca2+ in bath medium) evoked by depolarization to +20mV from119881

ℎ= minus90mV showing the sustained

component of Ca2+ conductance due to the C-tail anchoring (C) ACh-stimulation of theM1AChR to unbind the 1205721C C-tail caused activation

of IP3-dependent Ca2+ release (panel (c)) but did not show substantial activation of CREB transcription at 119881

ℎ= minus90mV (panel (a)) (D)

CREB-dependent transcription was activated only in response to depolarization applied to membrane channel with the 1205721C77 C-terminal tail

released from the plasma membrane to assume functional voltage-dependent conformation

intracellular Ca2+ released from the intracellular stores orCa2+ caging compounds does not accelerate significantly theinactivation of Cav12 [71] Thus apo-CaM associated withLA binds predominantly Ca2+ ions permeating through thepore A Ca2+-dependent transfer of CaM from LA to the IQ-motif opens the ldquolockrdquo and initiates a large rearrangement ofthe C-terminal tail This in turn facilitates inactivation of thechannel The Ca2+CaM complex with the IQ-motif is thentransferred by themobile C-tail to a downstream target of theCa2+-signaling cascade (such as CaMKII [72]) where Ca2+ isreleased as an activating stimulus while CaM switches back

to LA and returns the C-tail to the resting position availablefor the next cycle of Ca2+-signal transduction

Activation of CREB-dependent transcription by the L-type 119868Ca ismediated throughmultiple cell signaling pathwaysUsing FRET probes of CREB activity and 2D wavelet trans-form analysis we applied principles of quantitative biologyto detail the mechanism of Ca2+-activated CREB-dependenttranscription within localized regions (microdomains) ofthe nucleus We reached this goal by applying continuouswavelet analysis in two dimensions with a 2D wavelet asa deconvolution algorithm for FRET microscopy image

10 ISRN Biochemistry

Control

(A)

05

0

1

05

00 20 40 60 800 20 40 60 800 20 40 60 80

(a) (b) (c) (d)

20 40 60 800

0

20

40

60

80 1

C 3 5 10 mincAMP

C 2 10 mincAMP

C 3 10 min

cAMP

C 1 3 10 min

cAMP

5

cAMP stable (5)

(B)

119868Ca (3 min) cAMP (5 min) cAMP (10 min)

119868Ca cAMP (65) 119868Ca stable (15)

119868Ca119868Ca

119868Ca 119868Ca

119868Ca cAMP transient (15)

Figure 8 Microdomain analysis on the additive effect of 119868Ca and cAMP on CREB signaling in COS1 cells expressing the recombinantldquoatheroscleroticrdquo120572

1C77 channel (A) FRET signal within the nucleus during selected time points during 119868Ca stimulation and cAMP applicationOutlined are four types of signaling microdomains identified using 2D Mexican hat wavelet Red circles represent stable microdomains thatpersist through both 119868Ca and cAMP application White and yellow circles showmicrodomains stable activated by cAMP and 119868Ca respectivelyGreen circles represent transient microdomains of CREB signaling activation Axes show pixel numbers (B) Typical appearances of the fourtypes of CREB signaling microdomains activated by 119868Ca andor cAMP application and recorded in their maximal development in relationto the time of applied stimuli C control recorded before stimulation Color bars in (A) and (B) represent FRET values normalized to themaximum

analysis [73] Continuous wavelet analysis is a mathematicaltechnique that allows us to analyze a signal over severaldifferent frequencies across the entire signal [73 74] It isespecially useful for finding heterogeneity in a signal becauseit can easily find where the pattern (ie frequency) of asignal changes In these experiments we for the first timeobtained evidence of CREB signaling microdomains withinthe nucleus that respond differentially to 119868Ca stimulationand cAMP (Figure 8(A)) Results of the study revealed thatCREB-dependent transcriptional signaling occurs in dis-crete signaling microdomains underlying the architecture ofnuclear signaling Continuous activation ofCREB-dependenttranscriptional signaling by cAMP and Ca2+ resulted in agradual increase of the number of microdomains Four dif-ferent categories of cAMP and Ca2+-induced CREB signalingmicrodomains were characterized in COS1 cells expressingrecombinant Cav12 with the ldquoatheroscleroticrdquo 120572

1C77 splice

variant (Figure 8) In up to 65 of the microdomainstranscription was activated in additive manner by cAMPand Ca2+ Approximately 15 of signaling domains wereactivated only by 119868Ca and 5 of domains were activatedonly by cAMP Finally 15 of the domains were transientand activated by both cAMP and 119868Ca (Figure 8(B)) [75]A similar spatiotemporal organization of CREB-dependentsignaling was observed in spontaneously beating neonatalrat cardiomyocytes Although COS1 cells that were used inour experiments shown in Figures 7 and 8 are naturallydeprived of Cav12 [76] they inherited the ability to replicatethe Cav12-dependent activation of CREB signaling with theexogenous recombinant channel Thus this experimentalapproach fits the task which in my opinion is the mostimportant unresolved issue for the coupling of Cav12 toCREB signaling does the splice variation of 120572

1C affect thespatiotemporal organization of CREB-dependent signaling

ISRN Biochemistry 11

in a way that may affect cell proliferation and other crucialfunction

7 Conclusions

Association of Cav12 with regulation of transcription cellproliferation and its pathophysiology as in the case ofatherosclerosis requires detailed investigation of the roles ofthe naturally occurring 120572

1C splice variants It will limit thetraditionally intuitive approach to Cav12 in physiology andhelp to define new principle approaches to the treatment ofvarious Cav12 channelopathy-related dysfunctions above allcardiovascular diseases Humgenex Inc provides consultingand logistic support on a broad range of issues reported inthis review

Abbreviations

CaM CalmodulinCBD Calmodulin-binding domainCDI Ca2+-dependent inactivationDHP DihydropyridineECFP Enhanced cyan fluorescent proteinEYFP Enhanced yellow fluorescent proteinFALI Fluorophore-assisted light inactivationFRET Fluorescent resonance energy transfer[3H]PMD 26-Dimethyl-3-methoxycarbonyl-5-([23-

3H2]-n-propoxycarbonyl)-4-(21015840-

difluoromethoxyphenyl)-14-dihydropyridine

119868Ca Calcium currentPH Pleckstrin homology domainVSM Vascular smooth muscle

References

[1] Y S Lee M M Sayeed and R D Wurster ldquoInhibition ofcell growth and intracellular Ca2+ mobilization in humanbrain tumor cells by Ca2+ channel antagonistsrdquo Molecular andChemical Neuropathology vol 22 no 2 pp 81ndash95 1994

[2] K Sato J Ishizuka C W Cooper et al ldquoInhibitory effect ofcalcium channel blockers on growth of pancreatic cancer cellsrdquoPancreas vol 9 no 2 pp 193ndash202 1994

[3] V Bertrand M J Bastie N Vaysse and L Pradayrol ldquoInhibi-tion of gastrin-induced proliferation of AR4-2J cells by calciumchannel antagonistsrdquo International Journal of Cancer vol 56no 3 pp 427ndash432 1994

[4] JM Taylor andRU Simpson ldquoInhibition of cancer cell growthby calcium channel antagonists in the athymic mouserdquo CancerResearch vol 52 no 9 pp 2413ndash2418 1992

[5] M G Cattaneo M Gullo and L M Vicentini ldquoC2+ and Ca2+channel antagonists in the control of human small cell lung car-cinoma cell proliferationrdquo European Journal of Pharmacologyvol 247 no 3 pp 325ndash331 1993

[6] C Chen M J Corbley T M Roberts and P Hess ldquoVoltage-sensitive calcium channels in normal and transformed 3T3fibroblastsrdquo Science vol 239 no 4843 pp 1024ndash1026 1988

[7] P C Dartsch M Ritter M Gschwentner H J Lang andF Lang ldquoEffects of calcium channel blockers on NIH 3T3

fibroblasts expressing the Ha-ras oncogenerdquo European Journalof Cell Biology vol 67 no 4 pp 372ndash378 1995

[8] M Biel R Hullin S Freundner et al ldquoTissue-specific expres-sion of high-voltage-activated dihydropyridine-sensitive L-typecalcium channelsrdquo European Journal of Biochemistry vol 200no 1 pp 81ndash88 1991

[9] DM Rodman K Reese J Harral et al ldquoLow-voltage-activated(T-type) calcium channels control proliferation of human pul-monary artery myocytesrdquo Circulation Research vol 96 no 8pp 864ndash872 2005

[10] A Panner L L Cribbs G M Zainelli T C Origitano S Singhand R DWurster ldquoVariation of T-type calcium channel proteinexpression affects cell division of cultured tumor cellsrdquo CellCalcium vol 37 no 2 pp 105ndash119 2005

[11] C Chen and P Hess ldquoCalcium channels in mouse 3T3 andhuman fibroblastsrdquo Biophysical Journal vol 51 p 226a 1987

[12] N M Soldatov ldquoMolecular diversity of L-type Ca2+ channeltranscripts in human fibroblastsrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 89 no10 pp 4628ndash4632 1992

[13] A Fujii H Matsumoto S Nakao H Teshigawara and YAkimoto ldquoEffect of calcium-channel blockers on cell prolifera-tion DNA synthesis and collagen synthesis of cultured gingivalfibroblasts derived fromhumannifedipine responders and non-respondersrdquo Archives of Oral Biology vol 39 no 2 pp 99ndash1041994

[14] N M Soldatov ldquoGenomic structure of human L-type Ca2+channelrdquo Genomics vol 22 no 1 pp 77ndash87 1994

[15] V P Shevchenko N F Myasoedov N M Soldatov G JDuburs V V Kastron and I P Skrastins ldquoSynthesis andevaluation of biological activity of two novel tritium-labeledderivatives of riodipinerdquo Journal of Labelled Compounds andRadiopharmaceuticals vol 27 no 6 pp 721ndash730 1989

[16] S M Dudkin S N Gnedoj N N Chernyuk and N MSoldatov ldquo14-dihydropyridine receptor associated with Ca2+channels in human embryonic fibroblastsrdquo FEBS Letters vol233 no 2 pp 352ndash354 1988

[17] A DePover M A Matlib S W Lee et al ldquoSpecific bindingof [3H]nitrendipine to membranes from coronary arteriesand heart in relation to pharmacological effects Paradoxicalstimulation by diltiazemrdquo Biochemical and Biophysical ResearchCommunications vol 108 no 1 pp 110ndash117 1982

[18] N M Soldatov S N Gnedoj N N Chernyuk I A Britanovaand S M Dudkin ldquoMitogenesis and 14-dihydropyridinereceptor associated with Ca2+-channels in human embryonicfibroblastsrdquo Journal of Membrane Biology vol 5 pp 1161ndash11671988 (Russian)

[19] D Lipscombe D V Madison M Poenie H Reuter R Y Tsienand R W Tsien ldquoSpatial distribution of calcium channels andcytosolic calcium transients in growth cones and cell bodies ofsympathetic neuronsrdquo Proceedings of the National Academy ofSciences of the United States of America vol 85 no 7 pp 2398ndash2402 1988

[20] G S Harms L Cognet P H M Lommerse et al ldquoSingle-molecule imaging of L-type CA2+ channels in live cellsrdquoBiophysical Journal vol 81 no 5 pp 2639ndash2646 2001

[21] V di Biase G J Obermair Z Szabo et al ldquoStable membraneexpression of postsynaptic Cav12 calcium channel clustersis independent of interactions with AKAP79150 and PDZproteinsrdquo Journal of Neuroscience vol 28 no 51 pp 13845ndash13855 2008

12 ISRN Biochemistry

[22] M F Navedo E P Cheng C Yuan et al ldquoIncreased coupledgating of L-type Ca2+ channels during hypertension and timo-thy syndromerdquoCirculationResearch vol 106 no 4 pp 748ndash7562010

[23] E Kobrinsky P Abrahimi S Q Duong et al ldquoEffect of Cav120573subunits on structural organization of Cav12 calcium channelsrdquoPLoS One vol 4 no 5 Article ID e5587 2009

[24] Q Z Lao E Kobrinsky Z Liu and N M Soldatov ldquoOligomer-ization of Cav120573 subunits is an essential correlate of Ca

2+ channelactivityrdquoThe FASEB Journal vol 24 no 12 pp 5013ndash5023 2010

[25] E Kobrinsky S Tiwari V A Maltsev et al ldquoDifferential roleof the 1205721C subunit tails in regulation of the Cav12 channelby membrane potential 120573 subunits and Ca2+ ionsrdquo Journal ofBiological Chemistry vol 280 no 13 pp 12474ndash12485 2005

[26] S Tiwari Y Zhang J Heller D R Abernethy and N MSoldatov ldquoArtherosclerosis-related molecular alteration of thehuman Cav12 calcium channel 1205721C subunitrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 103 no 45 pp 17024ndash17029 2006

[27] I Splawski K W Timothy L M Sharpe et al ldquoCav12 calciumchannel dysfunction causes a multisystem disorder includingarrhythmia and autismrdquo Cell vol 119 no 1 pp 19ndash31 2004

[28] Z Z TangM C Liang S Lu et al ldquoTranscript scanning revealsnovel and extensive splice variations in human L-type voltage-gated calcium channel Cav12 1205721 subunitrdquo Journal of BiologicalChemistry vol 279 no 43 pp 44335ndash44343 2004

[29] P Liao T F Yong M C Liang D T Yue and T W SoongldquoSplicing for alternative structures of Cav12 Ca2+ channels incardiac and smooth musclesrdquo Cardiovascular Research vol 68no 2 pp 197ndash203 2005

[30] Y Blumenstein N Kanevsky G Sahar R Barzilai T Ivaninaand N Dascal ldquoA novel long N-terminal isoform of human L-type Ca2+ channel is up-regulated by protein kinase Crdquo Journalof Biological Chemistry vol 277 no 5 pp 3419ndash3423 2002

[31] N M Soldatov A Bouron and H Reuter ldquoDifferent voltage-dependent inhibition by dihydropyridines of human Ca2+channel splice variantsrdquo Journal of Biological Chemistry vol270 no 18 pp 10540ndash10543 1995

[32] D P Faxon M A Creager S C Smith et al ldquoAtheroscleroticvascular disease conference executive summary atheroscle-rotic vascular disease conference proceeding for healthcareprofessionals from a special writing group of the AmericanHeart Associationrdquo Circulation vol 109 no 21 pp 2595ndash26042004

[33] R Ross ldquoAtherosclerosismdashan inflammatory diseaserdquo The NewEngland Journal of Medicine vol 340 no 2 pp 115ndash126 1999

[34] H Roy S Bhardwaj and S Yla-Herttuala ldquoMolecular geneticsof atherosclerosisrdquo Human Genetics vol 125 no 5-6 pp 467ndash491 2009

[35] M Biel P Ruth E Bosse et al ldquoPrimary structure and func-tional expression of a high voltage activated calcium channelfrom rabbit lungrdquo FEBS Letters vol 269 no 2 pp 409ndash4121990

[36] X Cheng J Liu M Asuncion-Chin et al ldquoA novel Cav12N terminus expressed in smooth muscle cells of resistancesize arteries modifies channel regulation by auxiliary subunitsrdquoJournal of Biological Chemistry vol 282 no 40 pp 29211ndash292212007

[37] W J Koch P T Ellinor and A Schwartz ldquocDNA cloning ofa dihydropyridine-sensitive calcium channel from rat aortaevidence for the existence of alternatively spliced formsrdquo Journalof Biological Chemistry vol 265 no 29 pp 17786ndash17791 1990

[38] S Richard D Neveu G Carnac P Bodin P Travo and J Nar-geot ldquoDifferential expression of voltage-gated Ca2+-currents incultivated aortic myocytesrdquo Biochimica et Biophysica Acta vol1160 no 1 pp 95ndash104 1992

[39] A Schwartz ldquoCalcium antagonists review and perspective onmechanism of actionrdquo American Journal of Cardiology vol 64no 17 pp 3Indash9I 1989

[40] R P Mason ldquoMechanisms of plaque stabilization for thedihydropyridine calcium channel blocker amlodipine review ofthe evidencerdquo Atherosclerosis vol 165 no 2 pp 191ndash199 2002

[41] A Ruiz-Torres R Lozano J Melon and R Carraro ldquoL-calciumchannel blockade induced by diltiazem inhibits proliferationmigration and F-actin membrane rearrangements in humanvascular smooth muscle cells stimulated with insulin and IGF-1rdquo International Journal of Clinical Pharmacology andTherapeu-tics vol 41 no 9 pp 386ndash391 2003

[42] H Koshiyama S Tanaka and J Minamikawa ldquoEffect ofcalcium channel blocker amlodipine on the intimal-medialthickness of carotid arterial wall in type 2 diabetesrdquo Journal ofCardiovascular Pharmacology vol 33 no 6 pp 894ndash896 1999

[43] T Yamashita S KawashimaMOzaki et al ldquoA calcium channelblocker benidipine inhibits intimal thickening in the carotidartery of mice by increasing nitric oxide productionrdquo Journal ofHypertension vol 19 no 3 pp 451ndash458 2001

[44] R P Mason P Marche and T H Hintze ldquoNovel vascularbiology of third-generation L-type calcium channel antagonistsancillary actions of amlodipinerdquo Arteriosclerosis Thrombosisand Vascular Biology vol 23 no 12 pp 2155ndash2163 2003

[45] O Stepien J GogusevD L Zhu et al ldquoAmlodipine inhibition ofserum- thrombin- or fibroblast growth factor- induced vascu-lar smooth-muscle cell proliferationrdquo Journal of CardiovascularPharmacology vol 31 no 5 pp 786ndash793 1998

[46] V Ruiz-Velasco M B Mayer E W Inscho and L J HymelldquoModulation of dihydropyridine receptors in vascular smoothmuscle cells by membrane potential and cell proliferationrdquoEuropean Journal of Pharmacology vol 268 no 3 pp 311ndash3181994

[47] J Nilsson M Sjolund L Palmberg A M von Euler B Jonzonand J Thyberg ldquoThe calcium antagonist nifedipine inhibitsarterial smooth muscle cell proliferationrdquo Atherosclerosis vol58 no 1ndash3 pp 109ndash122 1985

[48] J Thyberg and L Palmberg ldquoThe calcium antagonist nisoldip-ine and the calmodulin antagonist W-7 synergistically inhibitinitiation of DNA synthesis in cultured arterial smooth musclecellsrdquo Biology of the Cell vol 60 no 2 pp 125ndash132 1987

[49] E Munro M Patel P Chan et al ldquoEffect of calcium channelblockers on the growth of human vascular smooth musclecells derived from saphenous vein and vascular graft stenosesrdquoJournal of Cardiovascular Pharmacology vol 23 no 5 pp 779ndash784 1994

[50] T A Kent A Jazayeri and J M Simard ldquoCalcium channelsand nifedipine inhibition of serotonin-induced [3H]thymidineincorporation in cultured cerebral smoothmuscle cellsrdquo Journalof Cerebral Blood Flow and Metabolism vol 12 no 1 pp 139ndash146 1992

[51] A Agrotis P J Little J Saltis and A Bobik ldquoDihydropyridineCa2+ channel antagonists inhibit the salvage pathway for DNAsynthesis in human vascular smooth muscle cellsrdquo EuropeanJournal of Pharmacology vol 244 no 3 pp 269ndash275 1993

[52] I Duque M R Puyol P Ruiz M Gonzalez-Rubio M LD Marques and D R Puyol ldquoCalcium channel blockers

ISRN Biochemistry 13

inhibit hydrogen peroxide-induced proliferation of culturedrat mesangial cellsrdquo Journal of Pharmacology and ExperimentalTherapeutics vol 267 no 2 pp 612ndash616 1993

[53] T Kuga S Kobayashi Y Hirakawa H Kanaide and ATakeshita ldquoCell cycle-dependent expression of L- and T-typeCa2+ currents in rat aortic smooth muscle cells in primaryculturerdquo Circulation Research vol 79 no 1 pp 14ndash19 1996

[54] E M Graf M Bock J F Heubach et al ldquoTissue distribution ofa humanCav12 1205721 subunit splice variant with a 75 bp insertionrdquoCell Calcium vol 38 no 1 pp 11ndash21 2005

[55] P Liao D Yu S Lu et al ldquoSmoothmuscle-selective alternativelyspliced exon generates functional variation in Cav12 calciumchannelsrdquo Journal of Biological Chemistry vol 279 no 48 pp50329ndash50335 2004

[56] O Skalli P Ropraz and A Trzeciak ldquoA monoclonal antibodyagainst 120572-smooth muscle actin a new probe for smooth muscledifferentiationrdquo Journal of Cell Biology vol 103 no 6 pp 2787ndash2796 1986

[57] T M Doherty K Asotra L A Fitzpatrick et al ldquoCalcificationin atherosclerosis bone biology and chronic inflammation atthe arterial crossroadsrdquo Proceedings of the National Academyof Sciences of the United States of America vol 100 no 20 pp11201ndash11206 2003

[58] G K Owens ldquoRegulation of differentiation of vascular smoothmuscle cellsrdquo Physiological Reviews vol 75 no 3 pp 487ndash5171995

[59] J P Bannister A Adebiyi G Zhao et al ldquoSmooth musclecell 1205722120575-1 subunits are essential for vasoregulation by Cav12channelsrdquo Circulation Research vol 105 no 10 pp 948ndash9552009

[60] S SonkusareM Fraer J DMarsh andN J Rusch ldquoDisruptingcalcium channel expression to lower blood pressure newtargeting of awell-known channelrdquoMolecular Interventions vol6 no 6 pp 304ndash310 2006

[61] S Telemaque S Sonkusare TGrain et al ldquoDesign ofmutant1205732subunits as decoy molecules to reduce the expression of func-tional Ca2+ channels in cardiac cellsrdquo Journal of Pharmacologyand Experimental Therapeutics vol 325 no 1 pp 37ndash46 2008

[62] A Goyenvalle A Vulin F Fougerousse et al ldquoRescue of dys-trophic muscle through U7 snRNA-mediated exon skippingrdquoScience vol 306 no 5702 pp 1796ndash1799 2004

[63] D BHalling P Aracena-Parks and S L Hamilton ldquoRegulationof voltage-gated Ca2+ channels by calmodulinrdquo Sciencersquos STKEvol 2005 no 315 p re15 2005

[64] K S Lee E Marban and R W Tsien ldquoInactivation of calciumchannels in mammalian heart cells joint dependence on mem-brane potential and intracellular calciumrdquo Journal of Physiologyvol 364 pp 395ndash411 1985

[65] C Shi and N M Soldatov ldquoMolecular determinants of voltage-dependent slow inactivation of the Ca2+ channelrdquo Journal ofBiological Chemistry vol 277 no 9 pp 6813ndash6821 2002

[66] E Kobrinsky E Schwartz D R Abernethy and NM SoldatovldquoVoltage-gated mobility of the Ca2+ channel cytoplasmic tailsand its regulatory rolerdquo Journal of Biological Chemistry vol 278no 7 pp 5021ndash5028 2003

[67] N M Soldatov ldquoCa2+ channel moving tail link between Ca2+-induced inactivation and Ca2+ signal transductionrdquo Trends inPharmacological Sciences vol 24 no 4 pp 167ndash171 2003

[68] D J Klemm P A Watson M G Frid et al ldquocAMP responseelement-binding protein content is a molecular determinantof smooth muscle cell proliferation and migrationrdquo Journal ofBiological Chemistry vol 276 no 49 pp 46132ndash46141 2001

[69] E Kobrinsky L Stevens Y Kazmi DWray andNM SoldatovldquoMolecular rearrangements of the Kv21 potassium channeltermini associated with voltage gatingrdquo Journal of BiologicalChemistry vol 281 no 28 pp 19233ndash19240 2006

[70] BMMayr G Canettieri andMRMontminy ldquoDistinct effectsof cAMP and mitogenic signals on CREB-binding proteinrecruitment impart specificity to target gene activation viaCREBrdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 98 no 19 pp 10936ndash10941 2001

[71] M Morad and N Soldatov ldquoCalcium channel inactivationpossible role in signal transduction and Ca2+ signalingrdquo CellCalcium vol 38 no 3-4 pp 223ndash231 2005

[72] DGWheeler C F Barrett R DGroth P Safa andRW TsienldquoCaMKII locally encodes L-type channel activity to signal tonuclear CREB in excitation-transcription couplingrdquo Journal ofCell Biology vol 183 no 5 pp 849ndash863 2008

[73] D E Mager E Kobrinsky A Masoudieh A Maltsev D RAbernethy andNM Soldatov ldquoAnalysis of functional signalingdomains from fluorescence imaging and the two-dimensionalcontinuous wavelet transformrdquo Biophysical Journal vol 93 no8 pp 2900ndash2910 2007

[74] E Kobrinsky D E Mager S A Bentil S I Murata DR Abernethy and N M Soldatov ldquoIdentification of plasmamembrane macro- and microdomains from wavelet analysis ofFRET microscopyrdquo Biophysical Journal vol 88 no 5 pp 3625ndash3634 2005

[75] E Kobrinsky S Q Duong A Sheydina and N M Solda-tov ldquoMicrodomain organization and frequency-dependence ofCREB-dependent transcriptional signaling in heart cellsrdquo TheFASEB Journal vol 25 no 5 pp 1544ndash1555 2011

[76] A Ravindran Q Z Lao J B Harry P Abrahimi E Kobrinskyand N M Soldatov ldquoCalmodulin-dependent gating of Cav12calcium channels in the absence of Cav120573 subunitsrdquo Proceedingsof the National Academy of Sciences of the United States ofAmerica vol 105 no 23 pp 8154ndash8159 2008

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

ISRN Biochemistry 9

10

0

2

10

100 pA125 ms

(a) (a) (a)

(b)(c)

(b)(c)

(b)(c)

(B) (C) (D)

C-tail Anchored ReleasedReleased

0 10No FRET No FRET

(d) (d) (d)

Fluo-4

PH

CYN

C

C

NY

C

Y EYFP

C ECFP

PH Plekstrin homologydomain

PHPHY

C

NC

IQ

LA

LAIQ

(A)

Fluo-4 Fluo-4

FRET FRET FRET

CRE

Gene expressionCREBCREB P

CBP

AC

GPCR

PKA

cAMP

CaM

Nucleus

cAMP

Plasmamembrane

Ca2+ Ca2+

Ca2+

+20mV +20mV

minus90mV minus90mV

1205721C77

1198651198650

Cav 12

Ca2+

minus90mVACh (5 mM)

4 120583

Figure 7 Evidence that Ca2+ signal transduction by Cav12 to activate CREB-dependent transcription is mediated by the voltage-gatedmobility of the 120572

1C C-tail carrying the CaM-caged Ca2+ and is not directly associated with the increase of [Ca2+]i CREB activation wasexamined under perforated patch conditions in COS1 cells expressing recombinant atherosclerotic 120572

1C77 channel (A) Phase-contrast imageof the COS1 cell with a shadow of patch pipette The cell was expressing the 120572

1C77-PHC1205731a1205722120575-1 channel with the membrane-trapped 1205721C77

subunit C-tail type 1muscarinic Ach receptor (to release the anchored C-tail in response to activation by Ach) and EYFP-KID and ECFP-KIXdomains both supplemented with nuclear localization sequences Inset at the bottom Cav12- and cAMP-dependent cell signaling pathwaysmediating CREB-dependent transcription CBP CREB-binding protein CRE cAMP-response element (B) Plasma-membrane anchoring ofthe 1205721C77 C-tail (see schematic diagram) inhibits CREB activation in spite of robust 119868Ca evoked by depolarization (a) A 100-ms images of

FRET between EYFP-KID and ECFP-KIX were recorded at the end of the 12th +20-mV depolarization step applied every 10 s for 1 s from119881ℎ=

minus90mV (b) A corresponding representative image of corrected FRET ratio (in a ldquo3Drdquo MetaMorph transformation) recorded within 50-mswindows at minus90 and at the end of the last test pulse of the applied train of pulses (c) An increase of [Ca2+]i detected by Ca

2+ indicator Fluo4(d) Representative trace of 119868Ca (20mM Ca2+ in bath medium) evoked by depolarization to +20mV from119881

ℎ= minus90mV showing the sustained

component of Ca2+ conductance due to the C-tail anchoring (C) ACh-stimulation of theM1AChR to unbind the 1205721C C-tail caused activation

of IP3-dependent Ca2+ release (panel (c)) but did not show substantial activation of CREB transcription at 119881

ℎ= minus90mV (panel (a)) (D)

CREB-dependent transcription was activated only in response to depolarization applied to membrane channel with the 1205721C77 C-terminal tail

released from the plasma membrane to assume functional voltage-dependent conformation

intracellular Ca2+ released from the intracellular stores orCa2+ caging compounds does not accelerate significantly theinactivation of Cav12 [71] Thus apo-CaM associated withLA binds predominantly Ca2+ ions permeating through thepore A Ca2+-dependent transfer of CaM from LA to the IQ-motif opens the ldquolockrdquo and initiates a large rearrangement ofthe C-terminal tail This in turn facilitates inactivation of thechannel The Ca2+CaM complex with the IQ-motif is thentransferred by themobile C-tail to a downstream target of theCa2+-signaling cascade (such as CaMKII [72]) where Ca2+ isreleased as an activating stimulus while CaM switches back

to LA and returns the C-tail to the resting position availablefor the next cycle of Ca2+-signal transduction

Activation of CREB-dependent transcription by the L-type 119868Ca ismediated throughmultiple cell signaling pathwaysUsing FRET probes of CREB activity and 2D wavelet trans-form analysis we applied principles of quantitative biologyto detail the mechanism of Ca2+-activated CREB-dependenttranscription within localized regions (microdomains) ofthe nucleus We reached this goal by applying continuouswavelet analysis in two dimensions with a 2D wavelet asa deconvolution algorithm for FRET microscopy image

10 ISRN Biochemistry

Control

(A)

05

0

1

05

00 20 40 60 800 20 40 60 800 20 40 60 80

(a) (b) (c) (d)

20 40 60 800

0

20

40

60

80 1

C 3 5 10 mincAMP

C 2 10 mincAMP

C 3 10 min

cAMP

C 1 3 10 min

cAMP

5

cAMP stable (5)

(B)

119868Ca (3 min) cAMP (5 min) cAMP (10 min)

119868Ca cAMP (65) 119868Ca stable (15)

119868Ca119868Ca

119868Ca 119868Ca

119868Ca cAMP transient (15)

Figure 8 Microdomain analysis on the additive effect of 119868Ca and cAMP on CREB signaling in COS1 cells expressing the recombinantldquoatheroscleroticrdquo120572

1C77 channel (A) FRET signal within the nucleus during selected time points during 119868Ca stimulation and cAMP applicationOutlined are four types of signaling microdomains identified using 2D Mexican hat wavelet Red circles represent stable microdomains thatpersist through both 119868Ca and cAMP application White and yellow circles showmicrodomains stable activated by cAMP and 119868Ca respectivelyGreen circles represent transient microdomains of CREB signaling activation Axes show pixel numbers (B) Typical appearances of the fourtypes of CREB signaling microdomains activated by 119868Ca andor cAMP application and recorded in their maximal development in relationto the time of applied stimuli C control recorded before stimulation Color bars in (A) and (B) represent FRET values normalized to themaximum

analysis [73] Continuous wavelet analysis is a mathematicaltechnique that allows us to analyze a signal over severaldifferent frequencies across the entire signal [73 74] It isespecially useful for finding heterogeneity in a signal becauseit can easily find where the pattern (ie frequency) of asignal changes In these experiments we for the first timeobtained evidence of CREB signaling microdomains withinthe nucleus that respond differentially to 119868Ca stimulationand cAMP (Figure 8(A)) Results of the study revealed thatCREB-dependent transcriptional signaling occurs in dis-crete signaling microdomains underlying the architecture ofnuclear signaling Continuous activation ofCREB-dependenttranscriptional signaling by cAMP and Ca2+ resulted in agradual increase of the number of microdomains Four dif-ferent categories of cAMP and Ca2+-induced CREB signalingmicrodomains were characterized in COS1 cells expressingrecombinant Cav12 with the ldquoatheroscleroticrdquo 120572

1C77 splice

variant (Figure 8) In up to 65 of the microdomainstranscription was activated in additive manner by cAMPand Ca2+ Approximately 15 of signaling domains wereactivated only by 119868Ca and 5 of domains were activatedonly by cAMP Finally 15 of the domains were transientand activated by both cAMP and 119868Ca (Figure 8(B)) [75]A similar spatiotemporal organization of CREB-dependentsignaling was observed in spontaneously beating neonatalrat cardiomyocytes Although COS1 cells that were used inour experiments shown in Figures 7 and 8 are naturallydeprived of Cav12 [76] they inherited the ability to replicatethe Cav12-dependent activation of CREB signaling with theexogenous recombinant channel Thus this experimentalapproach fits the task which in my opinion is the mostimportant unresolved issue for the coupling of Cav12 toCREB signaling does the splice variation of 120572

1C affect thespatiotemporal organization of CREB-dependent signaling

ISRN Biochemistry 11

in a way that may affect cell proliferation and other crucialfunction

7 Conclusions

Association of Cav12 with regulation of transcription cellproliferation and its pathophysiology as in the case ofatherosclerosis requires detailed investigation of the roles ofthe naturally occurring 120572

1C splice variants It will limit thetraditionally intuitive approach to Cav12 in physiology andhelp to define new principle approaches to the treatment ofvarious Cav12 channelopathy-related dysfunctions above allcardiovascular diseases Humgenex Inc provides consultingand logistic support on a broad range of issues reported inthis review

Abbreviations

CaM CalmodulinCBD Calmodulin-binding domainCDI Ca2+-dependent inactivationDHP DihydropyridineECFP Enhanced cyan fluorescent proteinEYFP Enhanced yellow fluorescent proteinFALI Fluorophore-assisted light inactivationFRET Fluorescent resonance energy transfer[3H]PMD 26-Dimethyl-3-methoxycarbonyl-5-([23-

3H2]-n-propoxycarbonyl)-4-(21015840-

difluoromethoxyphenyl)-14-dihydropyridine

119868Ca Calcium currentPH Pleckstrin homology domainVSM Vascular smooth muscle

References

[1] Y S Lee M M Sayeed and R D Wurster ldquoInhibition ofcell growth and intracellular Ca2+ mobilization in humanbrain tumor cells by Ca2+ channel antagonistsrdquo Molecular andChemical Neuropathology vol 22 no 2 pp 81ndash95 1994

[2] K Sato J Ishizuka C W Cooper et al ldquoInhibitory effect ofcalcium channel blockers on growth of pancreatic cancer cellsrdquoPancreas vol 9 no 2 pp 193ndash202 1994

[3] V Bertrand M J Bastie N Vaysse and L Pradayrol ldquoInhibi-tion of gastrin-induced proliferation of AR4-2J cells by calciumchannel antagonistsrdquo International Journal of Cancer vol 56no 3 pp 427ndash432 1994

[4] JM Taylor andRU Simpson ldquoInhibition of cancer cell growthby calcium channel antagonists in the athymic mouserdquo CancerResearch vol 52 no 9 pp 2413ndash2418 1992

[5] M G Cattaneo M Gullo and L M Vicentini ldquoC2+ and Ca2+channel antagonists in the control of human small cell lung car-cinoma cell proliferationrdquo European Journal of Pharmacologyvol 247 no 3 pp 325ndash331 1993

[6] C Chen M J Corbley T M Roberts and P Hess ldquoVoltage-sensitive calcium channels in normal and transformed 3T3fibroblastsrdquo Science vol 239 no 4843 pp 1024ndash1026 1988

[7] P C Dartsch M Ritter M Gschwentner H J Lang andF Lang ldquoEffects of calcium channel blockers on NIH 3T3

fibroblasts expressing the Ha-ras oncogenerdquo European Journalof Cell Biology vol 67 no 4 pp 372ndash378 1995

[8] M Biel R Hullin S Freundner et al ldquoTissue-specific expres-sion of high-voltage-activated dihydropyridine-sensitive L-typecalcium channelsrdquo European Journal of Biochemistry vol 200no 1 pp 81ndash88 1991

[9] DM Rodman K Reese J Harral et al ldquoLow-voltage-activated(T-type) calcium channels control proliferation of human pul-monary artery myocytesrdquo Circulation Research vol 96 no 8pp 864ndash872 2005

[10] A Panner L L Cribbs G M Zainelli T C Origitano S Singhand R DWurster ldquoVariation of T-type calcium channel proteinexpression affects cell division of cultured tumor cellsrdquo CellCalcium vol 37 no 2 pp 105ndash119 2005

[11] C Chen and P Hess ldquoCalcium channels in mouse 3T3 andhuman fibroblastsrdquo Biophysical Journal vol 51 p 226a 1987

[12] N M Soldatov ldquoMolecular diversity of L-type Ca2+ channeltranscripts in human fibroblastsrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 89 no10 pp 4628ndash4632 1992

[13] A Fujii H Matsumoto S Nakao H Teshigawara and YAkimoto ldquoEffect of calcium-channel blockers on cell prolifera-tion DNA synthesis and collagen synthesis of cultured gingivalfibroblasts derived fromhumannifedipine responders and non-respondersrdquo Archives of Oral Biology vol 39 no 2 pp 99ndash1041994

[14] N M Soldatov ldquoGenomic structure of human L-type Ca2+channelrdquo Genomics vol 22 no 1 pp 77ndash87 1994

[15] V P Shevchenko N F Myasoedov N M Soldatov G JDuburs V V Kastron and I P Skrastins ldquoSynthesis andevaluation of biological activity of two novel tritium-labeledderivatives of riodipinerdquo Journal of Labelled Compounds andRadiopharmaceuticals vol 27 no 6 pp 721ndash730 1989

[16] S M Dudkin S N Gnedoj N N Chernyuk and N MSoldatov ldquo14-dihydropyridine receptor associated with Ca2+channels in human embryonic fibroblastsrdquo FEBS Letters vol233 no 2 pp 352ndash354 1988

[17] A DePover M A Matlib S W Lee et al ldquoSpecific bindingof [3H]nitrendipine to membranes from coronary arteriesand heart in relation to pharmacological effects Paradoxicalstimulation by diltiazemrdquo Biochemical and Biophysical ResearchCommunications vol 108 no 1 pp 110ndash117 1982

[18] N M Soldatov S N Gnedoj N N Chernyuk I A Britanovaand S M Dudkin ldquoMitogenesis and 14-dihydropyridinereceptor associated with Ca2+-channels in human embryonicfibroblastsrdquo Journal of Membrane Biology vol 5 pp 1161ndash11671988 (Russian)

[19] D Lipscombe D V Madison M Poenie H Reuter R Y Tsienand R W Tsien ldquoSpatial distribution of calcium channels andcytosolic calcium transients in growth cones and cell bodies ofsympathetic neuronsrdquo Proceedings of the National Academy ofSciences of the United States of America vol 85 no 7 pp 2398ndash2402 1988

[20] G S Harms L Cognet P H M Lommerse et al ldquoSingle-molecule imaging of L-type CA2+ channels in live cellsrdquoBiophysical Journal vol 81 no 5 pp 2639ndash2646 2001

[21] V di Biase G J Obermair Z Szabo et al ldquoStable membraneexpression of postsynaptic Cav12 calcium channel clustersis independent of interactions with AKAP79150 and PDZproteinsrdquo Journal of Neuroscience vol 28 no 51 pp 13845ndash13855 2008

12 ISRN Biochemistry

[22] M F Navedo E P Cheng C Yuan et al ldquoIncreased coupledgating of L-type Ca2+ channels during hypertension and timo-thy syndromerdquoCirculationResearch vol 106 no 4 pp 748ndash7562010

[23] E Kobrinsky P Abrahimi S Q Duong et al ldquoEffect of Cav120573subunits on structural organization of Cav12 calcium channelsrdquoPLoS One vol 4 no 5 Article ID e5587 2009

[24] Q Z Lao E Kobrinsky Z Liu and N M Soldatov ldquoOligomer-ization of Cav120573 subunits is an essential correlate of Ca

2+ channelactivityrdquoThe FASEB Journal vol 24 no 12 pp 5013ndash5023 2010

[25] E Kobrinsky S Tiwari V A Maltsev et al ldquoDifferential roleof the 1205721C subunit tails in regulation of the Cav12 channelby membrane potential 120573 subunits and Ca2+ ionsrdquo Journal ofBiological Chemistry vol 280 no 13 pp 12474ndash12485 2005

[26] S Tiwari Y Zhang J Heller D R Abernethy and N MSoldatov ldquoArtherosclerosis-related molecular alteration of thehuman Cav12 calcium channel 1205721C subunitrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 103 no 45 pp 17024ndash17029 2006

[27] I Splawski K W Timothy L M Sharpe et al ldquoCav12 calciumchannel dysfunction causes a multisystem disorder includingarrhythmia and autismrdquo Cell vol 119 no 1 pp 19ndash31 2004

[28] Z Z TangM C Liang S Lu et al ldquoTranscript scanning revealsnovel and extensive splice variations in human L-type voltage-gated calcium channel Cav12 1205721 subunitrdquo Journal of BiologicalChemistry vol 279 no 43 pp 44335ndash44343 2004

[29] P Liao T F Yong M C Liang D T Yue and T W SoongldquoSplicing for alternative structures of Cav12 Ca2+ channels incardiac and smooth musclesrdquo Cardiovascular Research vol 68no 2 pp 197ndash203 2005

[30] Y Blumenstein N Kanevsky G Sahar R Barzilai T Ivaninaand N Dascal ldquoA novel long N-terminal isoform of human L-type Ca2+ channel is up-regulated by protein kinase Crdquo Journalof Biological Chemistry vol 277 no 5 pp 3419ndash3423 2002

[31] N M Soldatov A Bouron and H Reuter ldquoDifferent voltage-dependent inhibition by dihydropyridines of human Ca2+channel splice variantsrdquo Journal of Biological Chemistry vol270 no 18 pp 10540ndash10543 1995

[32] D P Faxon M A Creager S C Smith et al ldquoAtheroscleroticvascular disease conference executive summary atheroscle-rotic vascular disease conference proceeding for healthcareprofessionals from a special writing group of the AmericanHeart Associationrdquo Circulation vol 109 no 21 pp 2595ndash26042004

[33] R Ross ldquoAtherosclerosismdashan inflammatory diseaserdquo The NewEngland Journal of Medicine vol 340 no 2 pp 115ndash126 1999

[34] H Roy S Bhardwaj and S Yla-Herttuala ldquoMolecular geneticsof atherosclerosisrdquo Human Genetics vol 125 no 5-6 pp 467ndash491 2009

[35] M Biel P Ruth E Bosse et al ldquoPrimary structure and func-tional expression of a high voltage activated calcium channelfrom rabbit lungrdquo FEBS Letters vol 269 no 2 pp 409ndash4121990

[36] X Cheng J Liu M Asuncion-Chin et al ldquoA novel Cav12N terminus expressed in smooth muscle cells of resistancesize arteries modifies channel regulation by auxiliary subunitsrdquoJournal of Biological Chemistry vol 282 no 40 pp 29211ndash292212007

[37] W J Koch P T Ellinor and A Schwartz ldquocDNA cloning ofa dihydropyridine-sensitive calcium channel from rat aortaevidence for the existence of alternatively spliced formsrdquo Journalof Biological Chemistry vol 265 no 29 pp 17786ndash17791 1990

[38] S Richard D Neveu G Carnac P Bodin P Travo and J Nar-geot ldquoDifferential expression of voltage-gated Ca2+-currents incultivated aortic myocytesrdquo Biochimica et Biophysica Acta vol1160 no 1 pp 95ndash104 1992

[39] A Schwartz ldquoCalcium antagonists review and perspective onmechanism of actionrdquo American Journal of Cardiology vol 64no 17 pp 3Indash9I 1989

[40] R P Mason ldquoMechanisms of plaque stabilization for thedihydropyridine calcium channel blocker amlodipine review ofthe evidencerdquo Atherosclerosis vol 165 no 2 pp 191ndash199 2002

[41] A Ruiz-Torres R Lozano J Melon and R Carraro ldquoL-calciumchannel blockade induced by diltiazem inhibits proliferationmigration and F-actin membrane rearrangements in humanvascular smooth muscle cells stimulated with insulin and IGF-1rdquo International Journal of Clinical Pharmacology andTherapeu-tics vol 41 no 9 pp 386ndash391 2003

[42] H Koshiyama S Tanaka and J Minamikawa ldquoEffect ofcalcium channel blocker amlodipine on the intimal-medialthickness of carotid arterial wall in type 2 diabetesrdquo Journal ofCardiovascular Pharmacology vol 33 no 6 pp 894ndash896 1999

[43] T Yamashita S KawashimaMOzaki et al ldquoA calcium channelblocker benidipine inhibits intimal thickening in the carotidartery of mice by increasing nitric oxide productionrdquo Journal ofHypertension vol 19 no 3 pp 451ndash458 2001

[44] R P Mason P Marche and T H Hintze ldquoNovel vascularbiology of third-generation L-type calcium channel antagonistsancillary actions of amlodipinerdquo Arteriosclerosis Thrombosisand Vascular Biology vol 23 no 12 pp 2155ndash2163 2003

[45] O Stepien J GogusevD L Zhu et al ldquoAmlodipine inhibition ofserum- thrombin- or fibroblast growth factor- induced vascu-lar smooth-muscle cell proliferationrdquo Journal of CardiovascularPharmacology vol 31 no 5 pp 786ndash793 1998

[46] V Ruiz-Velasco M B Mayer E W Inscho and L J HymelldquoModulation of dihydropyridine receptors in vascular smoothmuscle cells by membrane potential and cell proliferationrdquoEuropean Journal of Pharmacology vol 268 no 3 pp 311ndash3181994

[47] J Nilsson M Sjolund L Palmberg A M von Euler B Jonzonand J Thyberg ldquoThe calcium antagonist nifedipine inhibitsarterial smooth muscle cell proliferationrdquo Atherosclerosis vol58 no 1ndash3 pp 109ndash122 1985

[48] J Thyberg and L Palmberg ldquoThe calcium antagonist nisoldip-ine and the calmodulin antagonist W-7 synergistically inhibitinitiation of DNA synthesis in cultured arterial smooth musclecellsrdquo Biology of the Cell vol 60 no 2 pp 125ndash132 1987

[49] E Munro M Patel P Chan et al ldquoEffect of calcium channelblockers on the growth of human vascular smooth musclecells derived from saphenous vein and vascular graft stenosesrdquoJournal of Cardiovascular Pharmacology vol 23 no 5 pp 779ndash784 1994

[50] T A Kent A Jazayeri and J M Simard ldquoCalcium channelsand nifedipine inhibition of serotonin-induced [3H]thymidineincorporation in cultured cerebral smoothmuscle cellsrdquo Journalof Cerebral Blood Flow and Metabolism vol 12 no 1 pp 139ndash146 1992

[51] A Agrotis P J Little J Saltis and A Bobik ldquoDihydropyridineCa2+ channel antagonists inhibit the salvage pathway for DNAsynthesis in human vascular smooth muscle cellsrdquo EuropeanJournal of Pharmacology vol 244 no 3 pp 269ndash275 1993

[52] I Duque M R Puyol P Ruiz M Gonzalez-Rubio M LD Marques and D R Puyol ldquoCalcium channel blockers

ISRN Biochemistry 13

inhibit hydrogen peroxide-induced proliferation of culturedrat mesangial cellsrdquo Journal of Pharmacology and ExperimentalTherapeutics vol 267 no 2 pp 612ndash616 1993

[53] T Kuga S Kobayashi Y Hirakawa H Kanaide and ATakeshita ldquoCell cycle-dependent expression of L- and T-typeCa2+ currents in rat aortic smooth muscle cells in primaryculturerdquo Circulation Research vol 79 no 1 pp 14ndash19 1996

[54] E M Graf M Bock J F Heubach et al ldquoTissue distribution ofa humanCav12 1205721 subunit splice variant with a 75 bp insertionrdquoCell Calcium vol 38 no 1 pp 11ndash21 2005

[55] P Liao D Yu S Lu et al ldquoSmoothmuscle-selective alternativelyspliced exon generates functional variation in Cav12 calciumchannelsrdquo Journal of Biological Chemistry vol 279 no 48 pp50329ndash50335 2004

[56] O Skalli P Ropraz and A Trzeciak ldquoA monoclonal antibodyagainst 120572-smooth muscle actin a new probe for smooth muscledifferentiationrdquo Journal of Cell Biology vol 103 no 6 pp 2787ndash2796 1986

[57] T M Doherty K Asotra L A Fitzpatrick et al ldquoCalcificationin atherosclerosis bone biology and chronic inflammation atthe arterial crossroadsrdquo Proceedings of the National Academyof Sciences of the United States of America vol 100 no 20 pp11201ndash11206 2003

[58] G K Owens ldquoRegulation of differentiation of vascular smoothmuscle cellsrdquo Physiological Reviews vol 75 no 3 pp 487ndash5171995

[59] J P Bannister A Adebiyi G Zhao et al ldquoSmooth musclecell 1205722120575-1 subunits are essential for vasoregulation by Cav12channelsrdquo Circulation Research vol 105 no 10 pp 948ndash9552009

[60] S SonkusareM Fraer J DMarsh andN J Rusch ldquoDisruptingcalcium channel expression to lower blood pressure newtargeting of awell-known channelrdquoMolecular Interventions vol6 no 6 pp 304ndash310 2006

[61] S Telemaque S Sonkusare TGrain et al ldquoDesign ofmutant1205732subunits as decoy molecules to reduce the expression of func-tional Ca2+ channels in cardiac cellsrdquo Journal of Pharmacologyand Experimental Therapeutics vol 325 no 1 pp 37ndash46 2008

[62] A Goyenvalle A Vulin F Fougerousse et al ldquoRescue of dys-trophic muscle through U7 snRNA-mediated exon skippingrdquoScience vol 306 no 5702 pp 1796ndash1799 2004

[63] D BHalling P Aracena-Parks and S L Hamilton ldquoRegulationof voltage-gated Ca2+ channels by calmodulinrdquo Sciencersquos STKEvol 2005 no 315 p re15 2005

[64] K S Lee E Marban and R W Tsien ldquoInactivation of calciumchannels in mammalian heart cells joint dependence on mem-brane potential and intracellular calciumrdquo Journal of Physiologyvol 364 pp 395ndash411 1985

[65] C Shi and N M Soldatov ldquoMolecular determinants of voltage-dependent slow inactivation of the Ca2+ channelrdquo Journal ofBiological Chemistry vol 277 no 9 pp 6813ndash6821 2002

[66] E Kobrinsky E Schwartz D R Abernethy and NM SoldatovldquoVoltage-gated mobility of the Ca2+ channel cytoplasmic tailsand its regulatory rolerdquo Journal of Biological Chemistry vol 278no 7 pp 5021ndash5028 2003

[67] N M Soldatov ldquoCa2+ channel moving tail link between Ca2+-induced inactivation and Ca2+ signal transductionrdquo Trends inPharmacological Sciences vol 24 no 4 pp 167ndash171 2003

[68] D J Klemm P A Watson M G Frid et al ldquocAMP responseelement-binding protein content is a molecular determinantof smooth muscle cell proliferation and migrationrdquo Journal ofBiological Chemistry vol 276 no 49 pp 46132ndash46141 2001

[69] E Kobrinsky L Stevens Y Kazmi DWray andNM SoldatovldquoMolecular rearrangements of the Kv21 potassium channeltermini associated with voltage gatingrdquo Journal of BiologicalChemistry vol 281 no 28 pp 19233ndash19240 2006

[70] BMMayr G Canettieri andMRMontminy ldquoDistinct effectsof cAMP and mitogenic signals on CREB-binding proteinrecruitment impart specificity to target gene activation viaCREBrdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 98 no 19 pp 10936ndash10941 2001

[71] M Morad and N Soldatov ldquoCalcium channel inactivationpossible role in signal transduction and Ca2+ signalingrdquo CellCalcium vol 38 no 3-4 pp 223ndash231 2005

[72] DGWheeler C F Barrett R DGroth P Safa andRW TsienldquoCaMKII locally encodes L-type channel activity to signal tonuclear CREB in excitation-transcription couplingrdquo Journal ofCell Biology vol 183 no 5 pp 849ndash863 2008

[73] D E Mager E Kobrinsky A Masoudieh A Maltsev D RAbernethy andNM Soldatov ldquoAnalysis of functional signalingdomains from fluorescence imaging and the two-dimensionalcontinuous wavelet transformrdquo Biophysical Journal vol 93 no8 pp 2900ndash2910 2007

[74] E Kobrinsky D E Mager S A Bentil S I Murata DR Abernethy and N M Soldatov ldquoIdentification of plasmamembrane macro- and microdomains from wavelet analysis ofFRET microscopyrdquo Biophysical Journal vol 88 no 5 pp 3625ndash3634 2005

[75] E Kobrinsky S Q Duong A Sheydina and N M Solda-tov ldquoMicrodomain organization and frequency-dependence ofCREB-dependent transcriptional signaling in heart cellsrdquo TheFASEB Journal vol 25 no 5 pp 1544ndash1555 2011

[76] A Ravindran Q Z Lao J B Harry P Abrahimi E Kobrinskyand N M Soldatov ldquoCalmodulin-dependent gating of Cav12calcium channels in the absence of Cav120573 subunitsrdquo Proceedingsof the National Academy of Sciences of the United States ofAmerica vol 105 no 23 pp 8154ndash8159 2008

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

10 ISRN Biochemistry

Control

(A)

05

0

1

05

00 20 40 60 800 20 40 60 800 20 40 60 80

(a) (b) (c) (d)

20 40 60 800

0

20

40

60

80 1

C 3 5 10 mincAMP

C 2 10 mincAMP

C 3 10 min

cAMP

C 1 3 10 min

cAMP

5

cAMP stable (5)

(B)

119868Ca (3 min) cAMP (5 min) cAMP (10 min)

119868Ca cAMP (65) 119868Ca stable (15)

119868Ca119868Ca

119868Ca 119868Ca

119868Ca cAMP transient (15)

Figure 8 Microdomain analysis on the additive effect of 119868Ca and cAMP on CREB signaling in COS1 cells expressing the recombinantldquoatheroscleroticrdquo120572

1C77 channel (A) FRET signal within the nucleus during selected time points during 119868Ca stimulation and cAMP applicationOutlined are four types of signaling microdomains identified using 2D Mexican hat wavelet Red circles represent stable microdomains thatpersist through both 119868Ca and cAMP application White and yellow circles showmicrodomains stable activated by cAMP and 119868Ca respectivelyGreen circles represent transient microdomains of CREB signaling activation Axes show pixel numbers (B) Typical appearances of the fourtypes of CREB signaling microdomains activated by 119868Ca andor cAMP application and recorded in their maximal development in relationto the time of applied stimuli C control recorded before stimulation Color bars in (A) and (B) represent FRET values normalized to themaximum

analysis [73] Continuous wavelet analysis is a mathematicaltechnique that allows us to analyze a signal over severaldifferent frequencies across the entire signal [73 74] It isespecially useful for finding heterogeneity in a signal becauseit can easily find where the pattern (ie frequency) of asignal changes In these experiments we for the first timeobtained evidence of CREB signaling microdomains withinthe nucleus that respond differentially to 119868Ca stimulationand cAMP (Figure 8(A)) Results of the study revealed thatCREB-dependent transcriptional signaling occurs in dis-crete signaling microdomains underlying the architecture ofnuclear signaling Continuous activation ofCREB-dependenttranscriptional signaling by cAMP and Ca2+ resulted in agradual increase of the number of microdomains Four dif-ferent categories of cAMP and Ca2+-induced CREB signalingmicrodomains were characterized in COS1 cells expressingrecombinant Cav12 with the ldquoatheroscleroticrdquo 120572

1C77 splice

variant (Figure 8) In up to 65 of the microdomainstranscription was activated in additive manner by cAMPand Ca2+ Approximately 15 of signaling domains wereactivated only by 119868Ca and 5 of domains were activatedonly by cAMP Finally 15 of the domains were transientand activated by both cAMP and 119868Ca (Figure 8(B)) [75]A similar spatiotemporal organization of CREB-dependentsignaling was observed in spontaneously beating neonatalrat cardiomyocytes Although COS1 cells that were used inour experiments shown in Figures 7 and 8 are naturallydeprived of Cav12 [76] they inherited the ability to replicatethe Cav12-dependent activation of CREB signaling with theexogenous recombinant channel Thus this experimentalapproach fits the task which in my opinion is the mostimportant unresolved issue for the coupling of Cav12 toCREB signaling does the splice variation of 120572

1C affect thespatiotemporal organization of CREB-dependent signaling

ISRN Biochemistry 11

in a way that may affect cell proliferation and other crucialfunction

7 Conclusions

Association of Cav12 with regulation of transcription cellproliferation and its pathophysiology as in the case ofatherosclerosis requires detailed investigation of the roles ofthe naturally occurring 120572

1C splice variants It will limit thetraditionally intuitive approach to Cav12 in physiology andhelp to define new principle approaches to the treatment ofvarious Cav12 channelopathy-related dysfunctions above allcardiovascular diseases Humgenex Inc provides consultingand logistic support on a broad range of issues reported inthis review

Abbreviations

CaM CalmodulinCBD Calmodulin-binding domainCDI Ca2+-dependent inactivationDHP DihydropyridineECFP Enhanced cyan fluorescent proteinEYFP Enhanced yellow fluorescent proteinFALI Fluorophore-assisted light inactivationFRET Fluorescent resonance energy transfer[3H]PMD 26-Dimethyl-3-methoxycarbonyl-5-([23-

3H2]-n-propoxycarbonyl)-4-(21015840-

difluoromethoxyphenyl)-14-dihydropyridine

119868Ca Calcium currentPH Pleckstrin homology domainVSM Vascular smooth muscle

References

[1] Y S Lee M M Sayeed and R D Wurster ldquoInhibition ofcell growth and intracellular Ca2+ mobilization in humanbrain tumor cells by Ca2+ channel antagonistsrdquo Molecular andChemical Neuropathology vol 22 no 2 pp 81ndash95 1994

[2] K Sato J Ishizuka C W Cooper et al ldquoInhibitory effect ofcalcium channel blockers on growth of pancreatic cancer cellsrdquoPancreas vol 9 no 2 pp 193ndash202 1994

[3] V Bertrand M J Bastie N Vaysse and L Pradayrol ldquoInhibi-tion of gastrin-induced proliferation of AR4-2J cells by calciumchannel antagonistsrdquo International Journal of Cancer vol 56no 3 pp 427ndash432 1994

[4] JM Taylor andRU Simpson ldquoInhibition of cancer cell growthby calcium channel antagonists in the athymic mouserdquo CancerResearch vol 52 no 9 pp 2413ndash2418 1992

[5] M G Cattaneo M Gullo and L M Vicentini ldquoC2+ and Ca2+channel antagonists in the control of human small cell lung car-cinoma cell proliferationrdquo European Journal of Pharmacologyvol 247 no 3 pp 325ndash331 1993

[6] C Chen M J Corbley T M Roberts and P Hess ldquoVoltage-sensitive calcium channels in normal and transformed 3T3fibroblastsrdquo Science vol 239 no 4843 pp 1024ndash1026 1988

[7] P C Dartsch M Ritter M Gschwentner H J Lang andF Lang ldquoEffects of calcium channel blockers on NIH 3T3

fibroblasts expressing the Ha-ras oncogenerdquo European Journalof Cell Biology vol 67 no 4 pp 372ndash378 1995

[8] M Biel R Hullin S Freundner et al ldquoTissue-specific expres-sion of high-voltage-activated dihydropyridine-sensitive L-typecalcium channelsrdquo European Journal of Biochemistry vol 200no 1 pp 81ndash88 1991

[9] DM Rodman K Reese J Harral et al ldquoLow-voltage-activated(T-type) calcium channels control proliferation of human pul-monary artery myocytesrdquo Circulation Research vol 96 no 8pp 864ndash872 2005

[10] A Panner L L Cribbs G M Zainelli T C Origitano S Singhand R DWurster ldquoVariation of T-type calcium channel proteinexpression affects cell division of cultured tumor cellsrdquo CellCalcium vol 37 no 2 pp 105ndash119 2005

[11] C Chen and P Hess ldquoCalcium channels in mouse 3T3 andhuman fibroblastsrdquo Biophysical Journal vol 51 p 226a 1987

[12] N M Soldatov ldquoMolecular diversity of L-type Ca2+ channeltranscripts in human fibroblastsrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 89 no10 pp 4628ndash4632 1992

[13] A Fujii H Matsumoto S Nakao H Teshigawara and YAkimoto ldquoEffect of calcium-channel blockers on cell prolifera-tion DNA synthesis and collagen synthesis of cultured gingivalfibroblasts derived fromhumannifedipine responders and non-respondersrdquo Archives of Oral Biology vol 39 no 2 pp 99ndash1041994

[14] N M Soldatov ldquoGenomic structure of human L-type Ca2+channelrdquo Genomics vol 22 no 1 pp 77ndash87 1994

[15] V P Shevchenko N F Myasoedov N M Soldatov G JDuburs V V Kastron and I P Skrastins ldquoSynthesis andevaluation of biological activity of two novel tritium-labeledderivatives of riodipinerdquo Journal of Labelled Compounds andRadiopharmaceuticals vol 27 no 6 pp 721ndash730 1989

[16] S M Dudkin S N Gnedoj N N Chernyuk and N MSoldatov ldquo14-dihydropyridine receptor associated with Ca2+channels in human embryonic fibroblastsrdquo FEBS Letters vol233 no 2 pp 352ndash354 1988

[17] A DePover M A Matlib S W Lee et al ldquoSpecific bindingof [3H]nitrendipine to membranes from coronary arteriesand heart in relation to pharmacological effects Paradoxicalstimulation by diltiazemrdquo Biochemical and Biophysical ResearchCommunications vol 108 no 1 pp 110ndash117 1982

[18] N M Soldatov S N Gnedoj N N Chernyuk I A Britanovaand S M Dudkin ldquoMitogenesis and 14-dihydropyridinereceptor associated with Ca2+-channels in human embryonicfibroblastsrdquo Journal of Membrane Biology vol 5 pp 1161ndash11671988 (Russian)

[19] D Lipscombe D V Madison M Poenie H Reuter R Y Tsienand R W Tsien ldquoSpatial distribution of calcium channels andcytosolic calcium transients in growth cones and cell bodies ofsympathetic neuronsrdquo Proceedings of the National Academy ofSciences of the United States of America vol 85 no 7 pp 2398ndash2402 1988

[20] G S Harms L Cognet P H M Lommerse et al ldquoSingle-molecule imaging of L-type CA2+ channels in live cellsrdquoBiophysical Journal vol 81 no 5 pp 2639ndash2646 2001

[21] V di Biase G J Obermair Z Szabo et al ldquoStable membraneexpression of postsynaptic Cav12 calcium channel clustersis independent of interactions with AKAP79150 and PDZproteinsrdquo Journal of Neuroscience vol 28 no 51 pp 13845ndash13855 2008

12 ISRN Biochemistry

[22] M F Navedo E P Cheng C Yuan et al ldquoIncreased coupledgating of L-type Ca2+ channels during hypertension and timo-thy syndromerdquoCirculationResearch vol 106 no 4 pp 748ndash7562010

[23] E Kobrinsky P Abrahimi S Q Duong et al ldquoEffect of Cav120573subunits on structural organization of Cav12 calcium channelsrdquoPLoS One vol 4 no 5 Article ID e5587 2009

[24] Q Z Lao E Kobrinsky Z Liu and N M Soldatov ldquoOligomer-ization of Cav120573 subunits is an essential correlate of Ca

2+ channelactivityrdquoThe FASEB Journal vol 24 no 12 pp 5013ndash5023 2010

[25] E Kobrinsky S Tiwari V A Maltsev et al ldquoDifferential roleof the 1205721C subunit tails in regulation of the Cav12 channelby membrane potential 120573 subunits and Ca2+ ionsrdquo Journal ofBiological Chemistry vol 280 no 13 pp 12474ndash12485 2005

[26] S Tiwari Y Zhang J Heller D R Abernethy and N MSoldatov ldquoArtherosclerosis-related molecular alteration of thehuman Cav12 calcium channel 1205721C subunitrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 103 no 45 pp 17024ndash17029 2006

[27] I Splawski K W Timothy L M Sharpe et al ldquoCav12 calciumchannel dysfunction causes a multisystem disorder includingarrhythmia and autismrdquo Cell vol 119 no 1 pp 19ndash31 2004

[28] Z Z TangM C Liang S Lu et al ldquoTranscript scanning revealsnovel and extensive splice variations in human L-type voltage-gated calcium channel Cav12 1205721 subunitrdquo Journal of BiologicalChemistry vol 279 no 43 pp 44335ndash44343 2004

[29] P Liao T F Yong M C Liang D T Yue and T W SoongldquoSplicing for alternative structures of Cav12 Ca2+ channels incardiac and smooth musclesrdquo Cardiovascular Research vol 68no 2 pp 197ndash203 2005

[30] Y Blumenstein N Kanevsky G Sahar R Barzilai T Ivaninaand N Dascal ldquoA novel long N-terminal isoform of human L-type Ca2+ channel is up-regulated by protein kinase Crdquo Journalof Biological Chemistry vol 277 no 5 pp 3419ndash3423 2002

[31] N M Soldatov A Bouron and H Reuter ldquoDifferent voltage-dependent inhibition by dihydropyridines of human Ca2+channel splice variantsrdquo Journal of Biological Chemistry vol270 no 18 pp 10540ndash10543 1995

[32] D P Faxon M A Creager S C Smith et al ldquoAtheroscleroticvascular disease conference executive summary atheroscle-rotic vascular disease conference proceeding for healthcareprofessionals from a special writing group of the AmericanHeart Associationrdquo Circulation vol 109 no 21 pp 2595ndash26042004

[33] R Ross ldquoAtherosclerosismdashan inflammatory diseaserdquo The NewEngland Journal of Medicine vol 340 no 2 pp 115ndash126 1999

[34] H Roy S Bhardwaj and S Yla-Herttuala ldquoMolecular geneticsof atherosclerosisrdquo Human Genetics vol 125 no 5-6 pp 467ndash491 2009

[35] M Biel P Ruth E Bosse et al ldquoPrimary structure and func-tional expression of a high voltage activated calcium channelfrom rabbit lungrdquo FEBS Letters vol 269 no 2 pp 409ndash4121990

[36] X Cheng J Liu M Asuncion-Chin et al ldquoA novel Cav12N terminus expressed in smooth muscle cells of resistancesize arteries modifies channel regulation by auxiliary subunitsrdquoJournal of Biological Chemistry vol 282 no 40 pp 29211ndash292212007

[37] W J Koch P T Ellinor and A Schwartz ldquocDNA cloning ofa dihydropyridine-sensitive calcium channel from rat aortaevidence for the existence of alternatively spliced formsrdquo Journalof Biological Chemistry vol 265 no 29 pp 17786ndash17791 1990

[38] S Richard D Neveu G Carnac P Bodin P Travo and J Nar-geot ldquoDifferential expression of voltage-gated Ca2+-currents incultivated aortic myocytesrdquo Biochimica et Biophysica Acta vol1160 no 1 pp 95ndash104 1992

[39] A Schwartz ldquoCalcium antagonists review and perspective onmechanism of actionrdquo American Journal of Cardiology vol 64no 17 pp 3Indash9I 1989

[40] R P Mason ldquoMechanisms of plaque stabilization for thedihydropyridine calcium channel blocker amlodipine review ofthe evidencerdquo Atherosclerosis vol 165 no 2 pp 191ndash199 2002

[41] A Ruiz-Torres R Lozano J Melon and R Carraro ldquoL-calciumchannel blockade induced by diltiazem inhibits proliferationmigration and F-actin membrane rearrangements in humanvascular smooth muscle cells stimulated with insulin and IGF-1rdquo International Journal of Clinical Pharmacology andTherapeu-tics vol 41 no 9 pp 386ndash391 2003

[42] H Koshiyama S Tanaka and J Minamikawa ldquoEffect ofcalcium channel blocker amlodipine on the intimal-medialthickness of carotid arterial wall in type 2 diabetesrdquo Journal ofCardiovascular Pharmacology vol 33 no 6 pp 894ndash896 1999

[43] T Yamashita S KawashimaMOzaki et al ldquoA calcium channelblocker benidipine inhibits intimal thickening in the carotidartery of mice by increasing nitric oxide productionrdquo Journal ofHypertension vol 19 no 3 pp 451ndash458 2001

[44] R P Mason P Marche and T H Hintze ldquoNovel vascularbiology of third-generation L-type calcium channel antagonistsancillary actions of amlodipinerdquo Arteriosclerosis Thrombosisand Vascular Biology vol 23 no 12 pp 2155ndash2163 2003

[45] O Stepien J GogusevD L Zhu et al ldquoAmlodipine inhibition ofserum- thrombin- or fibroblast growth factor- induced vascu-lar smooth-muscle cell proliferationrdquo Journal of CardiovascularPharmacology vol 31 no 5 pp 786ndash793 1998

[46] V Ruiz-Velasco M B Mayer E W Inscho and L J HymelldquoModulation of dihydropyridine receptors in vascular smoothmuscle cells by membrane potential and cell proliferationrdquoEuropean Journal of Pharmacology vol 268 no 3 pp 311ndash3181994

[47] J Nilsson M Sjolund L Palmberg A M von Euler B Jonzonand J Thyberg ldquoThe calcium antagonist nifedipine inhibitsarterial smooth muscle cell proliferationrdquo Atherosclerosis vol58 no 1ndash3 pp 109ndash122 1985

[48] J Thyberg and L Palmberg ldquoThe calcium antagonist nisoldip-ine and the calmodulin antagonist W-7 synergistically inhibitinitiation of DNA synthesis in cultured arterial smooth musclecellsrdquo Biology of the Cell vol 60 no 2 pp 125ndash132 1987

[49] E Munro M Patel P Chan et al ldquoEffect of calcium channelblockers on the growth of human vascular smooth musclecells derived from saphenous vein and vascular graft stenosesrdquoJournal of Cardiovascular Pharmacology vol 23 no 5 pp 779ndash784 1994

[50] T A Kent A Jazayeri and J M Simard ldquoCalcium channelsand nifedipine inhibition of serotonin-induced [3H]thymidineincorporation in cultured cerebral smoothmuscle cellsrdquo Journalof Cerebral Blood Flow and Metabolism vol 12 no 1 pp 139ndash146 1992

[51] A Agrotis P J Little J Saltis and A Bobik ldquoDihydropyridineCa2+ channel antagonists inhibit the salvage pathway for DNAsynthesis in human vascular smooth muscle cellsrdquo EuropeanJournal of Pharmacology vol 244 no 3 pp 269ndash275 1993

[52] I Duque M R Puyol P Ruiz M Gonzalez-Rubio M LD Marques and D R Puyol ldquoCalcium channel blockers

ISRN Biochemistry 13

inhibit hydrogen peroxide-induced proliferation of culturedrat mesangial cellsrdquo Journal of Pharmacology and ExperimentalTherapeutics vol 267 no 2 pp 612ndash616 1993

[53] T Kuga S Kobayashi Y Hirakawa H Kanaide and ATakeshita ldquoCell cycle-dependent expression of L- and T-typeCa2+ currents in rat aortic smooth muscle cells in primaryculturerdquo Circulation Research vol 79 no 1 pp 14ndash19 1996

[54] E M Graf M Bock J F Heubach et al ldquoTissue distribution ofa humanCav12 1205721 subunit splice variant with a 75 bp insertionrdquoCell Calcium vol 38 no 1 pp 11ndash21 2005

[55] P Liao D Yu S Lu et al ldquoSmoothmuscle-selective alternativelyspliced exon generates functional variation in Cav12 calciumchannelsrdquo Journal of Biological Chemistry vol 279 no 48 pp50329ndash50335 2004

[56] O Skalli P Ropraz and A Trzeciak ldquoA monoclonal antibodyagainst 120572-smooth muscle actin a new probe for smooth muscledifferentiationrdquo Journal of Cell Biology vol 103 no 6 pp 2787ndash2796 1986

[57] T M Doherty K Asotra L A Fitzpatrick et al ldquoCalcificationin atherosclerosis bone biology and chronic inflammation atthe arterial crossroadsrdquo Proceedings of the National Academyof Sciences of the United States of America vol 100 no 20 pp11201ndash11206 2003

[58] G K Owens ldquoRegulation of differentiation of vascular smoothmuscle cellsrdquo Physiological Reviews vol 75 no 3 pp 487ndash5171995

[59] J P Bannister A Adebiyi G Zhao et al ldquoSmooth musclecell 1205722120575-1 subunits are essential for vasoregulation by Cav12channelsrdquo Circulation Research vol 105 no 10 pp 948ndash9552009

[60] S SonkusareM Fraer J DMarsh andN J Rusch ldquoDisruptingcalcium channel expression to lower blood pressure newtargeting of awell-known channelrdquoMolecular Interventions vol6 no 6 pp 304ndash310 2006

[61] S Telemaque S Sonkusare TGrain et al ldquoDesign ofmutant1205732subunits as decoy molecules to reduce the expression of func-tional Ca2+ channels in cardiac cellsrdquo Journal of Pharmacologyand Experimental Therapeutics vol 325 no 1 pp 37ndash46 2008

[62] A Goyenvalle A Vulin F Fougerousse et al ldquoRescue of dys-trophic muscle through U7 snRNA-mediated exon skippingrdquoScience vol 306 no 5702 pp 1796ndash1799 2004

[63] D BHalling P Aracena-Parks and S L Hamilton ldquoRegulationof voltage-gated Ca2+ channels by calmodulinrdquo Sciencersquos STKEvol 2005 no 315 p re15 2005

[64] K S Lee E Marban and R W Tsien ldquoInactivation of calciumchannels in mammalian heart cells joint dependence on mem-brane potential and intracellular calciumrdquo Journal of Physiologyvol 364 pp 395ndash411 1985

[65] C Shi and N M Soldatov ldquoMolecular determinants of voltage-dependent slow inactivation of the Ca2+ channelrdquo Journal ofBiological Chemistry vol 277 no 9 pp 6813ndash6821 2002

[66] E Kobrinsky E Schwartz D R Abernethy and NM SoldatovldquoVoltage-gated mobility of the Ca2+ channel cytoplasmic tailsand its regulatory rolerdquo Journal of Biological Chemistry vol 278no 7 pp 5021ndash5028 2003

[67] N M Soldatov ldquoCa2+ channel moving tail link between Ca2+-induced inactivation and Ca2+ signal transductionrdquo Trends inPharmacological Sciences vol 24 no 4 pp 167ndash171 2003

[68] D J Klemm P A Watson M G Frid et al ldquocAMP responseelement-binding protein content is a molecular determinantof smooth muscle cell proliferation and migrationrdquo Journal ofBiological Chemistry vol 276 no 49 pp 46132ndash46141 2001

[69] E Kobrinsky L Stevens Y Kazmi DWray andNM SoldatovldquoMolecular rearrangements of the Kv21 potassium channeltermini associated with voltage gatingrdquo Journal of BiologicalChemistry vol 281 no 28 pp 19233ndash19240 2006

[70] BMMayr G Canettieri andMRMontminy ldquoDistinct effectsof cAMP and mitogenic signals on CREB-binding proteinrecruitment impart specificity to target gene activation viaCREBrdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 98 no 19 pp 10936ndash10941 2001

[71] M Morad and N Soldatov ldquoCalcium channel inactivationpossible role in signal transduction and Ca2+ signalingrdquo CellCalcium vol 38 no 3-4 pp 223ndash231 2005

[72] DGWheeler C F Barrett R DGroth P Safa andRW TsienldquoCaMKII locally encodes L-type channel activity to signal tonuclear CREB in excitation-transcription couplingrdquo Journal ofCell Biology vol 183 no 5 pp 849ndash863 2008

[73] D E Mager E Kobrinsky A Masoudieh A Maltsev D RAbernethy andNM Soldatov ldquoAnalysis of functional signalingdomains from fluorescence imaging and the two-dimensionalcontinuous wavelet transformrdquo Biophysical Journal vol 93 no8 pp 2900ndash2910 2007

[74] E Kobrinsky D E Mager S A Bentil S I Murata DR Abernethy and N M Soldatov ldquoIdentification of plasmamembrane macro- and microdomains from wavelet analysis ofFRET microscopyrdquo Biophysical Journal vol 88 no 5 pp 3625ndash3634 2005

[75] E Kobrinsky S Q Duong A Sheydina and N M Solda-tov ldquoMicrodomain organization and frequency-dependence ofCREB-dependent transcriptional signaling in heart cellsrdquo TheFASEB Journal vol 25 no 5 pp 1544ndash1555 2011

[76] A Ravindran Q Z Lao J B Harry P Abrahimi E Kobrinskyand N M Soldatov ldquoCalmodulin-dependent gating of Cav12calcium channels in the absence of Cav120573 subunitsrdquo Proceedingsof the National Academy of Sciences of the United States ofAmerica vol 105 no 23 pp 8154ndash8159 2008

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

ISRN Biochemistry 11

in a way that may affect cell proliferation and other crucialfunction

7 Conclusions

Association of Cav12 with regulation of transcription cellproliferation and its pathophysiology as in the case ofatherosclerosis requires detailed investigation of the roles ofthe naturally occurring 120572

1C splice variants It will limit thetraditionally intuitive approach to Cav12 in physiology andhelp to define new principle approaches to the treatment ofvarious Cav12 channelopathy-related dysfunctions above allcardiovascular diseases Humgenex Inc provides consultingand logistic support on a broad range of issues reported inthis review

Abbreviations

CaM CalmodulinCBD Calmodulin-binding domainCDI Ca2+-dependent inactivationDHP DihydropyridineECFP Enhanced cyan fluorescent proteinEYFP Enhanced yellow fluorescent proteinFALI Fluorophore-assisted light inactivationFRET Fluorescent resonance energy transfer[3H]PMD 26-Dimethyl-3-methoxycarbonyl-5-([23-

3H2]-n-propoxycarbonyl)-4-(21015840-

difluoromethoxyphenyl)-14-dihydropyridine

119868Ca Calcium currentPH Pleckstrin homology domainVSM Vascular smooth muscle

References

[1] Y S Lee M M Sayeed and R D Wurster ldquoInhibition ofcell growth and intracellular Ca2+ mobilization in humanbrain tumor cells by Ca2+ channel antagonistsrdquo Molecular andChemical Neuropathology vol 22 no 2 pp 81ndash95 1994

[2] K Sato J Ishizuka C W Cooper et al ldquoInhibitory effect ofcalcium channel blockers on growth of pancreatic cancer cellsrdquoPancreas vol 9 no 2 pp 193ndash202 1994

[3] V Bertrand M J Bastie N Vaysse and L Pradayrol ldquoInhibi-tion of gastrin-induced proliferation of AR4-2J cells by calciumchannel antagonistsrdquo International Journal of Cancer vol 56no 3 pp 427ndash432 1994

[4] JM Taylor andRU Simpson ldquoInhibition of cancer cell growthby calcium channel antagonists in the athymic mouserdquo CancerResearch vol 52 no 9 pp 2413ndash2418 1992

[5] M G Cattaneo M Gullo and L M Vicentini ldquoC2+ and Ca2+channel antagonists in the control of human small cell lung car-cinoma cell proliferationrdquo European Journal of Pharmacologyvol 247 no 3 pp 325ndash331 1993

[6] C Chen M J Corbley T M Roberts and P Hess ldquoVoltage-sensitive calcium channels in normal and transformed 3T3fibroblastsrdquo Science vol 239 no 4843 pp 1024ndash1026 1988

[7] P C Dartsch M Ritter M Gschwentner H J Lang andF Lang ldquoEffects of calcium channel blockers on NIH 3T3

fibroblasts expressing the Ha-ras oncogenerdquo European Journalof Cell Biology vol 67 no 4 pp 372ndash378 1995

[8] M Biel R Hullin S Freundner et al ldquoTissue-specific expres-sion of high-voltage-activated dihydropyridine-sensitive L-typecalcium channelsrdquo European Journal of Biochemistry vol 200no 1 pp 81ndash88 1991

[9] DM Rodman K Reese J Harral et al ldquoLow-voltage-activated(T-type) calcium channels control proliferation of human pul-monary artery myocytesrdquo Circulation Research vol 96 no 8pp 864ndash872 2005

[10] A Panner L L Cribbs G M Zainelli T C Origitano S Singhand R DWurster ldquoVariation of T-type calcium channel proteinexpression affects cell division of cultured tumor cellsrdquo CellCalcium vol 37 no 2 pp 105ndash119 2005

[11] C Chen and P Hess ldquoCalcium channels in mouse 3T3 andhuman fibroblastsrdquo Biophysical Journal vol 51 p 226a 1987

[12] N M Soldatov ldquoMolecular diversity of L-type Ca2+ channeltranscripts in human fibroblastsrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 89 no10 pp 4628ndash4632 1992

[13] A Fujii H Matsumoto S Nakao H Teshigawara and YAkimoto ldquoEffect of calcium-channel blockers on cell prolifera-tion DNA synthesis and collagen synthesis of cultured gingivalfibroblasts derived fromhumannifedipine responders and non-respondersrdquo Archives of Oral Biology vol 39 no 2 pp 99ndash1041994

[14] N M Soldatov ldquoGenomic structure of human L-type Ca2+channelrdquo Genomics vol 22 no 1 pp 77ndash87 1994

[15] V P Shevchenko N F Myasoedov N M Soldatov G JDuburs V V Kastron and I P Skrastins ldquoSynthesis andevaluation of biological activity of two novel tritium-labeledderivatives of riodipinerdquo Journal of Labelled Compounds andRadiopharmaceuticals vol 27 no 6 pp 721ndash730 1989

[16] S M Dudkin S N Gnedoj N N Chernyuk and N MSoldatov ldquo14-dihydropyridine receptor associated with Ca2+channels in human embryonic fibroblastsrdquo FEBS Letters vol233 no 2 pp 352ndash354 1988

[17] A DePover M A Matlib S W Lee et al ldquoSpecific bindingof [3H]nitrendipine to membranes from coronary arteriesand heart in relation to pharmacological effects Paradoxicalstimulation by diltiazemrdquo Biochemical and Biophysical ResearchCommunications vol 108 no 1 pp 110ndash117 1982

[18] N M Soldatov S N Gnedoj N N Chernyuk I A Britanovaand S M Dudkin ldquoMitogenesis and 14-dihydropyridinereceptor associated with Ca2+-channels in human embryonicfibroblastsrdquo Journal of Membrane Biology vol 5 pp 1161ndash11671988 (Russian)

[19] D Lipscombe D V Madison M Poenie H Reuter R Y Tsienand R W Tsien ldquoSpatial distribution of calcium channels andcytosolic calcium transients in growth cones and cell bodies ofsympathetic neuronsrdquo Proceedings of the National Academy ofSciences of the United States of America vol 85 no 7 pp 2398ndash2402 1988

[20] G S Harms L Cognet P H M Lommerse et al ldquoSingle-molecule imaging of L-type CA2+ channels in live cellsrdquoBiophysical Journal vol 81 no 5 pp 2639ndash2646 2001

[21] V di Biase G J Obermair Z Szabo et al ldquoStable membraneexpression of postsynaptic Cav12 calcium channel clustersis independent of interactions with AKAP79150 and PDZproteinsrdquo Journal of Neuroscience vol 28 no 51 pp 13845ndash13855 2008

12 ISRN Biochemistry

[22] M F Navedo E P Cheng C Yuan et al ldquoIncreased coupledgating of L-type Ca2+ channels during hypertension and timo-thy syndromerdquoCirculationResearch vol 106 no 4 pp 748ndash7562010

[23] E Kobrinsky P Abrahimi S Q Duong et al ldquoEffect of Cav120573subunits on structural organization of Cav12 calcium channelsrdquoPLoS One vol 4 no 5 Article ID e5587 2009

[24] Q Z Lao E Kobrinsky Z Liu and N M Soldatov ldquoOligomer-ization of Cav120573 subunits is an essential correlate of Ca

2+ channelactivityrdquoThe FASEB Journal vol 24 no 12 pp 5013ndash5023 2010

[25] E Kobrinsky S Tiwari V A Maltsev et al ldquoDifferential roleof the 1205721C subunit tails in regulation of the Cav12 channelby membrane potential 120573 subunits and Ca2+ ionsrdquo Journal ofBiological Chemistry vol 280 no 13 pp 12474ndash12485 2005

[26] S Tiwari Y Zhang J Heller D R Abernethy and N MSoldatov ldquoArtherosclerosis-related molecular alteration of thehuman Cav12 calcium channel 1205721C subunitrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 103 no 45 pp 17024ndash17029 2006

[27] I Splawski K W Timothy L M Sharpe et al ldquoCav12 calciumchannel dysfunction causes a multisystem disorder includingarrhythmia and autismrdquo Cell vol 119 no 1 pp 19ndash31 2004

[28] Z Z TangM C Liang S Lu et al ldquoTranscript scanning revealsnovel and extensive splice variations in human L-type voltage-gated calcium channel Cav12 1205721 subunitrdquo Journal of BiologicalChemistry vol 279 no 43 pp 44335ndash44343 2004

[29] P Liao T F Yong M C Liang D T Yue and T W SoongldquoSplicing for alternative structures of Cav12 Ca2+ channels incardiac and smooth musclesrdquo Cardiovascular Research vol 68no 2 pp 197ndash203 2005

[30] Y Blumenstein N Kanevsky G Sahar R Barzilai T Ivaninaand N Dascal ldquoA novel long N-terminal isoform of human L-type Ca2+ channel is up-regulated by protein kinase Crdquo Journalof Biological Chemistry vol 277 no 5 pp 3419ndash3423 2002

[31] N M Soldatov A Bouron and H Reuter ldquoDifferent voltage-dependent inhibition by dihydropyridines of human Ca2+channel splice variantsrdquo Journal of Biological Chemistry vol270 no 18 pp 10540ndash10543 1995

[32] D P Faxon M A Creager S C Smith et al ldquoAtheroscleroticvascular disease conference executive summary atheroscle-rotic vascular disease conference proceeding for healthcareprofessionals from a special writing group of the AmericanHeart Associationrdquo Circulation vol 109 no 21 pp 2595ndash26042004

[33] R Ross ldquoAtherosclerosismdashan inflammatory diseaserdquo The NewEngland Journal of Medicine vol 340 no 2 pp 115ndash126 1999

[34] H Roy S Bhardwaj and S Yla-Herttuala ldquoMolecular geneticsof atherosclerosisrdquo Human Genetics vol 125 no 5-6 pp 467ndash491 2009

[35] M Biel P Ruth E Bosse et al ldquoPrimary structure and func-tional expression of a high voltage activated calcium channelfrom rabbit lungrdquo FEBS Letters vol 269 no 2 pp 409ndash4121990

[36] X Cheng J Liu M Asuncion-Chin et al ldquoA novel Cav12N terminus expressed in smooth muscle cells of resistancesize arteries modifies channel regulation by auxiliary subunitsrdquoJournal of Biological Chemistry vol 282 no 40 pp 29211ndash292212007

[37] W J Koch P T Ellinor and A Schwartz ldquocDNA cloning ofa dihydropyridine-sensitive calcium channel from rat aortaevidence for the existence of alternatively spliced formsrdquo Journalof Biological Chemistry vol 265 no 29 pp 17786ndash17791 1990

[38] S Richard D Neveu G Carnac P Bodin P Travo and J Nar-geot ldquoDifferential expression of voltage-gated Ca2+-currents incultivated aortic myocytesrdquo Biochimica et Biophysica Acta vol1160 no 1 pp 95ndash104 1992

[39] A Schwartz ldquoCalcium antagonists review and perspective onmechanism of actionrdquo American Journal of Cardiology vol 64no 17 pp 3Indash9I 1989

[40] R P Mason ldquoMechanisms of plaque stabilization for thedihydropyridine calcium channel blocker amlodipine review ofthe evidencerdquo Atherosclerosis vol 165 no 2 pp 191ndash199 2002

[41] A Ruiz-Torres R Lozano J Melon and R Carraro ldquoL-calciumchannel blockade induced by diltiazem inhibits proliferationmigration and F-actin membrane rearrangements in humanvascular smooth muscle cells stimulated with insulin and IGF-1rdquo International Journal of Clinical Pharmacology andTherapeu-tics vol 41 no 9 pp 386ndash391 2003

[42] H Koshiyama S Tanaka and J Minamikawa ldquoEffect ofcalcium channel blocker amlodipine on the intimal-medialthickness of carotid arterial wall in type 2 diabetesrdquo Journal ofCardiovascular Pharmacology vol 33 no 6 pp 894ndash896 1999

[43] T Yamashita S KawashimaMOzaki et al ldquoA calcium channelblocker benidipine inhibits intimal thickening in the carotidartery of mice by increasing nitric oxide productionrdquo Journal ofHypertension vol 19 no 3 pp 451ndash458 2001

[44] R P Mason P Marche and T H Hintze ldquoNovel vascularbiology of third-generation L-type calcium channel antagonistsancillary actions of amlodipinerdquo Arteriosclerosis Thrombosisand Vascular Biology vol 23 no 12 pp 2155ndash2163 2003

[45] O Stepien J GogusevD L Zhu et al ldquoAmlodipine inhibition ofserum- thrombin- or fibroblast growth factor- induced vascu-lar smooth-muscle cell proliferationrdquo Journal of CardiovascularPharmacology vol 31 no 5 pp 786ndash793 1998

[46] V Ruiz-Velasco M B Mayer E W Inscho and L J HymelldquoModulation of dihydropyridine receptors in vascular smoothmuscle cells by membrane potential and cell proliferationrdquoEuropean Journal of Pharmacology vol 268 no 3 pp 311ndash3181994

[47] J Nilsson M Sjolund L Palmberg A M von Euler B Jonzonand J Thyberg ldquoThe calcium antagonist nifedipine inhibitsarterial smooth muscle cell proliferationrdquo Atherosclerosis vol58 no 1ndash3 pp 109ndash122 1985

[48] J Thyberg and L Palmberg ldquoThe calcium antagonist nisoldip-ine and the calmodulin antagonist W-7 synergistically inhibitinitiation of DNA synthesis in cultured arterial smooth musclecellsrdquo Biology of the Cell vol 60 no 2 pp 125ndash132 1987

[49] E Munro M Patel P Chan et al ldquoEffect of calcium channelblockers on the growth of human vascular smooth musclecells derived from saphenous vein and vascular graft stenosesrdquoJournal of Cardiovascular Pharmacology vol 23 no 5 pp 779ndash784 1994

[50] T A Kent A Jazayeri and J M Simard ldquoCalcium channelsand nifedipine inhibition of serotonin-induced [3H]thymidineincorporation in cultured cerebral smoothmuscle cellsrdquo Journalof Cerebral Blood Flow and Metabolism vol 12 no 1 pp 139ndash146 1992

[51] A Agrotis P J Little J Saltis and A Bobik ldquoDihydropyridineCa2+ channel antagonists inhibit the salvage pathway for DNAsynthesis in human vascular smooth muscle cellsrdquo EuropeanJournal of Pharmacology vol 244 no 3 pp 269ndash275 1993

[52] I Duque M R Puyol P Ruiz M Gonzalez-Rubio M LD Marques and D R Puyol ldquoCalcium channel blockers

ISRN Biochemistry 13

inhibit hydrogen peroxide-induced proliferation of culturedrat mesangial cellsrdquo Journal of Pharmacology and ExperimentalTherapeutics vol 267 no 2 pp 612ndash616 1993

[53] T Kuga S Kobayashi Y Hirakawa H Kanaide and ATakeshita ldquoCell cycle-dependent expression of L- and T-typeCa2+ currents in rat aortic smooth muscle cells in primaryculturerdquo Circulation Research vol 79 no 1 pp 14ndash19 1996

[54] E M Graf M Bock J F Heubach et al ldquoTissue distribution ofa humanCav12 1205721 subunit splice variant with a 75 bp insertionrdquoCell Calcium vol 38 no 1 pp 11ndash21 2005

[55] P Liao D Yu S Lu et al ldquoSmoothmuscle-selective alternativelyspliced exon generates functional variation in Cav12 calciumchannelsrdquo Journal of Biological Chemistry vol 279 no 48 pp50329ndash50335 2004

[56] O Skalli P Ropraz and A Trzeciak ldquoA monoclonal antibodyagainst 120572-smooth muscle actin a new probe for smooth muscledifferentiationrdquo Journal of Cell Biology vol 103 no 6 pp 2787ndash2796 1986

[57] T M Doherty K Asotra L A Fitzpatrick et al ldquoCalcificationin atherosclerosis bone biology and chronic inflammation atthe arterial crossroadsrdquo Proceedings of the National Academyof Sciences of the United States of America vol 100 no 20 pp11201ndash11206 2003

[58] G K Owens ldquoRegulation of differentiation of vascular smoothmuscle cellsrdquo Physiological Reviews vol 75 no 3 pp 487ndash5171995

[59] J P Bannister A Adebiyi G Zhao et al ldquoSmooth musclecell 1205722120575-1 subunits are essential for vasoregulation by Cav12channelsrdquo Circulation Research vol 105 no 10 pp 948ndash9552009

[60] S SonkusareM Fraer J DMarsh andN J Rusch ldquoDisruptingcalcium channel expression to lower blood pressure newtargeting of awell-known channelrdquoMolecular Interventions vol6 no 6 pp 304ndash310 2006

[61] S Telemaque S Sonkusare TGrain et al ldquoDesign ofmutant1205732subunits as decoy molecules to reduce the expression of func-tional Ca2+ channels in cardiac cellsrdquo Journal of Pharmacologyand Experimental Therapeutics vol 325 no 1 pp 37ndash46 2008

[62] A Goyenvalle A Vulin F Fougerousse et al ldquoRescue of dys-trophic muscle through U7 snRNA-mediated exon skippingrdquoScience vol 306 no 5702 pp 1796ndash1799 2004

[63] D BHalling P Aracena-Parks and S L Hamilton ldquoRegulationof voltage-gated Ca2+ channels by calmodulinrdquo Sciencersquos STKEvol 2005 no 315 p re15 2005

[64] K S Lee E Marban and R W Tsien ldquoInactivation of calciumchannels in mammalian heart cells joint dependence on mem-brane potential and intracellular calciumrdquo Journal of Physiologyvol 364 pp 395ndash411 1985

[65] C Shi and N M Soldatov ldquoMolecular determinants of voltage-dependent slow inactivation of the Ca2+ channelrdquo Journal ofBiological Chemistry vol 277 no 9 pp 6813ndash6821 2002

[66] E Kobrinsky E Schwartz D R Abernethy and NM SoldatovldquoVoltage-gated mobility of the Ca2+ channel cytoplasmic tailsand its regulatory rolerdquo Journal of Biological Chemistry vol 278no 7 pp 5021ndash5028 2003

[67] N M Soldatov ldquoCa2+ channel moving tail link between Ca2+-induced inactivation and Ca2+ signal transductionrdquo Trends inPharmacological Sciences vol 24 no 4 pp 167ndash171 2003

[68] D J Klemm P A Watson M G Frid et al ldquocAMP responseelement-binding protein content is a molecular determinantof smooth muscle cell proliferation and migrationrdquo Journal ofBiological Chemistry vol 276 no 49 pp 46132ndash46141 2001

[69] E Kobrinsky L Stevens Y Kazmi DWray andNM SoldatovldquoMolecular rearrangements of the Kv21 potassium channeltermini associated with voltage gatingrdquo Journal of BiologicalChemistry vol 281 no 28 pp 19233ndash19240 2006

[70] BMMayr G Canettieri andMRMontminy ldquoDistinct effectsof cAMP and mitogenic signals on CREB-binding proteinrecruitment impart specificity to target gene activation viaCREBrdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 98 no 19 pp 10936ndash10941 2001

[71] M Morad and N Soldatov ldquoCalcium channel inactivationpossible role in signal transduction and Ca2+ signalingrdquo CellCalcium vol 38 no 3-4 pp 223ndash231 2005

[72] DGWheeler C F Barrett R DGroth P Safa andRW TsienldquoCaMKII locally encodes L-type channel activity to signal tonuclear CREB in excitation-transcription couplingrdquo Journal ofCell Biology vol 183 no 5 pp 849ndash863 2008

[73] D E Mager E Kobrinsky A Masoudieh A Maltsev D RAbernethy andNM Soldatov ldquoAnalysis of functional signalingdomains from fluorescence imaging and the two-dimensionalcontinuous wavelet transformrdquo Biophysical Journal vol 93 no8 pp 2900ndash2910 2007

[74] E Kobrinsky D E Mager S A Bentil S I Murata DR Abernethy and N M Soldatov ldquoIdentification of plasmamembrane macro- and microdomains from wavelet analysis ofFRET microscopyrdquo Biophysical Journal vol 88 no 5 pp 3625ndash3634 2005

[75] E Kobrinsky S Q Duong A Sheydina and N M Solda-tov ldquoMicrodomain organization and frequency-dependence ofCREB-dependent transcriptional signaling in heart cellsrdquo TheFASEB Journal vol 25 no 5 pp 1544ndash1555 2011

[76] A Ravindran Q Z Lao J B Harry P Abrahimi E Kobrinskyand N M Soldatov ldquoCalmodulin-dependent gating of Cav12calcium channels in the absence of Cav120573 subunitsrdquo Proceedingsof the National Academy of Sciences of the United States ofAmerica vol 105 no 23 pp 8154ndash8159 2008

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

12 ISRN Biochemistry

[22] M F Navedo E P Cheng C Yuan et al ldquoIncreased coupledgating of L-type Ca2+ channels during hypertension and timo-thy syndromerdquoCirculationResearch vol 106 no 4 pp 748ndash7562010

[23] E Kobrinsky P Abrahimi S Q Duong et al ldquoEffect of Cav120573subunits on structural organization of Cav12 calcium channelsrdquoPLoS One vol 4 no 5 Article ID e5587 2009

[24] Q Z Lao E Kobrinsky Z Liu and N M Soldatov ldquoOligomer-ization of Cav120573 subunits is an essential correlate of Ca

2+ channelactivityrdquoThe FASEB Journal vol 24 no 12 pp 5013ndash5023 2010

[25] E Kobrinsky S Tiwari V A Maltsev et al ldquoDifferential roleof the 1205721C subunit tails in regulation of the Cav12 channelby membrane potential 120573 subunits and Ca2+ ionsrdquo Journal ofBiological Chemistry vol 280 no 13 pp 12474ndash12485 2005

[26] S Tiwari Y Zhang J Heller D R Abernethy and N MSoldatov ldquoArtherosclerosis-related molecular alteration of thehuman Cav12 calcium channel 1205721C subunitrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 103 no 45 pp 17024ndash17029 2006

[27] I Splawski K W Timothy L M Sharpe et al ldquoCav12 calciumchannel dysfunction causes a multisystem disorder includingarrhythmia and autismrdquo Cell vol 119 no 1 pp 19ndash31 2004

[28] Z Z TangM C Liang S Lu et al ldquoTranscript scanning revealsnovel and extensive splice variations in human L-type voltage-gated calcium channel Cav12 1205721 subunitrdquo Journal of BiologicalChemistry vol 279 no 43 pp 44335ndash44343 2004

[29] P Liao T F Yong M C Liang D T Yue and T W SoongldquoSplicing for alternative structures of Cav12 Ca2+ channels incardiac and smooth musclesrdquo Cardiovascular Research vol 68no 2 pp 197ndash203 2005

[30] Y Blumenstein N Kanevsky G Sahar R Barzilai T Ivaninaand N Dascal ldquoA novel long N-terminal isoform of human L-type Ca2+ channel is up-regulated by protein kinase Crdquo Journalof Biological Chemistry vol 277 no 5 pp 3419ndash3423 2002

[31] N M Soldatov A Bouron and H Reuter ldquoDifferent voltage-dependent inhibition by dihydropyridines of human Ca2+channel splice variantsrdquo Journal of Biological Chemistry vol270 no 18 pp 10540ndash10543 1995

[32] D P Faxon M A Creager S C Smith et al ldquoAtheroscleroticvascular disease conference executive summary atheroscle-rotic vascular disease conference proceeding for healthcareprofessionals from a special writing group of the AmericanHeart Associationrdquo Circulation vol 109 no 21 pp 2595ndash26042004

[33] R Ross ldquoAtherosclerosismdashan inflammatory diseaserdquo The NewEngland Journal of Medicine vol 340 no 2 pp 115ndash126 1999

[34] H Roy S Bhardwaj and S Yla-Herttuala ldquoMolecular geneticsof atherosclerosisrdquo Human Genetics vol 125 no 5-6 pp 467ndash491 2009

[35] M Biel P Ruth E Bosse et al ldquoPrimary structure and func-tional expression of a high voltage activated calcium channelfrom rabbit lungrdquo FEBS Letters vol 269 no 2 pp 409ndash4121990

[36] X Cheng J Liu M Asuncion-Chin et al ldquoA novel Cav12N terminus expressed in smooth muscle cells of resistancesize arteries modifies channel regulation by auxiliary subunitsrdquoJournal of Biological Chemistry vol 282 no 40 pp 29211ndash292212007

[37] W J Koch P T Ellinor and A Schwartz ldquocDNA cloning ofa dihydropyridine-sensitive calcium channel from rat aortaevidence for the existence of alternatively spliced formsrdquo Journalof Biological Chemistry vol 265 no 29 pp 17786ndash17791 1990

[38] S Richard D Neveu G Carnac P Bodin P Travo and J Nar-geot ldquoDifferential expression of voltage-gated Ca2+-currents incultivated aortic myocytesrdquo Biochimica et Biophysica Acta vol1160 no 1 pp 95ndash104 1992

[39] A Schwartz ldquoCalcium antagonists review and perspective onmechanism of actionrdquo American Journal of Cardiology vol 64no 17 pp 3Indash9I 1989

[40] R P Mason ldquoMechanisms of plaque stabilization for thedihydropyridine calcium channel blocker amlodipine review ofthe evidencerdquo Atherosclerosis vol 165 no 2 pp 191ndash199 2002

[41] A Ruiz-Torres R Lozano J Melon and R Carraro ldquoL-calciumchannel blockade induced by diltiazem inhibits proliferationmigration and F-actin membrane rearrangements in humanvascular smooth muscle cells stimulated with insulin and IGF-1rdquo International Journal of Clinical Pharmacology andTherapeu-tics vol 41 no 9 pp 386ndash391 2003

[42] H Koshiyama S Tanaka and J Minamikawa ldquoEffect ofcalcium channel blocker amlodipine on the intimal-medialthickness of carotid arterial wall in type 2 diabetesrdquo Journal ofCardiovascular Pharmacology vol 33 no 6 pp 894ndash896 1999

[43] T Yamashita S KawashimaMOzaki et al ldquoA calcium channelblocker benidipine inhibits intimal thickening in the carotidartery of mice by increasing nitric oxide productionrdquo Journal ofHypertension vol 19 no 3 pp 451ndash458 2001

[44] R P Mason P Marche and T H Hintze ldquoNovel vascularbiology of third-generation L-type calcium channel antagonistsancillary actions of amlodipinerdquo Arteriosclerosis Thrombosisand Vascular Biology vol 23 no 12 pp 2155ndash2163 2003

[45] O Stepien J GogusevD L Zhu et al ldquoAmlodipine inhibition ofserum- thrombin- or fibroblast growth factor- induced vascu-lar smooth-muscle cell proliferationrdquo Journal of CardiovascularPharmacology vol 31 no 5 pp 786ndash793 1998

[46] V Ruiz-Velasco M B Mayer E W Inscho and L J HymelldquoModulation of dihydropyridine receptors in vascular smoothmuscle cells by membrane potential and cell proliferationrdquoEuropean Journal of Pharmacology vol 268 no 3 pp 311ndash3181994

[47] J Nilsson M Sjolund L Palmberg A M von Euler B Jonzonand J Thyberg ldquoThe calcium antagonist nifedipine inhibitsarterial smooth muscle cell proliferationrdquo Atherosclerosis vol58 no 1ndash3 pp 109ndash122 1985

[48] J Thyberg and L Palmberg ldquoThe calcium antagonist nisoldip-ine and the calmodulin antagonist W-7 synergistically inhibitinitiation of DNA synthesis in cultured arterial smooth musclecellsrdquo Biology of the Cell vol 60 no 2 pp 125ndash132 1987

[49] E Munro M Patel P Chan et al ldquoEffect of calcium channelblockers on the growth of human vascular smooth musclecells derived from saphenous vein and vascular graft stenosesrdquoJournal of Cardiovascular Pharmacology vol 23 no 5 pp 779ndash784 1994

[50] T A Kent A Jazayeri and J M Simard ldquoCalcium channelsand nifedipine inhibition of serotonin-induced [3H]thymidineincorporation in cultured cerebral smoothmuscle cellsrdquo Journalof Cerebral Blood Flow and Metabolism vol 12 no 1 pp 139ndash146 1992

[51] A Agrotis P J Little J Saltis and A Bobik ldquoDihydropyridineCa2+ channel antagonists inhibit the salvage pathway for DNAsynthesis in human vascular smooth muscle cellsrdquo EuropeanJournal of Pharmacology vol 244 no 3 pp 269ndash275 1993

[52] I Duque M R Puyol P Ruiz M Gonzalez-Rubio M LD Marques and D R Puyol ldquoCalcium channel blockers

ISRN Biochemistry 13

inhibit hydrogen peroxide-induced proliferation of culturedrat mesangial cellsrdquo Journal of Pharmacology and ExperimentalTherapeutics vol 267 no 2 pp 612ndash616 1993

[53] T Kuga S Kobayashi Y Hirakawa H Kanaide and ATakeshita ldquoCell cycle-dependent expression of L- and T-typeCa2+ currents in rat aortic smooth muscle cells in primaryculturerdquo Circulation Research vol 79 no 1 pp 14ndash19 1996

[54] E M Graf M Bock J F Heubach et al ldquoTissue distribution ofa humanCav12 1205721 subunit splice variant with a 75 bp insertionrdquoCell Calcium vol 38 no 1 pp 11ndash21 2005

[55] P Liao D Yu S Lu et al ldquoSmoothmuscle-selective alternativelyspliced exon generates functional variation in Cav12 calciumchannelsrdquo Journal of Biological Chemistry vol 279 no 48 pp50329ndash50335 2004

[56] O Skalli P Ropraz and A Trzeciak ldquoA monoclonal antibodyagainst 120572-smooth muscle actin a new probe for smooth muscledifferentiationrdquo Journal of Cell Biology vol 103 no 6 pp 2787ndash2796 1986

[57] T M Doherty K Asotra L A Fitzpatrick et al ldquoCalcificationin atherosclerosis bone biology and chronic inflammation atthe arterial crossroadsrdquo Proceedings of the National Academyof Sciences of the United States of America vol 100 no 20 pp11201ndash11206 2003

[58] G K Owens ldquoRegulation of differentiation of vascular smoothmuscle cellsrdquo Physiological Reviews vol 75 no 3 pp 487ndash5171995

[59] J P Bannister A Adebiyi G Zhao et al ldquoSmooth musclecell 1205722120575-1 subunits are essential for vasoregulation by Cav12channelsrdquo Circulation Research vol 105 no 10 pp 948ndash9552009

[60] S SonkusareM Fraer J DMarsh andN J Rusch ldquoDisruptingcalcium channel expression to lower blood pressure newtargeting of awell-known channelrdquoMolecular Interventions vol6 no 6 pp 304ndash310 2006

[61] S Telemaque S Sonkusare TGrain et al ldquoDesign ofmutant1205732subunits as decoy molecules to reduce the expression of func-tional Ca2+ channels in cardiac cellsrdquo Journal of Pharmacologyand Experimental Therapeutics vol 325 no 1 pp 37ndash46 2008

[62] A Goyenvalle A Vulin F Fougerousse et al ldquoRescue of dys-trophic muscle through U7 snRNA-mediated exon skippingrdquoScience vol 306 no 5702 pp 1796ndash1799 2004

[63] D BHalling P Aracena-Parks and S L Hamilton ldquoRegulationof voltage-gated Ca2+ channels by calmodulinrdquo Sciencersquos STKEvol 2005 no 315 p re15 2005

[64] K S Lee E Marban and R W Tsien ldquoInactivation of calciumchannels in mammalian heart cells joint dependence on mem-brane potential and intracellular calciumrdquo Journal of Physiologyvol 364 pp 395ndash411 1985

[65] C Shi and N M Soldatov ldquoMolecular determinants of voltage-dependent slow inactivation of the Ca2+ channelrdquo Journal ofBiological Chemistry vol 277 no 9 pp 6813ndash6821 2002

[66] E Kobrinsky E Schwartz D R Abernethy and NM SoldatovldquoVoltage-gated mobility of the Ca2+ channel cytoplasmic tailsand its regulatory rolerdquo Journal of Biological Chemistry vol 278no 7 pp 5021ndash5028 2003

[67] N M Soldatov ldquoCa2+ channel moving tail link between Ca2+-induced inactivation and Ca2+ signal transductionrdquo Trends inPharmacological Sciences vol 24 no 4 pp 167ndash171 2003

[68] D J Klemm P A Watson M G Frid et al ldquocAMP responseelement-binding protein content is a molecular determinantof smooth muscle cell proliferation and migrationrdquo Journal ofBiological Chemistry vol 276 no 49 pp 46132ndash46141 2001

[69] E Kobrinsky L Stevens Y Kazmi DWray andNM SoldatovldquoMolecular rearrangements of the Kv21 potassium channeltermini associated with voltage gatingrdquo Journal of BiologicalChemistry vol 281 no 28 pp 19233ndash19240 2006

[70] BMMayr G Canettieri andMRMontminy ldquoDistinct effectsof cAMP and mitogenic signals on CREB-binding proteinrecruitment impart specificity to target gene activation viaCREBrdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 98 no 19 pp 10936ndash10941 2001

[71] M Morad and N Soldatov ldquoCalcium channel inactivationpossible role in signal transduction and Ca2+ signalingrdquo CellCalcium vol 38 no 3-4 pp 223ndash231 2005

[72] DGWheeler C F Barrett R DGroth P Safa andRW TsienldquoCaMKII locally encodes L-type channel activity to signal tonuclear CREB in excitation-transcription couplingrdquo Journal ofCell Biology vol 183 no 5 pp 849ndash863 2008

[73] D E Mager E Kobrinsky A Masoudieh A Maltsev D RAbernethy andNM Soldatov ldquoAnalysis of functional signalingdomains from fluorescence imaging and the two-dimensionalcontinuous wavelet transformrdquo Biophysical Journal vol 93 no8 pp 2900ndash2910 2007

[74] E Kobrinsky D E Mager S A Bentil S I Murata DR Abernethy and N M Soldatov ldquoIdentification of plasmamembrane macro- and microdomains from wavelet analysis ofFRET microscopyrdquo Biophysical Journal vol 88 no 5 pp 3625ndash3634 2005

[75] E Kobrinsky S Q Duong A Sheydina and N M Solda-tov ldquoMicrodomain organization and frequency-dependence ofCREB-dependent transcriptional signaling in heart cellsrdquo TheFASEB Journal vol 25 no 5 pp 1544ndash1555 2011

[76] A Ravindran Q Z Lao J B Harry P Abrahimi E Kobrinskyand N M Soldatov ldquoCalmodulin-dependent gating of Cav12calcium channels in the absence of Cav120573 subunitsrdquo Proceedingsof the National Academy of Sciences of the United States ofAmerica vol 105 no 23 pp 8154ndash8159 2008

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

ISRN Biochemistry 13

inhibit hydrogen peroxide-induced proliferation of culturedrat mesangial cellsrdquo Journal of Pharmacology and ExperimentalTherapeutics vol 267 no 2 pp 612ndash616 1993

[53] T Kuga S Kobayashi Y Hirakawa H Kanaide and ATakeshita ldquoCell cycle-dependent expression of L- and T-typeCa2+ currents in rat aortic smooth muscle cells in primaryculturerdquo Circulation Research vol 79 no 1 pp 14ndash19 1996

[54] E M Graf M Bock J F Heubach et al ldquoTissue distribution ofa humanCav12 1205721 subunit splice variant with a 75 bp insertionrdquoCell Calcium vol 38 no 1 pp 11ndash21 2005

[55] P Liao D Yu S Lu et al ldquoSmoothmuscle-selective alternativelyspliced exon generates functional variation in Cav12 calciumchannelsrdquo Journal of Biological Chemistry vol 279 no 48 pp50329ndash50335 2004

[56] O Skalli P Ropraz and A Trzeciak ldquoA monoclonal antibodyagainst 120572-smooth muscle actin a new probe for smooth muscledifferentiationrdquo Journal of Cell Biology vol 103 no 6 pp 2787ndash2796 1986

[57] T M Doherty K Asotra L A Fitzpatrick et al ldquoCalcificationin atherosclerosis bone biology and chronic inflammation atthe arterial crossroadsrdquo Proceedings of the National Academyof Sciences of the United States of America vol 100 no 20 pp11201ndash11206 2003

[58] G K Owens ldquoRegulation of differentiation of vascular smoothmuscle cellsrdquo Physiological Reviews vol 75 no 3 pp 487ndash5171995

[59] J P Bannister A Adebiyi G Zhao et al ldquoSmooth musclecell 1205722120575-1 subunits are essential for vasoregulation by Cav12channelsrdquo Circulation Research vol 105 no 10 pp 948ndash9552009

[60] S SonkusareM Fraer J DMarsh andN J Rusch ldquoDisruptingcalcium channel expression to lower blood pressure newtargeting of awell-known channelrdquoMolecular Interventions vol6 no 6 pp 304ndash310 2006

[61] S Telemaque S Sonkusare TGrain et al ldquoDesign ofmutant1205732subunits as decoy molecules to reduce the expression of func-tional Ca2+ channels in cardiac cellsrdquo Journal of Pharmacologyand Experimental Therapeutics vol 325 no 1 pp 37ndash46 2008

[62] A Goyenvalle A Vulin F Fougerousse et al ldquoRescue of dys-trophic muscle through U7 snRNA-mediated exon skippingrdquoScience vol 306 no 5702 pp 1796ndash1799 2004

[63] D BHalling P Aracena-Parks and S L Hamilton ldquoRegulationof voltage-gated Ca2+ channels by calmodulinrdquo Sciencersquos STKEvol 2005 no 315 p re15 2005

[64] K S Lee E Marban and R W Tsien ldquoInactivation of calciumchannels in mammalian heart cells joint dependence on mem-brane potential and intracellular calciumrdquo Journal of Physiologyvol 364 pp 395ndash411 1985

[65] C Shi and N M Soldatov ldquoMolecular determinants of voltage-dependent slow inactivation of the Ca2+ channelrdquo Journal ofBiological Chemistry vol 277 no 9 pp 6813ndash6821 2002

[66] E Kobrinsky E Schwartz D R Abernethy and NM SoldatovldquoVoltage-gated mobility of the Ca2+ channel cytoplasmic tailsand its regulatory rolerdquo Journal of Biological Chemistry vol 278no 7 pp 5021ndash5028 2003

[67] N M Soldatov ldquoCa2+ channel moving tail link between Ca2+-induced inactivation and Ca2+ signal transductionrdquo Trends inPharmacological Sciences vol 24 no 4 pp 167ndash171 2003

[68] D J Klemm P A Watson M G Frid et al ldquocAMP responseelement-binding protein content is a molecular determinantof smooth muscle cell proliferation and migrationrdquo Journal ofBiological Chemistry vol 276 no 49 pp 46132ndash46141 2001

[69] E Kobrinsky L Stevens Y Kazmi DWray andNM SoldatovldquoMolecular rearrangements of the Kv21 potassium channeltermini associated with voltage gatingrdquo Journal of BiologicalChemistry vol 281 no 28 pp 19233ndash19240 2006

[70] BMMayr G Canettieri andMRMontminy ldquoDistinct effectsof cAMP and mitogenic signals on CREB-binding proteinrecruitment impart specificity to target gene activation viaCREBrdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 98 no 19 pp 10936ndash10941 2001

[71] M Morad and N Soldatov ldquoCalcium channel inactivationpossible role in signal transduction and Ca2+ signalingrdquo CellCalcium vol 38 no 3-4 pp 223ndash231 2005

[72] DGWheeler C F Barrett R DGroth P Safa andRW TsienldquoCaMKII locally encodes L-type channel activity to signal tonuclear CREB in excitation-transcription couplingrdquo Journal ofCell Biology vol 183 no 5 pp 849ndash863 2008

[73] D E Mager E Kobrinsky A Masoudieh A Maltsev D RAbernethy andNM Soldatov ldquoAnalysis of functional signalingdomains from fluorescence imaging and the two-dimensionalcontinuous wavelet transformrdquo Biophysical Journal vol 93 no8 pp 2900ndash2910 2007

[74] E Kobrinsky D E Mager S A Bentil S I Murata DR Abernethy and N M Soldatov ldquoIdentification of plasmamembrane macro- and microdomains from wavelet analysis ofFRET microscopyrdquo Biophysical Journal vol 88 no 5 pp 3625ndash3634 2005

[75] E Kobrinsky S Q Duong A Sheydina and N M Solda-tov ldquoMicrodomain organization and frequency-dependence ofCREB-dependent transcriptional signaling in heart cellsrdquo TheFASEB Journal vol 25 no 5 pp 1544ndash1555 2011

[76] A Ravindran Q Z Lao J B Harry P Abrahimi E Kobrinskyand N M Soldatov ldquoCalmodulin-dependent gating of Cav12calcium channels in the absence of Cav120573 subunitsrdquo Proceedingsof the National Academy of Sciences of the United States ofAmerica vol 105 no 23 pp 8154ndash8159 2008

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology