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Cell Calcium 48 (2010) 183–194

Contents lists available at ScienceDirect

Cell Calcium

journa l homepage: www.e lsev ier .com/ locate /ceca

ormation of N-type (Cav2.2) voltage-gated calcium channel membraneicrodomains: Lipid raft association and clustering

hilip Robinson, Sarah Etheridge, Lele Song, Paul Armenise, Owen T. Jones ∗, Elizabeth M. Fitzgerald ∗∗

aculty of Life Sciences, University of Manchester, Core Technology Facility, 46 Grafton Street, Manchester, M13 9NT, United Kingdom

r t i c l e i n f o

rticle history:eceived 6 August 2010ccepted 12 August 2010

eywords:alcium channelargeting

a b s t r a c t

Voltage-gated calcium channels (Cavs) comprise a pore-forming �1 with auxiliary �2� and � subunitswhich modulate Cav function and surface expression. Cav�1 and �2� are present in signalling complexestermed lipid rafts but it is unclear whether �2� is obligatory for targeting Cavs to rafts or to what extentthis influences cell surface organisation of Cavs. Here, we have used imaging, biochemistry and elec-trophysiology to determine localisation and raft-partitioning of WT and functionally active HA-epitopetagged �2�-1 and Cav2.2 subunits expressed in COS-7 cells. We show that �2�-1 not only partitions intolipid rafts itself but also mediates raft-partitioning of Cav2.2/�1b complexes. Cav�2�-1, Cav2.2/�1b and

istribution Cav2.2/�1b/�2�-1 complexes are all organised into cell surface clusters although only in the presence of�2�-1 do they co-localise with raft markers, caveolin and flotillin. Such clusters persist in the presenceof 3-methyl-�-cyclodextrin even though the raft markers disperse. However, clustering is profoundlysensitive to disruption of the actin-based cytoskeleton by cytochalasin-D. We conclude that �2�-1, andlikely other �2� subunits, is necessary and sufficient for targeting Cavs to lipid rafts. However, forma-

g “hots, st

tion of clusters supportincontaining raft componen

. Introduction

Voltage-gated calcium channels (Cavs) represent a pivotal locusor coupling changes in cell membrane excitability to the intracellu-

ar calcium signalling events that underlie processes ranging fromeurotransmitter release to gene expression [1]. As such, dysfunc-ion in Cavs acquired through genetic manipulation [2], or naturally3], can cause severe phenotypes including blindness [4], myopa-

Abbreviations: Cav, voltage-gated calcium channel; DAPI, 4′ ,6-diamidino-2-henylindole; DMEM, Dulbecco’s modified Eagle’s medium; DRG, dorsal rootanglion; DRM, detergent-resistant membrane; ECM, extracellular matrix; GFP,reen fluorescent protein; GPI, glycophosphatidylinositol; HA, haemagglutinin;CA, intensity correlation analysis; ICQ, intensity correlation quotient; MBS, Mes-uffered saline; MEM, modified Eagle’s medium; M-�-CD, �-methylcyclodextrin;IDAS, metal ion-dependent adhesion site; PDM, product of the differences from theean; RIPA, radio immunoprecipitation assay; SNK, Student–Newman–Keuls; TBS,

ris-buffered saline; TTBS, TBS containing 0.1% Tween-20; VWA, Von Willebrandactor A; WT, wild type.∗ Corresponding author at: Faculty of Life Sciences, University of Manchester,

.234 Core Technology Facility, 46 Grafton Street, Manchester, M13 9NT, Unitedingdom.∗∗ Corresponding author at: Faculty of Life Sciences, University of Manchester,.14A Core Technology Facility, 46 Grafton Street, Manchester, M13 9NT, Unitedingdom. Tel.: +44 161 275 5495; fax: +44 161 275 5600.

E-mail addresses: Owen.t.jones@manchester.ac.uk (O.T. Jones),lizabeth.m.fitzgerald@manchester.ac.uk/ (E.M. Fitzgerald).

143-4160/$ – see front matter © 2010 Elsevier Ltd. All rights reserved.oi:10.1016/j.ceca.2010.08.006

tspots” of Cav activity requires aggregation of macromolecular complexesabilised by interactions with the cytoskeleton.

© 2010 Elsevier Ltd. All rights reserved.

thy [5] and epilepsy [6]. Conversely, pharmacological modulationof Cavs is a mainstay for treating heart disease and a growingnumber of other disorders, including epilepsy and neuropathicpain [7]. Nevertheless, despite their therapeutic potential, muchof the biology of Cavs remains poorly understood, especially atthe cellular level. Minimally, high voltage-activated Cavs are com-prised of a pore-forming �1 subunit (190–250 kDa) complexed as a1:1:1 heteromer with modulatory, cytoplasmic � (52–62 kDa) andtransmembranous �2� (125 kDa) subunits [1,8]. Molecular biolog-ical and electrophysiological studies have identified considerablediversity in the biophysical and pharmacological properties of Cavsowing to their extensive molecular heterogeneity. Each subunit isencoded by multiple genes (10 �1, 4 �, 4 �2�; 8), many with alter-native RNA splice variants, that exhibit specific, often overlapping,spatio-temporal patterns of cell expression [9]. Such differentia-tion in specific Cav subunit combinations enables the fine tuningof select functionalities of individual channels to the signallingdemands of discrete sub-cellular compartments [10]. Indeed, Cavsin diverse cell types often exhibit a non-random distribution com-mensurate with ‘hotspots’ of Cav activity detected in functionalassays [11,12]. Owing to the limited range over which calcium and

other regulatory signals usually occur, such hotspots are thoughtto be necessary for the compartmentalisation, amplification andmodulation of bi-directional signalling via and to Cavs [13–16]. Adefinition of the mechanisms that govern Cav expression and dis-tribution within cells is thus, not only key to understanding the

184 P. Robinson et al. / Cell Calcium 48 (2010) 183–194

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ig. 1. Schematic depiction of the primary structure of the Cav �2�-1 subunit. Aminotart site. Cleavage of the signal peptide (SP) at position 25 and additional cleavageSS) to a smaller � subunit, tethered to the plasma membrane via a single transmemre the conserved Von Willebrand Factor A (VWA) domain and the site into which t

hysiological roles of these channels, but also crucial to the devel-pment of a more rational pharmacology of Cavs [2,7,9,16–18].

In this context, one subunit, �2�, is now emerging as a key butoorly defined player [19]. Functionally, co-expression with �2�nhances the peak current densities of various Cav�1/� complexesy ∼2–4-fold [20–22] and modulates the voltage-dependence andates of current activation and inactivation [19]. Recent evidenceuggests that �2� subunits also promote anterograde traffickingnd cell surface stabilisation of Cav complexes [22–25]. However,iven the similarity of these effects with those of the � subunit, itas been unclear exactly why the �2� subunit is necessary. Clini-ally, �2� subunits are implicated in epilepsy [23], neuropathic pain26] and possibly as human tumour suppressors [27]. Importantly,2�-1 and �2�-2 have recently and unequivocally been shown to be

he primary targets of gabapentinoid drugs used in the treatmentf epilepsy and chronic/neuropathic pain [17,22]. However, theechanism by which gabapentinoids act on Cavs remains unclear

wing to the paucity of information on precisely how �2� subunitsunction.

During biosynthesis, �2� subunits are generated as largerecursor proteins which undergo post-translational cleavage, oxi-ation and glycosylation to yield mature subunits comprised ofisulphide-linked �2 and � glycopolypeptides. Originally thoughto have a classical type I transmembrane topology, recent evidenceuggests that membrane attachment of �2� is conferred by a GPInchor proposed to mediate its association with lipid rafts (Fig. 1)19,28]. In support of this �2� subunits have been shown to betrongly enriched in detergent insoluble cholesterol-rich lipid raftshen expressed either alone or with Cav2.x/� complexes, and to

o-immunoprecipitate with raft associated proteins of the SPFHstomatin/prohibitin/flotillin/HflK) family [19,28]. In intact cells,epletion or enhancement of cholesterol, a major raft constituent,as been shown to modulate Cav2.1 [19] and Cav2.2 [29] currents,uggesting a functional role for lipid raft association. However,lthough both Cav �1 and �2� subunits appear present in lipidafts, there is no evidence to date, for a direct role of �2� sub-nits in targeting Cav channels to rafts. Indeed, for at least onehannel, Cav1.2, raft targeting may be specified by the �1 subunit.n addition, there has been much controversy regarding the sizend organisational dynamics of lipid rafts in biological membranes.ndeed, there is now direct evidence that rafts may be comprisedf a limited number of lipids and proteins, at the margins of opti-al resolution by conventional microscopy. Thus, the complexesbserved in living cells may constitute higher order complexes ofaft components formed and stabilised through protein–protein,ather than lipid–protein, interactions. These observations raisewo key questions. Specifically, is the �2� subunit a pre-requisiteor the concentration of Cavs into lipid rafts and to what extent is

his integral to the organisation of Cav complexes at the cell surface?

To address the above questions, we have combined imaging,iochemical and electrophysiological approaches and determinedhe localisation and raft-partitioning of WT and functionally activeA-epitope tagged �2�-1 and Cav2.2 subunits in transiently trans-

re numbered (rat sequence: Genbank Accession NM 012919.2) from the translationidation events, yield an extracellular polypeptide (�2) linked via disulphide bondsspanning domain (TM) or glycophosphatidylinositol (GPI) anchor [28]. Also shownemagglutinin (HA) epitope tag was inserted.

fected COS-7 cells. We show that �2�-1 subunits not only partitioninto lipid rafts but crucially, also mediate the raft-partitioning ofCav2.2/�1b complexes. Depletion of membrane cholesterol with 3-methyl-�-cyclodextrin (M-�-CD) disrupts the targeting of �2�-1subunits and Cav2.2/�1b/�2�-1 complexes to lipid rafts. In imagingexperiments we find that �2�-1 subunits are present in clustersat the surface of both COS-7 cells and native dorsal root gan-glion neurons. Through extensive heterologous expression studieswe show that �2�-1, Cav2.2/�1b and Cav2.2/�1b/�2�-1 complexesare all organised into clusters at the cell surface. However, onlyin the presence of �2�-1-do these clusters co-localise with theraft marker proteins, caveolin and flotillin. Notably, such clus-ters persist following pre-treatment of cells with M-�-CD, eventhough the raft markers are dispersed. However, clustering isprofoundly sensitive to disruption of the actin-based cytoskele-ton following pre-treatment with cytochalasin-D. Finally, M-�-CDcauses a reduction in current density of Cav2.2/�1b/�2�-1 but notCav2.2/�1b complexes. Thus, we conclude that �2�-1, and likelyother �2� subunits, is both necessary and sufficient for the tar-geting of Cavs to lipid rafts. However, the formation of clustersto support physiologically relevant hotspots of calcium channelactivity is likely to require the formation of macromolecular aggre-gates containing raft components stabilised by interactions withthe cytoskeleton.

2. Materials and methods

2.1. Materials

The construct encoding wild-type rat Cav�2�-1 (Neuronal splicevariant; Genbank accession number: NM 012919.2) in pcDNA3.1was supplied by T.P. Snutch (Univ. British Columbia, Canada).Rabbit Cav2.2 in pMT2 (D14157), rat Cav�1b in pMT2 (X61394)and the mut-3 variant of GFP-pMT2 (U73901) were supplied byA.C. Dolphin (University College London, UK). The HA-Cav2.2 con-struct was a gift from E. Bourinet (Institut Génomique Fonctionelle,CNRS, Montpellier, France) [30]. The pcDNA3.1 plasmid wasobtained from Invitrogen, UK. Primary antibodies were obtainedfrom the following sources: anti-�2�-1 (Upstate/Millipore, UK),anti-caveolin (BD Biosciences, UK), anti-flotillin-1, anti-clathrin,anti-SNAP-25 and anti-GFP (Sigma–Aldrich, UK) and anti-HA(Covance, UK). Secondary antibodies were obtained as fol-lows: FITC-conjugated anti-rabbit and anti-mouse IgGs (JacksonImmunoresearch, UK), Cy5-conjugated anti-mouse and anti-rabbitIgG (Jackson Immunoresearch, UK) and horseradish peroxidase(HRP)-conjugated anti-rabbit and anti-mouse IgGs (Dako, UK). Allother reagents were obtained from Sigma–Aldrich, UK, unless oth-erwise stated.

2.2. Molecular biology

An �2�-1 construct bearing an HA-epitope tag between aminoacid residues I612 and K613, was generated using a three step

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P. Robinson et al. / Cell

trategy. First, two PCR reactions were performed using the �2�--WT construct as template and the following forward and reverserimers: Reaction 1: A2DHAPCR1-F (5′-CTG GAA CTC TAC CGG TCTCA ACG TCA C-3′) and A2DHAPCR1-R (5′-AGC GTA ATC TGG AACTC GTA TGG GTA TAT ATA GTA AAA ACT GTA GG–3′). Reaction: A2DHAPCR2-F (5′-TAC CCA TAC GAT GTT CCA GAT TAC GCTAA GCC AAA ATA GAA GAG AC-3′) and A2DHAPCR2-R (5′-CAATG CAG GCT TAA GAA GTT TTC CTT GGA-3′). The two PCR prod-cts, containing overlapping HA tag sequences, were then mixednd subjected to a third PCR reaction using the A2DHAPCR1-F and2DHAPCR2-R primers. The resulting 1021 bp PCR product was

hen cloned via its flanking sequences into AgeI and AflII (Roche,K) digested WT-�2�-1 using the In-Fusion Dry-Down PCR Cloningit (TakaraBio/Clontech, UK).

.3. Cell culture and transient transfection

COS-7 cells (European Cell Culture Collection, Health Pro-ection Agency, UK), were maintained at 37 ◦C, 5% CO2 inulbecco’s modified Eagle’s medium (DMEM) containing 10% foetalovine serum (Gibco/Invitrogen, Paisley, UK) plus 50 U ml−1 peni-illin/50 �g ml−1 streptomycin (PAA Laboratories, UK). Cells foruorescence microscopy or electrophysiology were seeded into 6-ell plates or 35 mm dishes and transfected with 2 �g total DNA.ells for biochemical experiments were seeded in 10 cm dishesnd transfected with 12 �g total DNA. Transient transfections wereerformed in serum-free DMEM at a cell confluency of 60–70%sing either FuGene 6 (Roche Applied Science, Burgess Hill, UK)or electrophysiology, or, Turbofect reagent (Fermentas, UK) foriochemistry/imaging (DNA:reagent ratio of 1:3 (w/v)). Transfec-ions with Cav2.2, Cav�1b and Cav�2�-1 used a ratio of 3:1:1 by

ass of DNA. Transfections omitting �2�-1 used empty pcDNA3.1ector as a substitute to maintain the equivalent mass of DNA.ells for fluorescence microscopy were cultured and transfecteds above, replacing the DMEM with �-MEM (Invitrogen/GIBCO,K). After transfection, cells were maintained at 37 ◦C, 5% CO2 for8 h until they were either fixed for fluorescence imaging or re-lated for electrophysiological analysis. Cells were re-plated using aon-enzymatic cell dissociation solution (Sigma–Aldrich, UK), andaintained at 29 ◦C for 1–4 h before making current recordings.dult rat dorsal root ganglion neurons were provided by Dr. N.ardiner (University of Manchester).

.4. Western immunoblotting

At 48 h post-transfection, COS-7 cells were washed in PBSnd lysed at 4 ◦C in a RIPA buffer with Complete MINI EDTA-ree protease inhibitor cocktail (Roche, UK). The cell lysatesere then passed through a 22-gauge syringe needle 10 times

o shear genomic DNA, and centrifuged at 1000 × gav. Super-atants were then incubated at 37 ◦C for 15 min with Laemmli

oading buffer containing 20 mM DTT and then heated to 95 ◦Cor 2 min. Sample proteins were resolved by SDS-PAGE on 10%ris–HCl gels for 80 min at 160 V (Mini-Protean cell, BioRad,K) and then transferred by electrophoresis (100 V for 2 h) ontoitrocellulose membranes (Whatman, UK). Air dried membranesere immersed overnight in blocking buffer (5% non-fat dryilk in Tris-buffered saline (TBS) with 0.1% Tween-20 (TTBS)),ashed three times with TTBS and then incubated with the

ppropriate primary antibody in TTBS for 1 h at 20 ◦C. The mem-

ranes were then re-washed with TTBS and incubated for 1 ht 20 ◦C with the appropriate secondary HRP-conjugated anti-ody (1:1000) in TTBS. After further washing with TTBS, theembranes were treated with Western Lightning enhanced

hemiluminescence reagent (PerkinElmer, UK) and immunore-

m 48 (2010) 183–194 185

active proteins detected by exposure to film (GE Life Sciences,UK).

2.5. Sucrose gradient fractionation

Transiently transfected COS-7 cells were washed in PBS andlysed 48 h post-transfection with MBS (Mes-buffered saline: 25 mMMes, pH 6.5, 150 mM NaCl) with 1% Triton-X-100 at 4 ◦C. For asingle experiment, 9 cm × 10 cm dishes were used and 150 �l ofMBS/Triton-X-100 was added to lyse the cells. Cells were scrapedoff the dish and passed through a 22-gauge needle 10 times toshear genomic DNA. 450 �l of lysate was reserved for use as a con-trol. The remaining 900 �l of lysate was mixed with 900 �l of 90%sucrose/MBS (w/v), placed in a 5 ml polypropylene centrifuge tube(Sorvall) and carefully overlaid with 1.5 ml of 30% sucrose/MBS,followed by 1.5 ml of 5% sucrose/MBS. Gradients were spun at38,500 rpm (140,000 × gav) in a Sorvall Discovery 100SE ultracen-trifuge using an AH-650 rotor for 16 h at 4 ◦C. Post-centrifugation,15 fractions were taken from top to bottom of the tube andanalysed in subsequent Western immunoblotting. To concentrateproteins, fractions were incubated with 25% trichloroacetic acid(final), at 4 ◦C for 30 min. Samples were centrifuged at 14,000 rpm(13,000 × gav) at 4 ◦C for 20 min and the pellets washed twice withice cold acetone, ensuring not to disrupt the pellets. Pellets weredried at 42 ◦C for 10 min before re-suspension in 50 �l of MBS andanalysed by Western immunoblotting.

2.6. Immunocytochemistry

To preclude fixation artefacts (see Section 3), all imaging exper-iments of surface expression were performed using a two-stepprotocol. In this protocol COS-7 cells (48 h post-transfection) werecooled on ice to 4 ◦C and after 10 min, treated with primary antibodydiluted in PBS. After 1 h at 4 ◦C, the coverslips were washed threetimes with PBS and the cells fixed with 4% (w/v) paraformaldehydefor 20 min at 20 ◦C. The cells were then treated with the appropriate(Cy5 or FITC) fluorophore-conjugated secondary antibody for 1 h at20 ◦C. In order to detect intracellular epitope expression, cells werepermeabilised post-fixation with 0.5% saponin for 10 min at 20 ◦C,prior to incubation with primary antibody. Nuclear staining wasperformed with DAPI (1 �g/ml) for 2 min at 20 ◦C, prior to mountingwith Prolong Gold Antifade reagent (Invitrogen/Molecular Probes).Staining for membrane cholesterol was performed with filipin III(10 �g/ml; Sigma) diluted 100-fold from 1 mg/ml DMSO stock, for1 h at 20 ◦C prior to mounting.

2.7. Fluorescence deconvolution microscopy and image analysis

Images of cells on coverslips were acquired on a Delta VisionRT (Applied Precision, Image Solutions, UK) restoration microscopeusing a 60× objective lens and appropriate wavelength filters. Theimages were collected using a Coolsnap HQ (Photometrics) cam-era with a Z optical spacing of 0.1 �m. Raw images were thendeconvolved using Softworx software and displayed as maximumprojections using NIH Image J (W.S. Rasband, National Institutesof Health, Bethesda, USA, Wright Cell Imaging facility bundle:http://www.uhnres.utoronto.ca/facilities/wcif.htm).

2.8. Whole-cell patch-clamp electrophysiology

COS-7 cells were transiently transfected with Cav2.2:�1b:�2�-1:mut3-GFP-pMT2 cDNA in a 3:1:1:0.2 mass ratio and cur-rent recordings made 48 h post-transfection. Electrophysiologicalrecordings were made from green fluorescent COS-7 cells. Thewhole-cell configuration of the patch clamp technique was used to

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86 P. Robinson et al. / Cell

ecord barium currents in the following solutions [31]. The inter-al solution contained (mM): caesium aspartate 140.0; EGTA 5.0;gCl2 2.0; CaCl2 0.1; Hepes 20.0; K2ATP 1.0; adjusted to pH 7.2ith CsOH and 310 mosm l−1 with sucrose. The external solution

ontained (mM): TEABr 160.0; MgCl2 1.0; KCl 5.0; NaHCO3 1.0;epes 10.0; glucose 4.0; tetrodotoxin 0.001; BaCl2 5; adjusted toH 7.4 with Tris-base and to 320 mosm l−1 with sucrose. All exper-

ments were performed at room temperature (20–22 ◦C). Patchipettes with resistance measured between 2 and 5 M� were rou-inely pulled from thin-walled borosilicate glass tubing (Intracel,K), fire-polished and coated with Sigmacote (Sigma–Aldrich, UK).n Axopatch 200B amplifier (Molecular Devices, Palo Alto, CA,SA) was used for recordings which were filtered at 2 Hz andigitised at 2–44 kHz using a Digidata 1328A A/D converter (Molec-lar Devices). Cells were held at a potential of −80 mV (Vh) wherehe holding current was less than −0.05 nA and series resistanceess than 10 M�. Current recordings were made with cell capac-tance compensated and leak currents subtracted using an online/4 leak subtraction protocol. Series resistance was compensatedy up to 80% and only cells that were adequately clamped weresed. Standard current–voltage protocols involved 120 ms sweepsrom command voltages −30 to +65 mV in 5 mV steps. Currentensity–voltage (I–V) relationships for each cell were fitted withBoltzmann function:

= g(V − Vrev)1 + exp(−(V − V50,act)/k)

here Vrev is the reversal potential, V50,act is the voltage for halfaximal activation of current, g is the conductance, and k is the

lope factor. Conductance–voltage relationships were derived fromndividual current density–voltage plots and fitted with a Boltz-

ann function of the form:

G

Gmax= 1

1 + exp(V50,act − V)/k

here Gmax is the maximum conductance. The peak conductance,, at each test potential was calculated from the corresponding peakurrent, I, as follows:

= I

V − Vrev

50, k and Vrev are as defined above.Data acquisition was performed using pCLAMP software (ver-

ion 9, Molecular Devices).

.9. Data analysis

All electrophysiological data, presented as the mean ± standardrror of the mean (S.E.M.) for n trials, were analysed usingCLAMP software (version 9, Molecular Devices) and Origin (ver-ion 7.0, Microcal, Northampton, MA, USA). Statistical analysisas carried out by Student’s t-test or ANOVA (one-way with

tudent–Newman–Keuls (SNK) post hoc correction), as appropriate,sing 95% confidence limits (SigmaStat software, Jandel Scientific).o-localisation of �2�-1 subunit with raft proteins was quanti-ed using intensity correlation analysis (ICA) to yield the intensityorrelation quotient (ICQs), as described by Li et al. [32]. Particlenalysis was performed using NIH Image J.

. Results

In order to define a role for the �2�-1 subunit in controllingav surface densities, we focused on the N-type (Cav2.2) channel,well-characterised neuronal subunit whose complexes with �1b

ubunits routinely elicit robust inward calcium currents in het-rologous expression systems [33,34]. For expression purposes, we

m 48 (2010) 183–194

used COS-7 cells, a highly defined fibroblast cell line which lacksendogenous Cav2.2 channels [33], contains components such ascaveolins required for the formation of lipid rafts [35] and whoselarge cell size and flatness facilitate imaging-based trafficking stud-ies [36].

3.1. Generation of a functional haemagglutinin (HA)epitope-tagged ˛2ı-1 subunit

In order to image the �2�-1 subunit and its complexes at the cellsurface, we exploited established immunochemical methodologiesbased upon recognition of an inserted exofacial haemagglutinin(HA) epitope tag [22,36,37]. The HA tag was introduced at an inter-nal site (L652-Q653) in �2�-1 at the equivalent position used byDavies et al. [22] for generating a functional HA-�2�-2 construct(Fig. 1). Whole-cell patch-clamp electrophysiology confirmed thatthe functionality of HA-�2�-1 was identical to that of WT �2�-1subunit (Supp. Fig. S1).

3.2. WT and HA-˛2ı-1 are expressed in puncta at the COS-7 cellsurface

Imaging of COS-7 cells transfected with WT �2�-1 cDNA alone(Fig. 2 and Supp. Fig. S3) revealed a non-uniform, punctate pat-tern of anti-�2�-1-labelling over the entire cell surface (Fig. 2A).Puncta (>800/cell) were only present in transfected cells and rangedin size from the limit of resolution (1 pixel2) to a maximum of≈0.5 �m in diameter. While most particles were of low intensityand size (inferred mode diameter of ≈0.01 �m), small particlesspanning a range of intensities, or, conversely large particles oflow intensity, were also observed (Supp. Fig. S2). Next, we exam-ined cells transfected with our HA-�2�-1 construct. As shown inFig. 2B, all HA-�2�-1-transfected cells exhibited a labelling pat-tern over the entire cell surface, comprised of puncta with similardimensions to those seen in cells transfected with WT-�2�-1. Takentogether, these data indicate that �2�-1 subunits, by themselves,are expressed primarily in small cell surface puncta.

3.3. HA-˛2ı-1 co-localises with the lipid raft markers caveolinand flotillin

We next compared labelling of HA-�2�-1 subunits with that oftwo established markers of lipid rafts, namely caveolin (Fig. 2C)[38,39] and flotillin (Fig. 2D) [40] and, as a control, with clathrin, amarker of clathrin-coated vesicles (Fig. 2E) [39] (see Supp. Fig. S4for details). Owing to their cytoplasmic disposition and our priorobservation of partial permeabilisation by fixative alone (≈25% ofcells at 2% fixative), detection of both of these markers required atwo-step approach whereby live cells were first treated with anti-HA antibody at 4 ◦C to detect just surface accessible HA-�2�-1, thenfixed, permeabilised and treated with either an anti-pan-caveolinantibody, recognising all three caveolin isoforms, or anti-flotillin-1. In transfected COS-7 cells, HA-�2�-1 labelling showed extensiveoverlap with the punctate labelling of both caveolin (Fig. 2C,Supp. Fig. S4) and flotillin-1 (Fig. 2D, Supp. Fig. S4). In contrast,HA-�2�-1 labelling showed little overlap with clathrin (Fig. 2E,Fig. S4). In support of this observation, intensity correlation analy-sis (ICA) [32], which resolves coincident variations in intensitiesabout the mean for both red and green channel pixels, yielded+ve ICQ scores (ICQ range: −0.5 (perfect segregation) < 0 > +0.5

(perfect co-localisation)), indicating strong co-localisation of HA-�2�-1 with caveolin (ICQ = +0.33 ± 0.015, n = 11) and flotillin-1(ICQ = +0.302 ± 0.019, n = 12), respectively (Fig. 2I). A weakly pos-itive ICQ value (ICQ = +0.087 ± 0.0072, n = 9), indicated significantlylower co-localisation of HA-�2�-1 and clathrin (Fig. 2I).

P. Robinson et al. / Cell Calcium 48 (2010) 183–194 187

Fig. 2. Clustering and co-localisation of the �2�-1 subunit with lipid raft markers caveolin and flotillin and its uncoupling by prior cell treatment with M-�-CD. In transfectedCOS-7 cells, both wild type �2�-1 (A) and HA-�2�-1 (B) subunits alone show a punctate cell surface distribution (arrowheads). In cells expressing the HA-�2�-1 subunit,anti-HA labelling (green) co-localises strongly with labelling (red) corresponding to the raft markers caveolin (C) and flotillin (D), but weakly with that for the non-raft markerclathrin (E) (red). Following pre-treatment of COS-7 cells expressing HA-�2�-1 with cyclodextrin–M-�-CD-labelling (red), corresponding to the raft markers caveolin (F) andflotillin (G) but not the non-raft marker clathrin (H), segregate from that of anti-HA (green), which remains punctate, throughout. COS-7 cells, transfected with WT anti-�2�-1(A) or HA-�2�-1 (C–H) cDNA, were labelled on ice with anti-HA antibody, then fixed, permeabilised and labelled with the corresponding anti-caveolin, -flotillin or -clathrina -�-CD( extrach non-2 s.

3r

c�c1eMndHaf

ntibodies. In the M-�-CD experiments, live cells were treated for 1 h with 10 mM MICQ) [32] values for co-localisation of HA-�2�-1 and caveolin, flotillin and clathrinigher degree of co-localisation (ICQ > 0 < +0.5) between HA-�2�-1 and raft versus0 �m (A and B); 10 �m (C–H). See Supp. Figs. S2–S4 for controls and further detail

.4. Uncoupling of the cell surface distribution of HA-˛2ı-1 andaft markers by cyclodextrin treatment

Given the association of HA-�2�-1 with the lipid raft markers,aveolin and flotillin, we next asked whether treatment with M--CD, an agent that disrupts lipid rafts via the sequestration ofholesterol, could also alter the cell surface distribution of HA-�2�-[38]. In these experiments, cells were transfected with either

mpty vector or HA-�2�-1 cDNA, treated for 1 h with or without-�-CD and processed for imaging of HA-�2�-1 and endoge-

ous caveolin, flotillin and clathrin, using the two-step approach

escribed above. As shown in Fig. 2 and Supp. Fig. S5, treatment ofA-�2�-1-transfected cells with M-�-CD revealed a pronouncedlteration in both caveolin (Fig. 2F) and flotillin (Fig. 2G) labellingrom the punctate patterns seen in untreated cells (Fig. 2C and D) to

prior to labelling with anti-HA. Panel I depicts mean intensity correlation quotientted from multiple images (n > 9 (>3 images for >3 experiments)). Note significantlyraft markers (P < 0.001 (***); one-way ANOVA; SNK correction (n ≥ 9)). Scale bars:

patterns comprised of large patches of intense labelling proximalto the cell nucleus, interspersed by diffuse labelling over the cellsurface. However, in these same cells, the anti-HA-�2�-1 labellingpatterns (Fig. 2F and G) remained similar to those in the absenceof M-�-CD (Fig. 2C and D). Similarly, M-�-CD had no effect on thedistribution of the non-raft marker, clathrin (Fig. 2E and H).

To explore the effects of M-�-CD in more detail, images weresubjected to quantitative particle analysis. As shown in Supp. Fig S2,M-�-CD treatment had no significant effect (P > 0.05) on the meanparticle size of HA-�2�-1 or clathrin puncta, but caused a signifi-cant (P < 0.001, ANOVA) increase in the mean size of both caveolin

and flotillin puncta. Similarly, M-�-CD had no effect on the pop-ulation distribution of HA-�2�-1 or clathrin particles (Supp. Fig.S2), but caused a decrease in the number of small particles andthe emergence of low numbers of large particles for both cave-

1 Calciu

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88 P. Robinson et al. / Cell

lin and flotillin (Supp. Fig. S2). This conclusion was supportedurther by examining the numbers of particles and their fractionalontributions to the overall particle area (coverage), thereby per-itting discrimination between coverage arising from many small

articles, or, from fewer larger particles (Supp. Fig. S2). In thebsence or presence of M-�-CD, the majority of individual HA-�2�-and clathrin particles covered <5% of the total particulate area.hile similar values were seen for both caveolin and flotillin in the

bsence of M-�-CD, when treated with M-�-CD, individual parti-les with high fractional coverage (up to 55%) were induced for bothhese raft markers.

To define the effect of M-�-CD on the co-localisation of HA-2�-1 with caveolin, flotillin and clathrin, images were subjected

o ICA (Fig. 2I). In HA-�2�-1-transfected cells, treatment with-�-CD yielded a mean ICQ value for the co-localisation of HA-

2�-1 and caveolin of +0.09 ± 0.006 (n = 22), significantly (P < 0.001,NOVA) lower than the value of +0.33 ± 0.015 (n = 11) found in

he absence of M-�-CD (above). Likewise, the ICQ value for theo-localisation of HA-�2�-1 and flotillin of +0.11 ± 0.011 (n = 10),as also significantly (P < 0.001, ANOVA) lower than that in the

bsence of M-�-CD (ICQ = +0.30 ± 0.019; n = 12) (above). As a con-rol, we also analysed the effect of M-�-CD on the distribution ando-localisation of HA-�2�-1 with clathrin (Fig. 2I) and obtained aean ICQ value of 0.12 ± 0.009 (n = 20), not significantly different

P > 0.05; ANOVA) from that found for non-M-�-CD-treated cellsICQ = +0.087 ± 0.0072).

Taken together, these data indicate that M-�-CD treatment

aused a specific re-distribution of caveolin and flotillin from smalluncta into large aggregates. In contrast, M-�-CD had no effect onhe size distribution of HA-�2�-1 puncta, even though these HA-2�-1 puncta co-localised strongly with both caveolin and flotillin

n non-M-�-CD-treated cells.

ig. 3. Localisation of HA-�2�-1 in detergent-resistant membranes. Transfected COS-7 ceradients containing 1% Triton-X-100, using antibodies to the lipid raft marker caveolinpecific transfection conditions, are depicted in both A and B as follows: i. mock (empty10 mM); iii. HA-�2�-1; iv. HA-�2�-1 transfected cells treated with M-�-CD. Note the prend the absence of HA-immunoreactivity (B blots i and ii) in mock-transfected cells. Imago the blots shown in panels A and B, were quantified by densitometry and the intensiA), respectively (mock (filled squares); mock + M-�-CD (open squares); HA-�2�-1 (filledholesterol (filled hexagons)). Schematic bars (top, panels A–D) indicate sucrose densitiey ‘T’ indicate immunodetection loading controls (i.e. aliquot of total lysate).

m 48 (2010) 183–194

3.5. HA-˛2ı-1 is present in detergent-resistant membranes and isdispersed by cyclodextrin treatment

To provide further support for the partitioning of �2�-1 intolipid rafts, we employed flotation equilibrium centrifugation ofcell lysates in sucrose density gradients containing 1% Triton-X-100 [22,39]. Surprisingly, cell lysis with 1% Triton-X-100, 50 mMNaCl, Mes pH 6.5 [22] afforded very low levels (<5% total) of HA-�2�-1 subunit. However, efficient extraction of HA-�2�-1 (≈50%)was obtained at higher (150 mM) salt concentrations in the samebuffer (data not shown). Following centrifugation, each gradientwas fractionated and fractions immunoblotted using antibodiesagainst HA-�2�-1 and the raft marker caveolin. As shown in Fig. 3A,endogenous caveolin (22 kDa isoform) was detected in both mock– (panel i) and HA-�2�-1 – (panel iii) transfected cells as a singlepeak in those fractions (3–6), corresponding to the 5–30% sucroseinterface. Bands corresponding to HA-�2�-1 were absent in the gra-dient fractions from mock-transfected cell lysates (Fig. 3B, panel i),but were clearly visible in those from the HA-�2�-1-transfectedcells (Fig. 3B, panel iii). In these latter lysates, the HA-�2�-1 sub-units had a distribution at the 5-30% interface, broadly overlappingthat of caveolin, with the remainder residing in fractions of higherdensity centered on the 30–45% sucrose interface. Based on quan-titative analysis, these data indicate that a substantial portion(≈30%) of HA-�2�-1 subunits are co-localised with caveolin indetergent-resistant membranes, with the remainder (≈70%) in thenon-raft fraction. To test this notion further, the gradient analysis

was repeated in the presence of 10 mM 3-methyl-�-cyclodextrin(M-�-CD), an agent that disrupts lipid rafts through the seques-tration of cholesterol [38]. As shown in Fig. 3C, treatment of mockor HA-�2�-1 transfected cell lysates with M-�-CD (panels ii andiv, respectively), caused a large shift in the proportion of endoge-

ll membranes were analysed via immunoblotting of fractions from sucrose density(A) or anti-HA (for HA-�2�-1) (B). Representative blots (i–iv), corresponding to

vector); ii. mock-transfected cells treated with the raft disrupting agent M-�-CDsence of endogenous caveolin (A blots i–iv) in mock or HA-�2�-1-transfected cellses were cropped from full length blots shown in Supp. Fig. S2. Bands correspondingties for each gradient fraction depicted in panels C (anti-caveolin) and (D) (anti-circles); HA-�2�-1 + M-�-CD (open circles); HA-�2�-1 + M-�-CD pre-treated with

s in original gradients aligned to corresponding fractions. Lanes in blots indicated

P. Robinson et al. / Cell Calcium 48 (2010) 183–194 189

Fig. 4. HA-�2�-1 targets GFP-Cav2.2/�1b channels to detergent-resistant membranes. COS-7 cells were transfected with either GFP-Cav2.2/�1b or GFP-Cav2.2/�1b/HA-�2�-1and membranes analysed via immunoblotting of fractions from sucrose density gradients containing 1% Triton-X-100, using antibodies to (A) caveolin, (B) anti-GFP (for GFP-Cav2.2) and (C) anti-HA (for HA-�2�-1). Representative blots (i–iv in panels A–C), correspond to the following transfection conditions: i. GFP-Cav2.2/�1b; ii. GFP-Cav2.2/�1b

treated with M-�-CD; iii. GFP-Cav2.2/�1b/HA-�2�-1; iv. GFP-Cav2.2/�1b/HA-�2�-1 treated with M-�-CD. Band intensities corresponding to each gradient fraction are depictedin panels D (anti-caveolin), E (anti-GFP) and F (anti-HA), respectively. Key: GFP-Cav2.2/�1b (filled squares); GFP-Cav2.2/�1b + M-�-CD (open squares); GFP-Cav2.2/�1b/HA-�2�-1 (top, pf ed wif ted by

ntSMncasa

3c

�miotctcc[ia(1iji(

(filled circles); GFP-Cav2.2/�1b/HA-�2�-1 + M-�-CD (open circles). Schematic barsractions. Note the presence of GFP-Cav2.2 in raft fractions (3–6) from cells transfectractions from cells transfected with GFP-Cav2.2/�1b (B, blot i). Lanes in blots indica

ous 22 kDa caveolin present at the 5–30% sucrose interface suchhat most (>90%) was now present at the denser 30–45% interface.ignificantly, upon treatment of HA-�2�-1-transfected cells with-�-CD, the HA-�2�-1 band was completely shifted from raft to

on-raft fractions (Fig. 3D). Pre-treatment of cells with an M-�-CD-holesterol complex, yielded similar distributions for both caveolinnd HA-�2�-1 as found for non-M-�-CD treated cells, indicatingpecificity of the effect of M-�-CD (Fig. 3A and B, panel v and Fig. 3Cnd D).

.6. The HA-˛2ı-1 subunit induces partitioning of Cav2.2omplexes into lipid rafts

Having obtained evidence for a partitioning of HA- and WT-2�-1 subunits in lipid rafts we addressed whether �2�-1 subunitsight confer a similar partitioning of the entire Cav2.2 complex

nto rafts. First, we compared the raft distributions of Cav2.2/�1br Cav2.2/�1b/HA-�2�-1 through flotation equilibrium centrifuga-ion of transfected COS-7 cell lysates in sucrose density gradientsontaining 1% Triton-X-100, as above. To facilitate immunodetec-ion of Cav2.2, transfections were performed using a fully functionalonstruct encoding Cav2.2 with the highly antigenic green fluores-ent protein (GFP) tag fused to its amino terminus (GFP-Cav2.2)34]. Following centrifugation, fractions from each gradient weremmunoblotted with antibodies to endogenous caveolin (Fig. 4A),nti-GFP (for GFP-Cav2.2) (Fig. 4B) and anti-HA (for HA-�2�-1)Fig. 4C). In lysates transfected with Cav2.2/�1b (i.e. no HA-�2�-

), most (>95%) endogenous caveolin was found at the 5–30%

nterface (Fig. 4A, panel i). In contrast, GFP-Cav2.2 appeared inust the dense (45%) sucrose fractions (11–13) (Fig. 4B, panel). No bands were evident in anti-HA immunoblotted fractionsFig. 4C, panel i) commensurate with the absence of the HA-�2�-1

anels A–F) indicate sucrose densities in original gradients aligned to correspondingth GFP-Cav2.2/�1b/HA-�2�-1 (B, blot iii (asterisk)) and its absence in corresponding

‘T’ indicate immunodetection loading controls (i.e. aliquot of total lysate).

construct. While treatment with M-�-CD caused a marked re-distribution of endogenous caveolin to denser fractions (Fig. 4A,panel ii, fractions 10–13), the GFP-Cav2.2 subunit remained inthe densest fractions (Fig. 4B, panel ii). In marked contrast, anal-ysis of lysates from cells transfected with Cav2.2/�1b/HA-�2�-1revealed the presence of ≈40% GFP-Cav2.2 (Fig. 4B panel iii) and≈70% HA-�2�-1 (Fig. 7C, panel iii) in the caveolin-rich (Fig. 7A,panel iii) 5–30% sucrose fractions (fractions 4–5) with the remain-der (GFP-Cav2.2 ≈60%; HA-�2�-1 ≈30%) in the denser (45% sucrose)non-raft fractions. Moreover, treatment of the lysates with M-�-CD caused a shift in the band distributions such that all theendogenous caveolin,≈90% GFP-Cav2.2 and all the HA-�2�-1 (paneliv, Fig. 4A–C), became enriched in the higher density, non-raftfractions.

3.7. Co-localisation of HA-Cav2.2/ˇ1b complexes with caveolinrequires the ˛2ı-1 subunit

Having defined the requirement for HA-�2�-1 in target-ing Cav2.2/�1b complexes to lipid rafts biochemically, we nextaddressed its role in specifying the cell surface distribution ofCav2.2/�1b complexes. To facilitate multicolour, surface labelling,cells were transfected with a Cav2.2 construct bearing an exo-facial HA tag, �1b and WT-�2�-1, as appropriate. As shown inFig. 5 and Supp. Fig. S6, cells expressing HA-Cav2.2/�1b manifesta punctate distribution of anti-HA labelling, but, very limited co-

localisation with caveolin (Fig. 5A). In contrast, cells expressingHA-Cav2.2/�1b and WT-�2�-1, exhibited extensive co-localisationof anti-HA with caveolin (Fig. 5B). In such transfectants, anti-WT-�2�-1 labelling also co-localised strongly with that of caveolin(Fig. 5C).

190 P. Robinson et al. / Cell Calcium 48 (2010) 183–194

Fig. 5. Co-localisation of HA-Cav2.2/�1b complexes with caveolin requires the �2�-1 subunit, but clustering is independent of raft co-localisation and requires the actin-basedcytoskeleton. COS-7 cells were transfected with HA-Cav2.2 and �1b in the absence (A and D) or presence (B, C, E, and F) of the WT-�2�-1 subunit. Panels A–F depict merged(anti-HA(Cav2.2) or anti-WT-�2�-1, green; anti-caveolin, red) images. Panels D–F correspond to cells pre-treated for 1 h with 10 mM M-�-CD. Note HA-Cav2.2 clusteringeven in the absence of WT-�2�-1, but lack of co-localisation with caveolin (A) (see Supp. Figs. S6 and S7 for details and analysis). In the presence of WT-�2�-1 (panelsB and C), HA-Cav2.2/�1b complexes show both clustering and strong co-localisation between HA-Cav2.2 (B) or WT-�2�-1 (C) and caveolin. Following M-�-CD treatment,caveolin becomes dispersed (D–F), leaving both HA-Cav2.2 (D-F) or WT-�2�-1 (F) clustered. The ICQ values corresponding to each transfection and treatment condition areshown in the inset lower right. Panels G-L depict the importance of the actin-based cytoskeleton in supporting clustering. Panel G: distribution of HA-�2�-1 (green) and theactin label, Tx-phalloidin (red), in HA-�2�-1-transfected COS-7 cells, prior to cytochalasin-D treatment. Note distribution of actin labelling at cell margin and filaments (seeFig. S8C for details). Panels H–J show the distribution of HA-�2�-1 (green) and Tx-phalloidin (actin, red) staining for COS-7 cells expressing HA-�2�-1 alone (H), HA-Cav2.2/�1b

(I) and HA-Cav2.2/�1b/WT-�2�-1 (J). Note the lack of HA-�2�-1 clusters, fragmentation of Tx-phalloidin labelling and dispersal of both labels to the cell margin (G, overlapin yellow). Panel K shows the pattern of anti-HA (Cav2.2) and anti-WT-�2�-1 labelling in HA-Cav2.2/�1b/WT-�2�-1 transfected cells, following pre-treatment with 2 �mcytochalasin-D. Note re-distribution of anti-HA labelling from a punctate pattern over the entire cell surface (e.g. Supp. Fig. S4), to one where puncta are found in or alignedtowards, large patches, especially over the centre of the cell above the nucleus. The corresponding ICQ values for HA-Cav2.2/�1b/WT-�2�-1 co-localisation, before and afterdrug treatment, are shown in panel M. Scale bars: 15 �m (A–F); 20 �m (G–K).

P. Robinson et al. / Cell Calcium 48 (2010) 183–194 191

-40 -20 0 20 40 60 80

-30

-20

-10

0

10

S-F/M-β-CD S-F

V (mV)

I (pA

.pF-1

) Control

-40 -20 0 20 40 60 80

-30

-20

-10

0

10

**

V (mV)

*I(pA

.pF-1

)

BA

1 nA

50 ms

Fig. 6. Effect of cholesterol depletion on Cav2.2/�1b currents in the presence and absence of the �2�-1 subunit. Average I–V plots for Cav2.2/�1b currents in the absence of�2�-1 (A) and in the presence of HA-�2�-1 (B). Cells were incubated for 1 h in serum-free media + M-�-CD (10 mM; S-F/M-�-CD, open circles) to deplete cholesterol. Controlcells were maintained in normal serum-containing medium (control, closed circles), in serum-free medium (S–F, open squares), or M-�-CD pre-treated with cholesterol for1 ive cuu V. Dat(

3a

ronchff((MHdspcMs1pWhcbi

3c

maolipIwtcc

h prior to recording of Cav2.2 currents (closed squares). Inset in (B): representatsing 120 ms depolarising steps in 5 mV intervals (−30 to +65 mV), from Vh, −80 m*P < 0.01). The cell number, n, for each treatment ranged between 8 and 10.

.8. Cyclodextrin treatment uncouples Cav2.2 complex clusteringnd caveolin co-localisation

Based on the above, we inferred that the �2�-1 subunit isequired for the co-localisation of Cav2.2/�1b complexes in cave-lin +ve clusters and detergent-rich membranes (Fig. 4), butot clustering per se. To examine this further, and test whethero-localisation of caveolin +ve clusters of Cav2.2/�1b/WT-�2�-1,ad similar features as those for �2�-1, COS-7 cells trans-

ected with HA-Cav2.2/�1b +/− WT-�2�-1 cDNAs, were treatedor 1 h with 10 mM M-�-CD, then processed for anti-caveolinred) (Fig. 5D–F, Supp. Fig. S7) and (in green) either anti-HAFig. 5D and E) or anti-WT-�2�-1 (Fig. 5F) labelling. Following

-�-CD treatment, cells expressing HA-Cav2.2/�1b (Fig. 5D) orA-Cav2.2/�1b/WT-�2�-1 (Fig. 5E and F) showed a marked re-istribution of caveolin labelling. In cells expressing HA-Cav2.2/�1b,urface anti-HA labelling retained the punctate distribution andoor co-localisation with caveolin seen in non-M-�-CD-treatedells (Fig. 5D, Supp. Fig. S7). However, and in contrast to non--�-CD treated cells, cells expressing HA-Cav2.2/�1b/WT-�2�-1

howed low co-localisation of anti-HA (Fig. 5E) or anti-WT-�2�-(Fig. 5F) labelling with that for caveolin, while retaining their

unctate distribution (see Supp. Fig. S7). Surprisingly, both anti-T-�2�-1 and anti-HA-(Cav2.2) labelling retained a similarly

igh degree of co-localisation as found in non-M-�-CD treatedells. Thus, M-�-CD appears to deplete caveolin from clusters ofoth �2�-1 and HA-Cav2.2/�1b/WT-�2�-1 without affecting their

ntegrity.

.9. HA-˛2ı-1 surface clustering requires an intact actinytoskeleton

Emerging models of lipid raft dynamics suggest that larger raftarker-positive membrane clusters, discerned by imaging, may

rise through a coalescence of nascent raft complexes and sec-ndary stabilisation by protein-protein interactions involving noteast the actin-based cytoskeleton. To test this notion, we exam-ned the distribution of HA-�2�-1 in the absence (Fig. 5G) orresence (Fig. 5H) of the actin-disrupting agent cytochalasin-D.

n control cells, actin labelling–detected with TxRed-phalloidin –as distributed throughout the cytoplasm, primarily in filamen-

ous structures (Supp. Fig. S8C) around the nucleus and at theell margins (Fig. 5G). In these cells, there was little evidence foro-localisation of HA-�2�-1 with actin (Supp. Fig. S8D) although

rrent traces from S–F control versus M-�-CD-treated cells. Currents were evokeda are shown as the mean ± S.E.M. Significant differences are indicated by asterisks

there was evidence for its concentration in cells displaying ruf-fles (Supp. Fig. S8E and F). However, as anticipated, pre-treatmentwith cytochalasin-D caused a reduction in peri-nuclear labelling,the number and length of the actin filaments and the appear-ance of aggregates within the cytoplasm. In all cells examined(n = 15), treatment with 2 �M cytochalasin-D led to almost com-plete removal of HA-�2�-1 surface labelling and caused a majorre-distribution of labelling towards the cell margins (arrowhead,Fig. 5H). In cells expressing HA-Cav2.2/�1b in the absence (Fig. 5I)or presence of WT-�2�-1 (Fig. 5J), cytochalasin-D caused a markedre-distribution and partial aggregation of particles detected withanti-HA (Fig. 5I). In both instances, particles often exhibited avectorial distribution (Fig. 5I and J arrowheads), unlike the morerandom distribution found in untreated cells (e.g. Fig. 5B, C, E,and F). Remarkably, cytochalasin-D treatment almost abolishedthe co-localisation of HA-Cav2.2/�1b with WT-�2�-1 (Fig. 5K andFig. S8K–M). Thus, treatment with cytochalasin-D not only inducesa re-arrangement of HA-�2� labelling throughout the cell but alsoits coupling to Cav2.2/�1b.

3.10. Cyclodextrin treatment reduces Cav2.2 current density

Having established that M-�-CD disrupted the presence of HA-�2�-1 and Cav2.2/�1b/HA-�2�-1 complexes in detergent-resistantmembranes, we examined whether cholesterol depletion mightalso exert functional effects on Cav2.2 channels. Since M-�-CD wasdissolved in serum-free medium, we first confirmed that 1 h incu-bation of cells in serum-free medium had no effect on currentdensity (Fig. 6) or the biophysical properties (data not shown) ofCav2.2 channels in the presence or absence of HA-�2�-1. Incubationof cells for 1 h with 10 mM M-�-CD had no effect on Cav2.2 currentsin the absence of HA-�2�-1 (Fig. 6A). However, in cells expressingCav2.2/�1b/HA-�2�-1, M-�-CD induced a decrease in current den-sity at all potentials tested (Fig. 6B), that was significant between0 and +10 mV. At Vpeak (+5 mV), Imax was reduced from −23.4 ± 1.3to −17.7 ± 2.6 pA pF−1 (≈25%; P < 0.05). No effects on the voltage-dependence of activation or current kinetics were observed. Theaverage values for �act and �inact at Vpeak were 2.3 ± 0.8 ms and

115 ± 27 ms, respectively in serum-starved controls compared with2.6 ± 0.6 ms and 129 ± 18 ms in the presence of M-�-CD. Thus, inagreement with other studies [22,30], disruption of lipid rafts viacholesterol depletion has functional consequences for Cav2.2 activ-ity.

192 P. Robinson et al. / Cell Calcium 48 (2010) 183–194

Fig. 7. Endogenous �2�-1 subunits have a punctate distribution in rat dorsal root ganglion (DRG) neurons and co-localise with the t-SNARE SNAP-25 and to a lesser extent,�-tubulin. Panel (A) depicts a montage prepared from nine separate image stacks for a DRG neuron (soma, red asterisk), with labelling as follows: anti-WT-�2�-1 (green),anti-SNAP-25 (red) and anti-�-tubulin (blue). Note the presence of anti-WT-�2�-1 labelling on both neuronal, i.e. �-tubulin +ve cells and non-neuronal cells (white asterisks.White arrowheads denote SNAP-25 +ve regions lacking anti-WT-�2�-1 labelling). (B) Enlargement of boxed region (box i in A). Note presence of punctate anti-WT-�2�-1l �-1 (gP regiono (A),

3s

twrt(ap[dbenlDab

abelling on DRG neurites (arrowheads). Panel (C) denotes labelling of anti-WT-�2

anel (E) denotes labelling of anti-WT-�2�-1 (green) and anti-�-tubulin (blue) withf anti-WT-�2�-1 with either and anti-SNAP-25 or anti-�-tubulin, Scale bars: 10 �m

.11. Endogenous ˛2ı-1 is expressed in puncta at the surface ofensory neurons

Although WT-�2�-1 and HA-�2�-1 exhibited a punctate dis-ribution in transfected cells, we considered it important to testhether �2�-1 had a similar distribution in a more physiologically

elevant cell context. To address this issue, we examined the dis-ribution of endogenous, i.e. WT-�2�-1 in rat dorsal root ganglionDRG) neurons. Cultures of DRGs exposed to nerve growth factorre an established sensory neuron preparation, relevant to neuro-athic pain, which express several Cav subtypes, including Cav2.234,41]. As shown in Fig. 7A–F, WT-�2�-1 shows a punctate surfaceistribution on the somata and neurites of DRG neurons, definedy expression of �-tubulin (arrowheads Fig. 7B). There was alsovidence for a punctate distribution of WT-�2�-1 in some non-euronal (�-tubulin −ve) cells. Interestingly, surface WT-�2�-1

abelling co-localised with the t-SNARE protein SNAP-25 (Fig. 7C,, and G), and to a lesser extent with �-tubulin (Fig. 7E–G). Particlenalysis revealed such puncta had a very similar population distri-ution to those seen in transfected COS-7 cells (data not shown).

reen) and anti-SNAP-25 (red) with regions of co-localisation shown in panel (D).s of co-localisation shown in panel (F). Panel (G): comparison of the co-localisation2 �m (B) and 2.5 �m (C–F).

4. Discussion

In this paper we have used a variety of independent techniquesto show that the Cav�2�-1 subunit is not only present withinlipid rafts itself but crucially, is also necessary for the partitioningof Cav2.2 �1/�1b subunits into DRMs and moreover, their co-localisation with lipid raft markers. However, �2�-1, while presentin small puncta, does not appear to be required for the formation ofCav2.2 �1/�1b-containing puncta which are present irrespective ofthe presence of �2�-1. Thus, �2�-1 appears to be a requisite for theassociation of Cav2.2�1/�1b with rafts but not for the formation ofchannel clusters per se. These data, therefore, conform to emergingmodels where raft formation occurs via extensive protein-proteininteractions, which can allow rafts to undergo transitions fromsmaller nuclei to larger and more stable clusters [39,42–44]. Indeed,the clusters we visualised by fluorescence imaging are much big-

ger than the rafts identified by alternative methods [39,45,46] butare commensurate with the clusters of raft markers seen in var-ious cells including COS-7s [35,47], and those generated by otherraft-associated membrane proteins, including ion channels [48,49].

P. Robinson et al. / Cell Calcium 48 (2010) 183–194 193

Fig. 8. Dynamic aggregation model for �2�-1 clustering. In this model clustering is rationalised on the basis of our experimental data showing: (a) independent expressionof �2�-1 subunits or �1/� complexes, (b) expression of a fraction of �2�-1 subunits in detergent-resistant membranes (DRMs), (c) the absence of �1/� complexes in DRMs,(d) the requirement of �2�-1 subunits for the expression of �1/� complexes in DRMs and (e) co-localisation of clusters with the raft markers flotillin and caveolin. Here,discernible clusters (puncta) are presented as being derived by the coalescence and stabilisation of more dynamic lipid rafts present in the membrane, but below the limits ofoptical resolution. Stabilisation is considered to arise in direct or indirect response to ECM-derived ligands (e.g. thrombospondin) through heterophilic interactions of �2�-1subunits (or Cav �1/�/�2� complexes) with other raft embedded proteins, including those attached to the cytoskeleton. Although it is assumed that Cav subunits assemble int r �1/�m re-disw d any

ircsGhtdorflHataocCpnbaaip[

vcnoeoCssad�t(s

he endoplasmic reticulum, the independent surface expression of �2�-1 subunits oodel by ‘?’). In this model, cholesterol depletion by M-�-CD is deemed to cause ahereas disruption of the (actin-based) cytoskeleton disperses both the clusters an

In this context, an interesting feature emerging from our imag-ng analysis is the high degree to which the �2�-1 subunit isequired for co-localisation of Cav2.2 channels with the raft markersaveolin and flotillin-1. Recent evidence has suggested that �2�-1ubunits are attached to the external leaflet of the lipid bilayer via aPI-anchor [28], known to concentrate proteins into rafts. Thus, itas been inferred that GPI-anchoring, itself, drives the concentra-ion of Cav�1/�1b into lipid rafts. However, if this were the case thenisruption of lipid rafts should re-distribute Cav�1/�1b complexesver the cell surface. Indeed, Dolphin and co-workers have recentlyeported a decrease in cell surface localisation of Cav2.2 (decreaseduorescence intensity) upon disruption of GPI-anchoring [28].owever, in contrast, we find the distribution of �2�-1 subunitlone or with Cav�1/�1b complexes is cyclodextrin-resistant, evenhough raft markers become re-distributed. Thus, additional inter-ctions must be required to sustain the cell surface puncta webserved. Indeed, our data showing that disruption of actin withytochalasin-D disperses puncta containing �2�-1 subunit or anyav�1/� subunits, raises the question of how a GPI-anchoredrotein could interact with the cytoskeleton? The most likely sce-ario is that clustering reflects stabilising, heterophilic, interactionsetween exofacial regions of �2�-1 (expressed alone or in associ-tion with Cav subunits) and transmembrane proteins which arettached to the cytoskeleton, such as integrins. Additional stabil-sing interactions could include those with purely extracellularroteins such as the �2�-1-interacting protein, thrombospondin44,50,51].

The question remains, however, what is the physiological rele-ance of raft association and clustering? In COS-7 cells the �2�-1luster sizes have a similar population distribution to those endoge-ous to DRG neurons and are, therefore, likely to be of similarrigins and physiological relevance in spite of the extensive differ-nces between the two cell phenotypes. In neurons, the presencef �2�-1 throughout the cell surface, combined with evidence thatav�1 subunits possess independent trafficking motifs [9,18,52],uggests that �2�-1 is unlikely to direct Cavs into specific regionsuch as axons and dendrites per se. More likely, is that �2� subunitsre involved in a secondary mechanism that shapes the surface

istribution of Cavs in response to epigenetic cues [9,44]. Indeed,2�-1 subunits have been reported to stabilise Cav expression at

he cell surface [24]. Moreover, deletion of the Drosophila �2� genestraightjacket) has been found to disrupt neuromuscular transmis-ion by dispersal of Cavs at active zones [53]. The ability of the

complexes raises the untested possibility of assembly at the cell surface (shown intribution of caveolin and flotillin leaving the stabilised, discernible, clusters intact,associated raft proteins.

�2�-1 subunits to form clusters is also consistent with, and mayrationalise, both the punctate distributions of Cavs and hotspotsof voltage-gated calcium influx hitherto identified in diverse celltypes [11,12]. Owing to the limited range of cytoplasmic calciumdiffusion, such hotspots would serve to amplify calcium signallingin response to local demand [13–15]. Importantly, the ability of�2�-1 subunits to drive Cavs into rafts would also allow the co-assembly of Cavs with the plethora of raft-associated signalling (e.g.G-proteins and protein kinases) and scaffolding proteins that areknown to modulate their activity [16,54]. Indeed, a multiplicity ofsignalling complexes may exist within and between different celltypes, thereby rationalising the differential effects of cholesteroldepletion on specific calcium currents [22,29,55].

Lastly, like others, we have assumed that co-assembly of �2�-1and �1 subunits occurs early in biogenesis [21,24,25,56]. However,our data show, unequivocally, that it is possible to have Cav2.2/�1bclusters with, or without, the �2�-1 subunit but, only those con-taining the �2�-1 subunit co-localise with raft proteins. Thus, someco-assembly may also occur in the plasma membrane (Fig. 8). Insuch a paradigm, any independent expression of �2�-1 subunitswould provide an elegant mechanism for regulating the relativeproportions of Cav complexes (�2�-1/Cav2.2/�1b and Cav2.2/�1b)with distinct biophysical and trafficking properties. This notionwould be consistent with the growing number of reports that show�2�-1 subunits can localise to the cell surface and exhibit functionsindependent of pore-forming Cav�1 subunits [50,51,53]. Irrespec-tive of where assembly occurs, the ability of the �2�-1 subunit toconcentrate Cavs into discrete membrane microdomains makes ituniquely placed in the modulation of Cav activity and also for fine-tuning the spatial and temporal expression of Cavs and thence Ca2+

signalling within cells.

Conflict of interest statement

None declared.

Acknowledgement

This work was supported by funds from the Medical ResearchCouncil, UK (Project Grant I.D. 81568 to EMF and OTJ).

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[55] S.M. Jacobo, M.L. Guerra, R.E. Jarrard, et al., The intracellular II-III loops of Cav1.2and Cav1.3 uncouple L-type voltage-gated Ca2+ channels from glucagon-likepeptide-1 potentiation of insulin secretion in INS-1 cells via displacement from

94 P. Robinson et al. / Cell

ppendix A. Supplementary data

Supplementary data associated with this article can be found, inhe online version, at doi:10.1016/j.ceca.2010.08.006.

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