Inhibition of TRP3 channels by lanthanides: block from the ... · membrane current was recorded...

Inhibition of TRP3 channels by lanthanides: block from the cytosolic side of the plasma membrane

Christian R. Halaszovich* , Christof Zitt* , Eberhard Jüngling, Andreas Lückhoff#

Institut für Physiologie

Universitätsklinikum der RWTH Aachen

Pauwelsstrasse 30

D-52074 Aachen, Germany

*These two authors contributed equally.

#to whom correspondence should be addressed:

Tel.: +49-241-80 88812

Fax: +49-241-8888 434

e-mail: [email protected]

Running title: Inhibition of TRP3 channels by lanthanides

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Copyright 2000 by The American Society for Biochemistry and Molecular Biology, Inc.

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Summary

The lanthanide ions La3+ and Gd3+ block Ca2+-permeable cation channels and have been used

as important tools to characterize channels of the TRP family. However, widely different

concentrations of La3+ and Gd3+ have reportedly been required for block of TRP3 channels in

various expression systems. The present study provides a possible explanation for this

discrepancy. After overexpression of TRP3 in CHO cells, whole-cell currents through TRP3

were reversibly inhibited by La3+ with an EC50 of 4 µM. For comparison, the organic blocker

SKF96365 required an EC50 of 8 µM. Gd3+ blocked with an EC50 of 0.1 µM but this block was

slow in onset and was not reversible after wash-out. When the two lanthanides were added to the

cytosolic side of inside-out patches, block was achieved with considerably lower concentrations

(EC50 for La3+: 0.02 µM; EC50 for Gd3+: 0.02 µM). Uptake of La3+ into the cytosol of CHO

cells was demonstrated with intracellular fura-2. We conclude that lanthanides block TRP3 more

potently from the cytosolic than from the extracellular side of the plasma membrane and that

uptake of lanthanides will largely affect the apparent EC50 values after extracellular application.

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Introduction

Elucidation of the mechanisms responsible for receptor-mediated Ca2+ influx in

electrically non-excitable cells remains a continuous challenge. Although it is generally accepted

that in many cells, the predominant signal for Ca2+ influx is the depletion of intracellular

calcium stores (1), the underlying mechanisms are not known in detail. Furthermore, the molecular

structure of the Ca2+ channels permitting Ca2+ entry across the plasma membrane has not been

clarified. Proteins of the recently discovered TRP1 family may be an essential part of these

channels because antisense constructs of several TRP cDNAs inhibit store-operated Ca2+ influx

(2-6). Heterologous expression of several members of the TRP family leads to the appearance of

Ca2+-permeable cation channels (7) but these exhibit properties not congruent with those of channels

mediating store-operated Ca2+ influx, particularly the Ca2+-selective ICRAC channels (8;9).

Moreover, overexpression of corresponding TRP orthologues in different cell types by different

research groups resulted in ion currents that obeyed different regulatory principles. For example,

TRP3 was initially characterized as a constitutively active, store-independent, Ca2+-regulated

channel (10) but was also described as being regulated by diacylglycerol (DAG) (11). Other studies

suggest a store-dependent mechanism for the activation of TRP3 (2;12;13). Additionally, an important

regulation of TRP3 occurs through its interaction with the InsP3 receptor (14;15). This interaction

occurs at a defined region of TRP3 localized within the C-terminal tail that extends into the

cytosol (16). A recent report (17) on this interaction indicates that overexpressed TRP3 channels are not

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activated by store-emptying alone.

In light of these discrepant results on the regulation of TRP3, a detailed functional and

biophysical characterization would be desirable for any study on heterologously expressed TRP

family members, to clarify whether identical channels are expressed in the respective expression

systems. In general, important tools for the characterization of ion channels are channel blockers.

Unfortunately, there are no specific inhibitors known for any particular member of the TRP

family. A widely used compound is SKF96365, but it is not specific because it inhibits Ca2+

entry channels at similar concentrations and effectivities as it inhibits other channels such as Cl-

and cation channels (18). Whether SKF96365 can be used to discriminate between various Ca2+

channels or between channels appearing after overexpression of members of the TRP family has

not been determined in detail.

In the absence of specific organic blockers, many researchers have resorted to the ions of

the lanthanides gadolinium (Gd3+) and lanthanum (La3+). They block a wide range of Ca2+-

permeable channels; however, the sensitivity to Gd3+ and La3+ may vary between different

channels such that the sensitivity to lanthanides has been used as part of the characterization of

overexpressed TRP channels. In the case of TRP3, however, discrepancies in this sensitivity

have been reported. Zhu et al. required 250 µM La3+ for an inhibition by 30-40% of Ca2+ entry

through TRP3 channels expressed in COS-M6 cells; a complete block was achieved with 1 mM

(2). The same authors induced a complete inhibition with 150 µM La3+ when TRP3 was expressed

in HEK293 cells (19). An EC50 of 24 µM was estimated for the inhibition of TRP3 by La3+ in

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cultured bovine pulmonary endothelial cells (20), in line with a report that 50 µM abolished TRP3

currents in porcine aortic endothelial cells (21). Considerably lower concentrations (10 µM) induced

a complete inhibition of TRP3 in COS-1 cells (12). Gd3+ inhibited TRP3-related Ca2+ entry

completely in HEK293 cells at a concentration of 200 µM, but 10 µM in the bath could be used

to discriminate Ca2+ entry pathways endogenous in these cells from that attributable to TRP3

which was still apparent in the presence of 10 µM Gd3+ in the bath (19).

When we tested the effects of the two lanthanides on TRP3 expressed in CHO cells, we

were surprised to notice that the concentrations required for block were markedly lower than

reported anywhere else in the literature. Although the effects of lanthanides are attributed to an

action strictly confined to the extracellular side of the plasma membrane (22), we examined block by

Gd3+ and La3+ on the cytosolic side of TRP3 channels in inside-out patches. Concentrations

were effective that were even lower than those required during extracellular application.

Furthermore, entry of La3+ was directly demonstrated in fura-2 loaded cells. We propose that

lanthanides may act on TRP3 from the intracellular side and that the extracellular concentrations

at which lanthanides inhibit Ca2+ entry may be largely influenced by their cell-specific uptake

rates.

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Experimental Procedures

Cell culture

CHO cells were obtained from the German Collection of Microorganisms and Cell

Cultures (DSMZ, Braunschweig, Germany) and cultured in Ham’s F12 medium supplemented

with 0.259 g/l N-acetyl-L-alanyl-L-glutamine and 10% FCS. Cells were seeded on glass

coverslips at a density of <103 cells/mm2. Expression vectors containing the genes of interest

were imported into the cells either by intranuclear microinjection as described (10) or by transfection

(TransFast Transfection Reagent, Promega, Mannheim, Germany). The solution for injection

contained 0,3 µg/µl of the reporter plasmid pEGFP-C1 (Clontech, Heidelberg, Germany) and

1.5 µg/µl of the expression plasmid pcDNA3 (Invitrogen, Leek, Netherlands) carrying the TRP3

cDNA. For transfection we used the TRP3-pcDNA3 vector in which the neomycin resistance

was replaced by the cDNA of EGFP. Thus TRP3 (under the control of CMV promotor) and

EGFP (under the control of SV40 promotor) were expressed from the same vector construct.

Transfection was performed following the instructions of the manufacturer. After injection resp.

transfection, cells were kept in culture medium for 18-27 h.

Electrophysiology

Patch-clamp experiments were performed in the whole-cell configuration as well as in

the inside-out configuration as described (23). CHO cells exhibiting EGFP fluorescence were chosen

for the experiments. For whole-cell measurements, the solutions contained (mM): standard bath

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(“N”): NaCl 140, MgCl2 1.2, CaCl2 1.2, glucose 10, HEPES 10; pH 7.4. NMDG (N-methyl-D-

glucamine) bath (“NMDG”): same as standard bath, but NMDG substituted for Na+. Pipette

solution: CsCl2 140, MgCl2 2, EGTA 0.1, ATP 0.3, GTP 0.03, HEPES 10; pH 7.2. For inside-

out patches the solutions contained (mM): bath: Na-isethionate 120, hemi-Ca-gluconate 11.86,

hemi-Mg-gluconate 2, EGTA 10, HEPES 10, glucose 10; pH 7.4; pipette: CsCl 120, hemi-Ca-

gluconate 3.6, hemi-Mg-gluconate 2, HEPES 10, glucose 10; pH 7.4. Osmolarity was adjusted

to 300±10 mosm/kg using mannitol; the osmolarity-difference between bath- and pipette-

solution was kept below 5 mosm/kg. 1-Oleoyl-2-acetyl-sn-glycerol (OAG) was first

dissolved in DMSO and then added to the bath solution. The final concentration of OAG in the

bath was 100 µM, the concentration of DMSO was 1%. SKF96365, GdCl3, or LaCl3 were added

to the bath solution at concentrations as indicated. The holding potential was set to -60 mV. The

membrane current was recorded with a HEKA EPC-9 patch-clamp amplifier using HEKA’s

PULSE software (HEKA Elektronik, Lambrecht/Pfalz, Germany). In whole-cell mode, currents

were filtered at 1 kHz, in inside-out mode at 3 kHz; the sampling rates were set appropriately.

NPo was calculated with IgorPro (Wavemetrics, Lake Oswego, USA).

Fura-2 measurements

Measurements were performed using a digital imaging system (T.I.L.L. Photonics,

Germany). Fura-2 was excited with light of the wavelength λex=360 nm, emission was

measured at λem=510 nm. For in-vitro experiments, fura-2 salt (Calbiochem) was dissolved in

nominally Ca- and Mg-free PBS ([fura-2]=20 µM). For in-vivo experiments, CHO cells were

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loaded with fura-2 by incubation in fura-2/AM (Calbiochem) as described (24); during

measurements, the cells were kept in nominally Ca- and Mg-free PBS. Additionally, spectra of

fura-2 in the presence of various concentrations of La3+ were obtained in a RF-5001PC spectro-

fluorophotometer (Shimadzu).

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Results

Expression of TRP3 in CHO cells resulted in markedly enhanced whole-cell cation

currents, in comparison to those in control CHO cells. Currents reached a maximum right after

obtaining the whole cell configuration and declined steadily over several minutes. Inward

currents were mostly carried by Na+ but also to a minor part by Ca2+ and disappeared when

extracellular Na+ was substituted with the large impermeant cation NMDG (Fig. 1). These

results are in agreement with our previous report on functional characterization of TRP3 (10).

[Place Fig. 1 here]

To analyze the effects of lanthanides and SKF96365, we first determined the inhibition of

currents by NMDG in each experiment. This defined the amplitude of cation currents.

Thereafter, the normal bath was restored. Then, the cells were exposed to various concentrations

of the inhibitors. In some experiments, the inhibitors were washed out to test for reversibility of

the block. To quantify inhibition in a situation when currents decline spontaneously, we

performed a graphical extrapolation of the currents from time periods when no inhibitor was

present to the time in the presence of the inhibitors, thereby obtaining current values expected to

be present if inhibitors had been absent. Actual currents in the presence of inhibitors were

divided by these extrapolated current values, yielding the relative inhibition.

The inhibition of TRP3 currents by La3+ (Fig. 1A) and SKF96365 (Fig. 1B) was clearly

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distinct from the spontaneous current decline because it occurred much faster and was dependent

on the concentration of the respective substance. An EC50 value of 4 µM was assessed for La3+

(Fig. 2A). This inhibition could be reversed by wash-out of La3+ (Fig. 1A). SKF96365 did not

completely block the currents even at the maximal concentrations applied (30 µM); the EC50

value was estimated to be 8 µM (Fig. 2B).

[Place Fig. 2 here]

Similarly as SKF96365 and La3+, Gd3+ at concentrations between 1 and 30 µM induced

a rapid inhibition of TRP3 currents (data not shown). This inhibition was complete since no

further current depression was evoked by NMDG. Washout of Gd3+ did not lead to a recovery of

the currents. When lower concentrations (0.1-0.3 µM) of Gd3+ were used (Fig. 1C), inhibition

of the currents occurred slowly although still distinctly faster than the spontaneous decline. The

absolute amplitude of the currents in the presence of these low concentrations was above that

after substitution of extracellular cations with NMDG, indicating a partial inhibition by Gd3+.

The EC50 was estimated to be 0.1 µM (Fig. 2C).

The peculiar kinetics of the Gd3+ effects could be explained if the action of the ion on

TRP3 channels occurred from the cytosolic side of the membrane after a slow entry of Gd3+ into

the cell. To test this hypothesis directly, we performed experiments with inside-out patches in

which the cytosolic side of the patches was exposed to Gd3+ concentrations from 30 nM to 1

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µM. As reported (10), TRP3 channels in inside-out patches from CHO cells are characterized by a

spontaneous channel activity right after obtaining the inside-out configuration and a decline of

the activity over 3-6 min. Since it has been reported that a “rigorous” wash abolishes channel

activity (14), exchange of the bath was performed by a slow exchange of the bath volume over 15-20

s which did not cause rapid changes in channel activities.

[Place Fig. 3 here]

Since channel activity was low in most patches and is difficult to quantify due to the short

open times (mean open time ≤ 0.2 ms according to (10)), effects of higher Gd3+ concentrations (i.e.

0.1 and 1 µM) were analyzed in patches stimulated with the membrane permeable DAG analogue

OAG. OAG considerably stimulated TRP3 channels for about 2 min (Fig. 3A), in accordance

with the report by Hofmann et al. (11). Addition of Gd3+ at 1 µM (5 out of 5) and 0.1 µM (10 out of

10) to those patches abolished channel activity within a few seconds (Fig. 3B). Wash-out of

Gd3+ did not restore channel activity even in the continuous presence of OAG (not shown).

[Place Fig. 4 here]

For a quantification of the concentration-dependence of Gd3+, unstimulated patches

were used because the time course of OAG-induced stimulations was too variable to allow

calculation of relative inhibitions. Each patch was consecutively exposed to two increasing

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concentrations of Gd3+ (0.03 and 0.1 µM or 0.3 and 1.0 µM) for time periods of 40-50 s each.

Afterwards, a Gd3+-free bath was reestablished. Gd3+ induced inhibitions of channel openings

as fast as the bath exchange was performed, in a concentration-dependent manner (Fig. 4, 5A).

Again, wash-out of Gd3+ failed to restore channel activity. The same result was obtained when

patches were exposed to only one concentration (0.1 µM) of Gd3+ for a short (20 s) time (data

not shown). To test whether this observation indicates a long-lasting inhibition of TRP3

channels by Gd3+ or, alternatively, may reflect spontaneous complete decline of channel

activity, the same protocol was performed with La3+ (0.03-1 µM) as inhibitor applied to the

cytosolic side of inside-out patches (Fig. 6). There was a concentration-dependent reduction of

TRP3 channel activity (Fig. 5B). In contrast to the experiments involving Gd3+, removal of

La3+ led to a restoration of channel activity that may be considered complete if the spontaneous

inactivation of the channels is taken into consideration (Fig. 6). Estimated EC50 values are 0.02

µM Gd3+ and 0.02 µM La3+.

[Place Fig. 5 here]

[Place Fig. 6 here]

To demonstrate that La3+ actually enters the cytosol of CHO cells, we performed

experiments with fura-2. A shift in the excitation spectrum of fura-2 by lanthanum has been

descibed (25), resulting in an increase in fluorescence at excitation wavelengths from 300 to 350 nm.

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At 360 nm, we found that La3+ led to a concentration-dependent decrease in the fluorescence of

fura-2 in vitro (Fig. 7A). At shorter excitation wavelengths, the previously described increase in

fluorescence was reproduced but we were unable to discriminate between the effects of La3+ and

those of contaminating Ca2+ (Fig. 7B). Therefore, we used an excitation wavelength of 360 nm

(the isosbestic wavelength for Ca2+ in the absence of La3+) to examine the effects of La3+ on

cells loaded with fura-2 after incubation with fura-2/AM. Addition of La3+ to the bath evoked a

rapid decrease in the fluorescence of the cells. This effect was reversible after wash of the cells

(Fig. 7C). Thus, CHO cells are capable to take up and to extrude La3+ at rates compatible with

the observed block of TRP3 currents in whole-cell experiments. We did not detect any sizeable

decrease of fura-2 fluorescence after addition of Gd3+ in vitro, therefore a similar demonstration

of Gd3+ uptake into CHO cells was not possible.

[Place Fig. 7 here]

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Discussion

The important findings of this study are that the cations Gd3+ and La3+ inhibit TRP3

currents when applied from the extracellular as well as from the intracellular side of the cell

membrane. Concentrations of either ion that evoked half-maximal inhibitions from outside were

consistently found to inhibit TRP3 channels completely when added to the cytosolic side of

inside-out patches. Intracellular block by lanthanides may be of particular relevance in CHO

cells because entry of La3+ was directly demonstrated. Therefore, the experimentally determined

potency of extracellular Gd3+ and La3+ on TRP3 channels will be strongly influenced by the

rate with which extracellular and intracellular concentrations reach an equilibrium. This may be

of general importance in attempts to characterize TRP3 and possibly other TRP channels after

heterologous expression. Discrepancies of results between various expression systems may

simply reflect differences in the uptake and extrusion rate of Gd3+ and La3+, rather than

differences in the expressed gene products.

When comparing the extracellular effects of Gd3+ with that of La3+, Gd3+-induced

current inhibitions were much slower in onset. Gd3+ block was slow even in comparison with

SKF96365, which otherwise proved to be an unsuitable pharmacological tool because of its high

EC50 that precluded preparation of solutions which would evoke a complete inhibition of TRP3.

In contrast to the results with extracellular Gd3+, Gd3+ applied to the cytosolic side of inside-

out patches evoked its effects without any delay other than that attributable to the time required

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for the bath exchange. The block by Gd3+ was irreversible over the observation times of this

study. This may indicate a long-lasting binding of Gd3+ to the TRP3 protein at low

concentrations.

Our results suggest that whenever entry of Gd3+ and La3+ into cells occurs, it will

certainly enhance blocking effects on TRP3. It may be asked whether inhibition of TRP3

currents by lanthanides is completely due to intracellular rather than extracellular action.

However, it cannot be deduced from our experiments whether entry is essentially required and to

what extent block from the outside takes place. An experimental protocol to exclude extracellular

effects would require outside-out patches. Unfortunately, we were unable to detect TRP3

channel activity in those patches. The reason for that failure may be the noise level, that was

considerably higher than in inside-out patches. Under these circumstances, analysis of channel

openings of TRP3 is impossible since the mean open time is extremely short. Even if

experiments with outside-out patches were successful, they might not definitely rule out a

strictly intracellular action of Gd3+, because transmembrane pathways for Gd3+ may be present

in isolated patches as well as in whole cell preparations. Taken together, we cannot exclude

extracellular effects of Gd3+, but if they are present, they are certainly weaker than the

intracellular ones.

In the case of La3+, the differences of equipotent extracellular and intracellular

concentrations were more pronounced than in experiments with Gd3+. Thus, even a relatively

small rate of La3+ uptake may account for La3+ block. At the same time, reversibility of the

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extracellular effects would be achieved by a modest extrusion rate.

Interestingly, lanthanides are likely to be chelated by Ca2+ chelators such as EGTA and

fura-2 (22), in spite of the fact that they differ from Ca2+ in their valency. In experiments where

La3+ inhibited Ca2+ entry at extracellular concentrations of 0.5 µM, addition of EGTA (10 µM) to

the bath completely abolished this effect (26). A reported (27) dissociation constant for the Gd-EGTA-

complex (log K = -17.5) would mean that practically all Gd3+ was bound to EGTA in our

experiments with inside-out patches when the EGTA concentration in the cytosolic bath was

10 mM. EGTA was also used in the pipette solution of whole-cell current measurements and

should therefore be present in the cytosol. Considerable chelation would moreover be expected

by cytosolic fura-2. Therefore, the blocking species in our experiments might be lanthanide

complexes rather then lanthanide ions. A similar mechanism has been proposed by Caldwell et

al. (27) for effects of Gd3+ in the presence of phosphate and bicarbonate (28-31). (Phosphate and

bicarbonate anions are known to form complexes with lanthanide ions (32).)

Effects of La3+ and of Gd3+ are frequently considered to be strictly confined to the

extracellular space because cell membranes are believed to be impermeable for these ions (22).

However, there are exceptions reported for red blood cells (33) as well as myocardial cells (34). In our

experiments, the fluorescence of intracellular fura-2 in CHO cells was decreased after addition

of La3+ to the bath, as was the fluorescence of fura-2 in vitro by La3+. As excitation wavelength

in the experiments with cells, we chose the isosbestic wavelength of fura-2 for Ca2+ in the

absence of La3+. Therefore, any decrease in the intracellular Ca2+ concentration that a block of

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Ca2+ influx might induce would minimally affect the fluorescence of fura-2. Furthermore, the

experiments were performed in nominally Ca-free bath (estimated Ca2+ concentration 50 µM) in

which block of Ca2+ channels is expected to have no instant effects on intracellular Ca2+

concentrations. Therefore, the experiments demonstrate that La3+ rapidly enters CHO cells and is

also quickly extruded from the cytosol after wash. Since these findings were obtained in wild-

type CHO cells, transmembrane movements of La3+ are not dependent on expression of TRP3.

The changes of fluorescence occurred as rapidly as block of TRP3 currents by La3+. Whereas

these experiments provide information about the time over which cytosolic concentrations of

La3+ change, they do not allow a quantification of these concentrations. The cytosolic concentration

of fura-2 would be important for the calculation of La3+-concentrations but is not known under

our experimental conditions. Nevertheless, it is safe to conclude that La3+ enters CHO cells in

concentrations relevant for block of TRP3 channels.

One of the disturbing points of the literature on members of the TRP family are the

apparent inconsistencies of results reported by different research groups. For studies that

performed an overexpression of TRP proteins, this raises the question whether cation channels

formed by the overexpressed gene products are identical. However, when channels are

characterized by means of block with lanthanides, our present study emphasizes that conflicting

results may be explained not by differences in the channel structure but by different

transmembrane transport rates of lanthanides. We propose that any quantification of block by

lanthanides should include a discrimination of extracellular and intracellular effects.

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Acknowledgements

This work was funded by the Deutsche Forschungsgemeinschaft SFB542. We thank

Ilinca Ionescu for technical assistance.

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32. Martell, A. E. and Smith, R. E. (1976) Critical Stability Constants. Inorganic Complexes, 1st Ed., Plenum Press, New York

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34. Peeters, G. A., Kohmoto, O., and Barry, W. H. (1989) Am.J.Physiol. 256, C351-C357

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Footnotes

Abbreviations used: CHO cells: Chinese hamster ovary cells, COS cells: African green monkey

kidney cells, DAG: diacylglycerol, EGTA: ethylene glycol-bis(β-aminoethyl ether)-

N,N,N’,N’-tetraacetic acid, HEK cells: human embryonic kidney cells, ICRAC: calcium release

activated calcium current, InsP3: inositol-1,4,5-trisphosphate, NMDG: N-methyl-D-

glucamine, NPo: number of channels in a patch multiplied by the open probability of a single

channel, OAG: 1-oleoyl-2-acetyl-sn-glycerol, PBS: Dulbecco’s phosphate-buffered saline,

SEM: standard error of the mean, TRP: transient receptor potential.

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Figure Legends

FIG 1: Effects of La3+, SKF96365, and Gd3+ on TRP3-currents in whole-cell patch-clamp

experiments. Cells injected or transfected with cDNA of TRP3 and EGFP were identified as

expressing by EGFP fluorescence. Cells were kept in normal bath solution (N). After

establishing the whole-cell configuration (wc), the bath was changed to a solution in which Na+

was substituted by NMDG. After restoring the normal bath (N), various concentrations of

channel blockers were applied as indicated. Panel A: La3+ at 3 µM and 5 µM (followed by a

wash-out when bath solution N was restored); B: SKF96365 at 10 µM and 30 µM; C: Gd3+ at

0.1 µM and 0.3 µM.

FIG 2: Concentration dependencies of block by La3+, SKF96365, and Gd3+ in whole-cell

patch-clamp experiments. The numbers of individual experiments for each concentration are

indicated. Error bars denote SEM. EC50 values were assessed by fitting the data with the

variable-slope sigmoidal dose-response function. The respective EC50 values are: La3+ 4 µM

(A); SKF96365 8 µM (B); Gd3+ 0.1 µM (C).

FIG 3: Stimulation of TRP3 channel activity by OAG and subsequent inhibition by Gd3+ in

inside-out patch-clamp experiments. Channel activity is expressed as NPo over time. The

holding potential was -60 mV. At the time points indicated by arrows, the bath was exchanged

to a solution containing 100 µM OAG (panel A, B) or 0.1 µM Gd3+ in addition to 100 µM OAG

(panel B). The insert shows a sample trace of the single channel recording. The time at which the

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sample was taken is indicated in panel A with an *.

FIG 4: Irreversible concentration-dependent block of TRP3 channel activity by Gd3+ in inside-

out patch-clamp experiments. A: Channel activity, expressed as NPo over time. At the arrows,

the bath solutions were changed as indicated. B-D: Sample tracings during basal activity (B) and

in the presence of 0.03 or 0.1 µM Gd3+ (C,D).

FIG 5: Concentration-dependent block by Gd3+ and La3+ in inside-out patch-clamp

experiments. (Each data point represents 2-3 experiments, error bars denote SEM.) EC50 values

were assessed by fitting the data with the variable-slope sigmoidal dose-response function. The

respective EC50 values are: Gd3+ 0.02 µM (A); La3+ 0.02 µM (B).

FIG 6: Reversible concentration-dependent block of TRP3 channel activity by La3+ in inside-

out patch-clamp experiments. Channel activity, expressed as NPo over time. At the arrows, the

bath solutions were changed as indicated.

FIG 7: La3+ uptake by CHO cells. A: Concentration-dependent decrease of fura-2 fluorescence

in vitro after application of La3+. The relative fluorescence 1.00 was obtained in the absence of

La3+. Fura-2 (20 µM) was dissolved in nominally Ca- and Mg-free PBS. B: Spectra of fura-2

in the presence of La3+. Spectra were obtained from a fura-2 solution (1 µM, dissolved in

distilled water) to which La3+ was cumulatively added to the final concentrations indicated.

Further addition of Ca2+ (100 µM) resulted in the spectrum shown as dashed line. A further

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spectrum was obtained after addition of EGTA (2 mM). C: Reversible decrease of fura-2

fluorescence in CHO cells after addition of La3+ (10 µM) to the bath. Similar results were

obtained in two further experiments.

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Christian R. Halaszovich, Christof Zitt, Eberhard Jüngling and Andreas Luckhoffplasma membrane

Inhibition of TRP3 channels by lanthanides: block from the cytosolic side of the

published online September 1, 2000J. Biol. Chem.

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