ATP/UTP activate cation-permeable channels with TRPC3/7 properties in rat cardiomyocytes

Julio Alvarez1,2, Alain Coulombe3, Olivier Cazorla1, Mehmet Ugur1, Jean-Michel Rauzier1, Janos

Magyar4, Eve-Lyne Mathieu5, Guylain Boulay5, Rafael Souto2, Patrice Bideaux1, Guillermo Salazar1,

François Rassendren6, Alain Lacampagne1, Jérémy Fauconnier1, Guy Vassort1

1 Inserm, U637, F-34925 Montpellier, France. Université Montpellier 1, UFR de Médecine, CHU

Arnaud de Villeneuve, F-34925 Montpellier, France

2Laboratorio de Electrofisiología, Instituto de Cardiología, La Habana, Cuba

3Inserm UMR S621, Université Pierre et Marie Curie, CHU Pitié-Salpêtrière, 91 bd de l’Hôpital, 75634

Paris, France

4Department of Physiology, University of Debrecen, P.O. Box 22, Debrecen, 4012, Hungary

5Pharmacology, University of Sherbrooke, Faculty of Medicine, Fleurimont, Québec J1H 5N4. Canada

6Department of Pharmacology, Institut de Génomique Fonctionnelle, CNRS UMR 5203, Inserm U661,

Université Montpellier I, Université Montpellier II, 141 rue de la Cardonille, 34396 Montpellier Cedex,

France.

Address for correspondence :

Dr Vassort Guy

INSERMU-637, Physiopathologie cardiovasculaire,

CHU Arnaud de Villeneuve

F-34295 MONTPELLIER, France

e-mail: vassort@montp.inserm.fr

T° 33467415248 Fax 3341674142

Running head : ATP/UTP activate TRPC3/7 channels in rat cardiomyocytes

Keywords: Purinergic receptor, Signal transduction, Infarction, Arrhythmia

Abstract: 247 words. Figures : 8. Table: none.

Articles in PresS. Am J Physiol Heart Circ Physiol (May 23, 2008). doi:10.1152/ajpheart.00135.2008

Copyright © 2008 by the American Physiological Society.

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ABSTRACT

Extracellular purines and pyrimidines have major effects on cardiac rhythm and contraction. ATP/UTP

are released during various physiopathological conditions such as ischemia and, despite degradation by

ectonucleotidases, their interstitial concentrations can markedly increase, a fact that is clearly associated

with arrhythmia. In the present whole-cell patch-clamp analysis on ventricular cardiomyocytes isolated

from various mammalian species, ATP and UTP elicited a sustained, non-selective cationic current, IATP.

UDP was ineffective while BzATP was active, suggesting that P2Y2 receptors are involved. IATP resulted

from the binding of ATP4- to P2Y2 purinoreceptors. IATP was maintained after ATP removal in the

presence of GTPγS and was inhibited by U-73122, a phospholipase C inhibitor. Single channel openings

are rather infrequent under basal conditions. ATP markedly increased opening probability, an effect

prevented by U-73122. Two main conductance levels of 14 and 23 pS were easily distinguished.

Similarly, in Fura 2-loaded cardiomyocytes Mn2+ quenching and Ba2+ influx were significant only in the

presence of ATP or UTP. Adult rat ventricular cardiomyocytes expressed TRPC1, 3, 4, 7 mRNA and the

TRPC3 and TRPC7 proteins that co-immunoprecipitated. Finally, the anti-TRPC3 antibody added to the

patch pipette solution inhibited IATP. In conclusion, activation of P2Y2 receptors, via a G-protein and

stimulation of PLCβ, induces the opening of heteromeric TRPC3/7 channels leading to a sustained, non-

specific cationic current. Such a depolarizing current could induce cell automaticity and trigger the

arrhythmic events during an early infarct when ATP/UTP release occurs. These results emphasize a

new, potentially deleterious, role of TRPC channel activation.

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INTRODUCTION

ATP, a high-energy phosphate donor, has been extensively studied since the early description of a role

for extracellular purines by Drury & Szent-Györgyi in 1929 (11). A low ATP concentration is present in

the interstitial space despite its degradation by ectonucleotidases; moreover, its level can markedly

increase during various physiopathological conditions (40). Particularly ATP and UTP are released

during ischemia from various cell types including cardiomyocytes (12) and this was shown to be

associated with arrhythmia (21, 42). However the mechanisms that lead to arrhythmia are unknown.

This is of importance since the early phase of arrhythmia during an ischemic period in patients is highly

deleterious and is not sensitive to presently known pharmacological agents.

Extracellular ATP activates both the ionotropic (ligand-gated) receptors, P2X receptor family

and the metabotropic (G-protein coupled) receptors, P2Y receptor families (40). The P2Y family is

divided into two structurally distinct subfamilies. The first is composed of P2Y1, P2Y2, P2Y4, P2Y6, and

P2Y11 receptors all coupled to Gq which promotes phospholipase C activation and diacylglycerol (DAG)

production. The others, P2Y12, P2Y13, P2Y14 are coupled to Gi, inhibiting adenylate cyclase (6, 14).

Among the first, P2Y2,4,6 could also be activated by UTP to various extents. P2-purinergic stimulation

has multiple effects on cardiac ionic currents (40). On cells clamped at the resting potential, a fast

application of ATP elicits a transient inward current that requires extracellular Mg2+ (8, 31, 32).

Furthermore during prolonged ATP application, in the presence of Mg2+ or not, after deactivation of the

initial transient inward current, a weak sustained inward current can be recorded (31, 35). The nature of

the channel protein that carries this later current is unknown.

Transient receptor potential, TRP channels were first described in the phototransduction system

of Drosophila melanogaster. Mammalian homologues encode channel proteins that have six

transmembrane domains and assemble into heterotetramers (9, 28, 41). TRP are widely distributed in

mammalian tissues and are involved in several cardiovascular functions and diseases (18, 25). Like P2X

purinoceptors, most TRP channels are non selective to cations and act to shift the membrane potential to

around 0 mV, thus depolarizing cells from resting potential and allowing Ca2+ influx and cell

automaticity. The TRPC subfamily is composed of seven members, TRPC1-7, with the TRPC3,6,7

subgroup being directly activated by diacylglycerol (24). TRPC3- and TRPC7-expressing cells were

both demonstrated to have both constitutively activated and ATP-enhanced inward currents that allow

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Ca2+ influx (17, 26). Recently, TRPC6 and TRPC6/7 have been identified as essential part of the α1-

adrenoceptor-activated cation currents in smooth muscle cells (19) while in heart TRPC3 and TRPC6

proteins are essential for angiotensin II-induced hypertrophy (7, 27) and TRPC3 to the potentiated

insulin-induced current (13).

In the present work at the cellular level, we show that ATP and UTP activate purinergic P2Y2

receptors. These receptors, via a G-protein-dependent activation of PLCβ, trigger a fast-activating,

sustained, non-selective cationic current through TRPC3/7 channels.

MATERIALS AND METHODS

Experimental models and chemicals. Experiments were performed on cardiomyocytes isolated

from adult male Wistar rats, or otherwise specified. Rats were killed by an i.v. injection of

pentobarbitone (100 mg kg-1). The investigation conforms with the Guide for the Care and Use of

Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23,

revised 1996). Cardiomyocytes were also isolated from post-myocardial infracted, PMI rats (1) and from

control and transgenic mice deficient for the P2X1, P2X4 or for both P2X1-P2X4 purinergic receptors

(36) using a similar procedure as well as from control dog and human according to (39). All materials

were purchased from Sigma-Aldrich, except SKF96365 (Calbiochem), U-73122 (Biomol), Fura-2 AM

(Molecular Probes, Eugene, OR). Anti-TRPC antibodies raised against putative intracellular epitopes

were obtained from Alomone Laboratories (Israel, anti-TRPC3 and anti-TRPC6 antibodies) and the anti-

TRPC7 antibody, a generous gift from Professor W.P. Schilling (Cleveland, US), was made against the

sequence 843EKFGKNLNKDHLRVN857 and was demonstrated to recognize no other TRPCs (15).

Electrophysiological experiments. Rat ventricular myocytes were enzymatically dissociated using a

method previously described (4) were kept in physiological solution: (mM): NaCl 117, KCl 5.4, CaCl2

1, MgCl2 1.0, glucose 10, HEPES 10, pH 7.4 at 22°-24°C and were used within 6-8 hours. Currents were

recorded using the "whole-cell" variant of the patch-clamp method at 22°-24°C. K+ currents were

blocked by Cs+. Compared to the physiological solution, the standard extracellular solution contained 20

CsCl, instead of KCl, and 2 CaCl2. The pipette ("intracellular") solution contained (mM): CsCl 130,

Na2-GTP 0.4, Na2-ATP 5, Na2-creatine phosphate 5, ethyleneglycol-bis-(-aminoethyl ether) N,N,N',N'-

tetraacetic acid (EGTA) 11, CaCl2 4.7 (free Ca2+ 108 nM); HEPES 10; pH adjusted to 7.2 with CsOH.

For the experiments cells were placed, in Petri dishes containing the same solution, on the stage of an

inverted microscope. A cell attached to the micropipette could be positioned on the extremity of each of

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six microcapillaries (i.d. 250 µm; Tygon microbore tubing; Norton Performance Plastics, Wayne, NJ,

USA) through which the different extracellular solutions were perfused by gravity at a rate of 0.1ml/mn.

Solution changes were accomplished within # 1 s. Alterations of the current at a holding potential of –80

mV were analysed every 4s. Currents were scaled to cell capacitance.

Single-channel recordings were performed with the classical cell-attached patch-clamp

configuration. Only rod-shaped adherent cells isolated from rat heart with clear striations, sharp edges,

without granulation and showing no spontaneous contractile activity, were chosen. Patch pipettes were

pulled from borosilicate glass capillaries (Corning Kovar Sealing code 7052, WPI, FL) using a

horizontal puller (DMZ-Universal Puller, Zeitz Instrument, Germany) and fire polished before use. The

pipette resistance was 5–10 MΩ. The currents were recorded using a patch-clamp amplifier (Axopatch

200B, Axon Instruments, CA) and filtered through an eight-pole Bessel low pass filter 920LPF

(Frequency devices) setting of 1 kHz (-3dB point). Data were digitized at 5 kHz with a Digidata 1200

(Axon Instruments) using Acquis1 software (G. Sadoc, CNRS, Gif/Yvette, France). Single channel

activity was recorded only when the seal resistance was equal or larger than 10 GΩ. All experiments

were conducted at room temperature (20-24°C). Elementary conductances were determined as

previously reported (10). Channel activity (mean patch current) was calculated by integrating current

flow during the channel openings and dividing the integral by the total sampling time. The K+-rich

medium in which cells were maintained before use, contained (mM): L-glutamic acid monopotassium

salt, 70; KCl, 25; KH2PO4, 10; MgCl2, 3; EGTA, 0.5; HEPES, 10; glucose, 20; taurine, 10; pH was

adjusted to 7.4 with KOH. The superfusion solution contained (mM): NaCl 135, KCl 4, MgCl2 2, CaCl2

2, HEPES 10 and glucose 20; pH adjusted to 7.4 with NaOH. The pipette solution contained (mM):

NaCl 135, KCl 4, CaCl2 2, HEPES 10, glucose 20; pH adjusted to 7.4 with NaOH. In some experiments

Na2ATP (100 µM) was added to the pipette solution.

Measurements of changes in [Ca2+]i. Rat ventricular cardiomyocytes bathed in the

physiological solution were loaded with Fura-2 AM (2.5 µM) for 30 min at 35°C and then allowed to

attach on a coverslip. Fluorescence images (2 to 4 rod-shaped cells in a field) were recorded and

analysed with a video image analysis system (MetaMorph 6.0/6.1, Universal Imaging Corporation). The

Fura-2 fluorescence image at an emission wavelength of 510 nm (bandwidth 20 nm) was followed at 22-

24°C by exciting Fura-2 alternatively at 340 and 380 nm (bandwidth 11 nm) to get a 340/380 ratio on a

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pixel/pixel basis. Ca2+ was removed (Ca2+-free solution) and later Ba2+ was added (2 mM, Ba2+-

solution).

Biochemical analyses. For Reverse Transcription (RT)-PCR, total RNA was extracted from

myocytes or brain using TRIzol (Invitrogen) according to the manufacturer's specifications and reverse

transcribed into cDNA using random hexamer (Amersham Pharmacia Biotech). An aliquot of the first

strand cDNA was used as a template for PCR and rat TRPCs were amplified using the following primer

sets: TRPC1 (forward, 5'-CAAGATTTTGGGAAATTTCTAG-3' and reverse, 5'-

TTTATCCTCATGATTTGCTAT-3'); TRPC3 (forward, 5'-TGACTTCTGTTGTGCTCAAATATG-3'),

reverse, 5'-CCTTCTGAAGTCTTCTCCTCCTGC-3'); TRPC4 (forward, 5'-

TCTGCAGATATCTCTGGGAAGAATGC-3', reverse, 5'-AAGCTTTGTTCGAGCAAATTTCC-3');

TRPC5 (forward, 5'-ATCTACTGCCTAGTACTACTGGCT-3', reverse 5'-

CAGCATGGTCGGCAATGAGCTG-3'), TRPC6 (forward, 5'-TCACTTGGAAGAACAGTGAAAGA-

3', reverse 5'-CATCCTCAATTTCCTGGAATGAAC-3'), and TRPC7 (forward, 5'-

ACCTTCACAGACTACCCCAAAC-3', reverse 5'-GCCAAATATGGACCAAAACAAGG-3'). The

resulting PCR product was analyzed by ethidium bromide-agarose gel electrophoresis and cloned into

the pCR®2.1-TOPO plasmid vector (Invitrogen) for sequencing analysis.

For Western blotting, adult rat cardiomyocytes isolated from left ventricles were quick-frozen,

then homogenized. Proteins were heated for 5 min at 60°C in SDS sample buffer (2% SDS, urea 8 M,

DTT 0.08 M, Tris 0.05 M, EDTA 1 mM, EGTA 1 mM, PMSF 0.5 mM, E64 10 µM and leupeptin 40

µM). Proteins were separated by 7.5% SDS-PAGE, followed by transfer to 0.45-µm PVDF membranes

using standard techniques. The membranes were blocked with 3% bovine serum albumin (BSA) in 0.1%

Tween-20 TBS (TBS-T). Membranes were labelled overnight with primary antibodies (anti-TRPC3 and

anti-TRPC7, 1:200) 0.1% BSA TBS-T and washed with TBS-T prior to labelling with 1:5.000

horseradish peroxidase conjugated anti-rabbit antibody. Immunodetection was revealed with West Pico

chemiluminescent substrate (Pierce Biotechnology). Quantification of signals was performed by

densitometry using an imaging system (Kodak Image Station 2000R).

For the immunoprecipitation experiments, all reactions were performed while tumbling at 4°C.

Cell protein extracts (1 mg) were pre-cleared for 1 H with Protein A Sepharose CL-4B beads

(Pharmacia) and then incubated for 1 H with 5 µl of antibody. Antibody-protein complexes were

captured by the addition of Protein A Sepharose and incubated 1 H to facilitate binding.

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Immunoprecipitated complexes were eluted from the beads using SDS sample buffer prior to SDS-

PAGE and immunoblotting. Blots were visualized as described above.

Statistical analysis. The values are the mean of n cells ± S.E.M. Statistical analysis was carried

out using paired (comparing effects of agents on same cell) or un paired (comparing effects between

cells) Student’s t tests with the level of significance set at P < 0.05.

RESULTS

P2Y2 purinergic receptor mediates the ATP/UTP effect in isolated cardiomyocytes. The

mechanisms by which ATP triggers cardiac tissue automaticity, namely the nature of the ATP-induced

sustained inward current, IATP and its signal transduction pathway were investigated at the cellular level.

The present study was mostly conducted in Mg2+-free extracellular solutions taking advantage that IATP

did not require the presence of Mg2+ for its activation. This allowed us to eliminate the initial transient

surge of inward current (31). As can be seen in figure 1A, in the presence of Mg2+, application of 1-mM

ATP triggered a fast downward change in the holding current that rapidly decreased. This transient

inward current was followed by a sustained inward current. In the absence of Mg2+, only a sustained

ATP-induced inward current, IATP was seen. Furthermore, IATP was significantly increased in the Mg2+-

free solution. IATP activated and deactivated within a minute on ATP application and withdrawal. IATP

amplitude increased stepwise with cumulative ATP-concentrations within the range from 30 µM to 3

mM (Fig. 1B). Under our experimental conditions that omitted Mg2+, IATP could be elicited by ATP and

UTP with similar concentration-dependency and amplitude-range on adult ventricular cardiomyocytes

isolated from control and transgenic mice deficient for the P2X1, P2X4 or for both P2X1-P2X4 purinergic

receptors, post-myocardial infarcted rats as well as from control dog and human (Fig. 1C).

Under our experimental conditions, 2’,3’-0-(benzoylbenzoyl)ATP, BzATP, adenosine-5’-0-

(thiotriphosphate), ATPγS, both applied at 100 µM, were slightly less effective (70% of the ATP effect,

n>5 cells). Furthermore, whenever used at 30 µM or 1 mM, the ATP analogues adenylyl-

imidodiphosphate, AMP-PNP and α,β-metATP (see also Fig. 6 in (32)) as well as ADP did not affect

the holding current. UDP was also ineffective to trigger IATP. IATP exhibited a specific pharmacology

that excluded it to be carried by any of the P2X receptors including P2X7 (38). First, BzATP described to

be more potent than ATP to activate P2X7 receptor induced a slightly weaker current. Second the strong

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P2X inhibitors, pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic acid, PPADS, oxidized ATP and

Brilliant Blue, did not significantly affect IATP. However, suramin, a relatively selective P2Y1-P2Y2

antagonist, added at 100 µM reduced by 45% the sustained current induced by 1-mM ATP (not shown

n>4). Moreover, in cells patched with a pipette that contained a solution with 500-µM GTPγS, IATP

occurred with similar kinetics and amplitude on ATP addition whereas, on ATP removal, the inward

current was maintained in agreement with the metabotropic nature of the receptor (see Fig. 4B).

Collectively, these data indicate that ATP and UTP elicited a sustained current through activation of the

purinergic P2Y2 -subtype receptors (40, 43).

To elucidate the nature of the P2Y2 ligand, ATP2- or ATP4-, (and UTP2- or UTP4-) we compared

the effects of two external solutions with similar calculated free ATP4- and Ca2+ activities but varying

the ATP and MgCl2 concentrations. Except for the initial surge of transient current related to the

presence of Mg2+, IATP had similar amplitude under these two experimental conditions indicating that

ATP4- was the P2Y2 specific agonist (Fig. 2A). At a constant 300-µM free external Ca2+ concentration,

the EC50 for the ATP4- effect determined in various ATP-containing, but Mg2+-free solutions was 58 µM

(Fig. 2B).

P2Y receptor activation triggers multiple signal transduction pathways in heart including the

production of DAG by various phospholipases (40). In the present experiments, activation of the

sustained inward current by ATP was prevented by U-73122, a common PLC inhibitor, not by its

inactive analogue U-73433 nor by propranolol and AACOCF3, respectively inhibitors of PLD and PLA2

cascades to produce DAG (2). Furthermore, IATP was neither modified by LY294002 and wortmannin,

nor by genistein respectively PLCγ, PI3K and tyrosine kinase inhibitors (Fig. 3). Altogether these data

indicate that PLCβ was stimulated by the P2Y2-receptors through a G-protein to produce DAG.

Characteristics of the ATP/UTP-induced inward current in rat cardiomyocytes. The IATP-

voltage relationship, recorded during a negative voltage ramp from +50 mV to –100 mV, exhibited a

weak voltage-dependence and showed a current reversal potential near 0 mV indicating that the channel

protein had a low selectivity for cations (Fig. 4A). Although a detailed analysis had not been performed

it was observed that the equimolar substitution of external Na+ by NMDG+ only slightly reduced (6 mV)

the reversal potential of the ATP-induced current (n=3).

To investigate the effect of various [Ca2+]o and avoid changing the active ATP4- concentration, a

GTPγS-containing solution was used in the pipette and ATP was applied for a short period, however

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sufficient to activate the then maintained IATP. At that point, reducing Ca2+ in the perfusing solution

markedly enhanced IATP. This effect was reversible, voltage-independent with a twofold increase in

chord conductance when reducing [Ca2+]O 20-fold and without significant effect on the reversal potential

(Fig. 4 B,C). This indicates that the channel was inhibited by external Ca2+ despite allows Ca2+ to pass

through. Furthermore, cyclopiazonic acid and FK506, reported to increase [Ca2+]i, reduced significantly

IATP (Fig. 4 D). To further characterize the channel properties various pharmacological compounds were

used, namely La3+ and Gd3+ and the imidazole derivative SKF96365 that were reported to inhibit Ca2+

permeable TRP channels, including the ATP-activated TRPC7 channel (3, 26). Likewise, these

compounds significantly reduced the ATP-induced current in rat ventricular cardiomyocytes. Flufenamic

acid, a common inhibitor of expressed homomeric TRPCs except TRPC6 (19) (but see (5)), slightly

increased it (Fig. 4D). Besides, several compounds known to alter various currents in cardiac tissues,

lidocaine (100 µM), amiodarone (10-100 µM), quinidine (10-100 µM) and glibenclamide (50 µM) as

well as isoproterenol (1 µM) did not significantly affect IATP (n ≥4).

Under control conditions in cell-attached patch-clamp, the patch membrane continuously held

at -80 mV demonstrated very rare single channel openings. However, when the pipette contained 100-

µM ATP, numerous openings were recorded (Fig. 5). The current reversed near 0 mV and showed no

rectification. The two most frequently observed current levels exhibited conductances of 14 and 23 pS.

In line with the above reported signal transduction cascade, channel activity was markedly reduced when

the cell was bathed in the presence of U-73122.

Further properties of the sustained cationic current were determined by

microspectrofluorescence analysis. The application of 30 µM ATP, in the absence of Mg2+, was

sufficient to trigger an influx of Mn2+ indicated by a marked quenching of the Fura-2 emissions

following excitations at 340 and 380 nm wavelengths while the signals varied in opposite direction on

ATP application in the control conditions as a consequence of Ca2+ influx and ATP-induced intracellular

Ca2+ release (8, 30). Quenching was only very weak under basal conditions in the presence of Mn2+

before ATP application (Fig. 6a). We also used Ba2+ as a surrogate for Ca2+ to estimate cation influx.

Adding Ba2+ to the Mg2+-free, Ca2+-free solution did not affect basal cell fluorescence. The further ATP

application induced a significant Ba2+ influx (Fig. 6B). Ba2+ influx rate was similar in the presence of

UTP while UDP was inefficient to elicit Ba2+ influx. The ATP-induced Ba2+ influx was in most part

inhibited by U-73122 and reduced by half in the presence of 30-µM FK506.

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TRPC3/7 channel proteins carry the ATP/UTP-induced inward current. The presence of

various TRPC mRNA was checked in isolated adult rat ventricular cardiomyocytes. The mRNA of

TRPC1, TRPC3, TRPC4 and TRPC7, but not TRPC5 and TRPC6, were detected. Western blots further

revealed the presence of TRPC3 and TRPC7 proteins having apparent MW around 90-95 kD. Further, it

was possible to immunoprecipitate TRPC7 with the anti-TRPC3 antibody suggesting that both proteins

contribute to heteromeric TRPC3/7 channels (Fig. 7). A band, whose nature was not further checked,

was always seen around 170 kD (n=3). Anti-TRPC6 antibody did not reveal any protein.

That TRPC3 contributes to the channel carrying the ATP-induced current was strengthened by

the fact that adding the anti-TRPC3 antibody to the pipette solution markedly reduced IATP elicited in a

rat ventricular cardiomyocyte under whole-cell patch-clamp. This inhibitory effect did not occur in the

presence of the antigenic peptide, nor was it observed when the anti-TRPC6 antibody was used instead

(Fig. 8).

DISCUSSION

Extracellular purines and pyrimidines are released during various physiopathological conditions

such as ischemia and are clearly associated with arrhythmia. Here we report a series of events that could

account for the triggering of arrhythmia by ATP-UTP. ATP (and UTP) in its free form binds P2Y2

purinoceptors, which via PLCβ activation, trigger a sustained non-selective cationic current occurring

through heteromeric TRPC3/7 channels.

TRPC channels are assumed to be composed of four subunits that assemble to form homo- or

heteromeric ion channels (16). The type of native channel formed depends on TRPC homologs

endogenously expressed. The TRPC3/6/7 subfamily, besides having a high amino acid identity, is

generally considered to be activated by a mechanism dependent on receptor-mediated activation of PLC

and DAG production as initially reported for the activation of expressed TRPC3 and TRPC7 by ATP

(17, 26). Recently, a similar cation channel with TRPC3/7 properties was shown to be activated by

endothelin-1 in rabbit coronary artery myocytes (29). In the present work in ventricular cardiomyocytes

both TRPC3 and TRPC7 are expressed and co-immunoprecipitate. They both contribute to the ATP-

induced current. Neither TRPC6 mRNA nor protein were observed at variance with TRPC6 protein

occurrence in the mouse sinoatrial node (20) and in neonatal rat (27). Besides the fact that our

biochemical analyses were all performed on isolated rat cardiomyocytes, these different observations

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could be attributable to different tissues or species expression, or to a shift in TRPC expression during

development as occurs in failing human heart (7). However, it is to be noted that the ATP/UTP-induced

current had similar amplitude and dependency in PMI rat cardiomyocytes, as well as in P2X4-deficient

mice. Indeed, these transgenic mice were investigated because the similarities of the current elicited by

ATP in wild type and P2X4-overexpressing transgenic mice led to suggest a specific role of P2X4-

mediated current in control mice (33). The contribution of TRPC3 to the cationic current carrying

channel is demonstrated by the fact that anti-TRPC3 antibody significantly reduced IATP. Such an anti-

TRPC3 antibody applied on the internal membrane face was previously shown to produce a pronounced

reduction of the TRPC3 properties in native constitutively active Ca2+-permeable channel in ear artery

(3) and in OAG-treated mouse cardiomyocytes (13). Heterologous expressed TRPC7 was early reported

to be activated by ATP (26). The contribution of TRPC7 to the ATP-evoked current in cardiomyocytes

is strengthened by the observation that external Ca2+ ions have an inhibitory effect on the current

amplitude as initially reported in heterologous expression system while it increases the TRPC6 carried

current (34). Besides the above mentioned inhibition by external Ca2+, an increase in [Ca2+]i after

applying CPA or FK506 reduced significantly IATP as initially reported for expressed TRPC6 and TRPC7

(34) and heteromultimeric TRPC3-TRPC6 and TRPC6-TRPC7 channels in A7r5 smooth muscle cells

(23). One cannot exclude however that the elevated [Ca2+]i activates PKC or that FK506 directly affect

the TRPC channel behaviour since various immunophilins were reported to bind to TRPC proteins, more

particularly FKBP12 with TRPC3 and TRPC7 proteins (37).

In addition to being activated by a mechanism dependent on receptor-mediated activation of

PLC and DAG production, TRPC3 and TRPC7, when overexpressed, often lead to increased basal Ca2+

level or increased Ba2+ and Mn2+ leak fluxes (17, 26, 45, 46) and even demonstrate activation by store-

depletion (22). In HEK-293 cells, TRPC1, TRPC3 and TRPC7 assemble to form native SOCs while

TRC3 and TRPC7 can simultaneously participate in forming native store-operated and native-DAG

stimulated channels (44). Recently also, it was suggested that TRPCs mediate store-operated Ca2+-

channel activity to regulate mouse pacemaker firing (20). In isolated rat ventricular cardiomyocytes,

heteromeric TRPC3/7 channels do not display constitutive nor SOC activities as indicated by very low

single channel openings in cell-attached patch and weak Mn2+ quenching and by lack of Ba2+ influx in

Ca2+-free medium.

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In this work, ATP activates a current that is maintained in the presence of GTPγS. UTP was

equally active while UDP, an efficient agonist of the P2Y6 purinoceptor, was ineffective. These

observations suggest the involvement of P2Y2 or P2Y4 receptor. The current was also activated by

BzATP and ATPγS, and showed much weaker inhibition by PPADS than by suramin. That further

indicates ATP/UTP bind to P2Y2 receptors on the basis of the pharmacological profiles since after re-

expression in oocytes rP2Y2, but not rP2Y4 receptors are activated by BzATP and ATPγS and more

sensitive to suramin (43). Furthermore, activation of the P2Y2 receptors via a G-protein leads to

activation of PLCβ as suggested by its inhibition by U-73122 but not by other PLC inhibitors. We also

clarify for the first time that only the free form of ATP, ATP4- is the agonist at P2Y2 receptors by

comparing the current amplitudes triggered by two solutions with similar calculated ATP4- and Ca2+

contents in the presence or not of Mg2+ since to our knowledge Mg2+ ions have not been reported to alter

TRPC3/7 channel conductance. Thus, the increase in the sustained current amplitude observed on

removing Mg2+ in figure 1.

It is worth noting that the apparent EC50 of ATP to activate IATP via P2Y2 receptors was 58 µM,

about 10-20 fold the values determined on re-expressed rP2Y2-receptor (2.7 and 3.6 µM for ATP and

UTP, respectively (43)) and the one observed for the ATP-induced modulations in Ca2+ and K+ currents

whose involved purinoceptor subtypes highly activated by 10-µM ATP, are still unknown (40). This

would allow physiological regulation before activation of this detrimental arrhythmogenic pathway. Our

data suggest that in adult normal cardiomyocyte, IATP activates only in specific conditions such as after a

large surge of ATP/UTP release during infarction, at odds with the multiple physiological modulations

of electrical and contractile activities induced by lower ATP and UTP concentrations (40, 42).

In conclusion, this work reveals a new, potentially deleterious, role of TRPC channels. Besides

the multiple modulatory effects of ATP on electrical and contractile activities mediated by various

pathways, we suggest that following the anomalous large release of ATP and UTP during early ischemic

events, P2Y2 purinergic receptors stimulated by the free forms ATP4-/UTP4- activate heteromeric

TRPC3/7 channels. The sustained inward current occurring through these channels induces cell

depolarization and Ca2+ overload and possibly triggers arrhythmia. It is worth noting that to some extent

other agonists that activate the DAG pathways and TRPC could have some proarrhythmic activities;

however, a specific arrhythmic effect of ATP should be considered as a consequence of its interstitial

and potentially large release.

- 13rev -

Grants

This work was supported, in most part, by the “Institut National de la Santé et de la Recherche

Médicale”. JL was supported by the French Embassy at La Habana Cuba; OC and AL are supported by

the “Centre National de la Recherche Scientifique”.

Disclosures none

- 14rev -

References

Figure Legends

Fig. 1. ATP and UTP activate a sustained inward current that is mediated by P2Y receptors. A: currents

elicited at HP -80 mV on application of 1-mM ATP in the presence or absence of Mg2+ on a rat

ventricular cardiomyocyte. The fast transient current required the presence of Mg2+ ions and was elicited

- 15rev -

only on fast ATP application. B: inward sustained currents, IATP elicited at HP –80 mV by applying

increasing concentrations of ATP. C: concentration-effect relationships of current density elicited by

ATP or UTP in the absence of Mg2+ on ventricular cardiomyocytes isolated from control (n ≥ 9) and

infarcted rats (PMI-rat, n ≥6). The inset shows current densities induced by two ATP concentrations in

control (n ≥ 4) and transgenic mice deficient for the P2X1, P2X4 or the P2X1-P2X4 purinoceptors (KO-

mice; n ≥3 for each single and double KO, pooled) and in dog (n ≥ 5) and human (n = 4) ventricular

myocytes. n = number of cardiomyocytes from at least two hearts.

Fig. 2. Free ATP4- is the agonist at the purinergic receptor. A: typical recording of the sustained inward

current elicited by ATP in two solutions that contained (mM) both Ca2+ 2, and either ATP 1 and 0 Mg2+,

or ATP 3 and Mg2+ 3.5 leading to similar estimated free ATP4- and Ca2+ activities (respectively, 150,

136 µM and 1.14, 1.18 mM) (representative of 5 similar experiments). B: dose-response curve of IATP

amplitude elicited by various ATP concentrations in a Mg2+-free, 300-µM Ca2+ solution (n=6). The

apparent half-efficient ATP concentration, EC50 was 558 µM corresponding to a calculated EC50-ATP4- of

58 µM. HP = -80 mV.

Fig. 3. The ATP-induced current requires the activation of the phospholipase Cβ. Incubating the

cardiomyocytes with the phospholipase D inhibitor, propranolol (200 µM), the phospholipase A2

inhibitor, AACOCF3 (50 µM, incubated 10-20 min), the phospholipase Cγ inhibitor LY294002 (100

µM, incubated 30 min) as well as with the broad spectrum tyrosine kinase inhibitor, genistein (20 µg/ml,

incubated 30 min at 37°C) had no effect on the inward current elicited at -80 mV on the application of

300-µM ATP in the absence of Mg2+. IATP was prevented only in the presence of the PLCβ inhibitor, U-

73122 (10 µM, incubated 10-15 min). Empty bars: respective control cells. Number of cells indicated

above each bar, * P < 0.05.

Fig. 4. Characteristics of the ATP-induced current. A: current-voltage relationship established after the

application of 300 µM ATP in the absence of Mg2+ during a ramp potential as indicated above. B: with

500-µM GTPγS in the pipette solution, the brief application of ATP induced a current that was not

reversible after ATP removal. Then, decreasing extracellular Ca2+ significantly increased IATP amplitude.

HP = -80 mV. C: mean sustained inward currents elicited under the above conditions. Mean estimated

- 16rev -

chord conductances are 3.7 ±0.8, 5.7±1.2 and 7.3±1.2 nS at 2, 0.6 and 0.1 mM extracellular Ca2+

concentrations respectively (n=5). D: the sustained ATP-induced current in Mg2+-free solution shared

pharmacological properties with the expressed TRPC7. Cyclopiazonic acid (CPA, 30 µM, incubated 10

min) and FK506 (25 µM, incubated 10-30 min), both thought to increase [Ca 2+]i inhibited the 300-µM

ATP-induced current. La3+ (100 µM), Gd2+ (100 µM) and SKF96375 (25 µM) induced a marked

reversible inhibition of IATP while flufenamic acid (FFA, 100µM) significantly potentiated the sustained

current. Values are represented as percent of control. Number of cells in bars for each experimental

condition, * P < 0.05.

Fig. 5. Single channel characteristics of the ATP-induced current. A: no opening was observed in a

patch from control rat cardiomyocyte while frequent openings were seen on another cell when the patch

pipette contained 100-µM Na2ATP. Downward deflections of the current trace represent inwardly

directed membrane currents at –80 mV. Note that bathing the cell in the presence of 10-µM U-73122

prevented channel openings. B: mean ATP-induced current in cell-attached patches (n as indicated) on

rat cardiomyocytes in control solution or in the presence of U-73122. C: mean single-channel current

amplitudes as a function of membrane potential for the two most frequently observed low levels of

current, determined from at least 14 membrane patches. The straight lines represent least-squares fits of

the data.

Fig. 6. Fluorescence analysis of the ATP-induced current. A: original recordings in a rat ventricular

cardiomyocyte loaded with Fura-2. Changes in fluorescence induced by 30-µM ATP were in opposite

direction after excitation at either 340 or 380 nm while there was a marked quenching effect after

excitation at both wavelengths when applying the same ATP concentration in the presence of 1-mM

Mn2+ (3 similar experiments). B: original tracings of the fluorescence ratio in three rat ventricular

cardiomyocytes sequentially submitted to Ca2+-free, 2-mM Ba2+-containing solution. A significant slow

increase in Ba2+-fluorescence was observed only after 1-mM ATP application. C: pooled data of the

maximal rate of increase in Ba2+-fluorescence induced by ATP, UTP and UDP all at 1 mM, or by 1-mM

ATP on cells incubated with 10 µM U-73122 or 25 µM FK506. Number of cells in bars for each

experimental condition, * P < 0.05.

- 17rev -

Fig. 7. Nature of the TRPC channel subunits. A: expression of TRPCs mRNA in cardiomyocytes

isolated from adult rat, compared to whole brain tissue, indicates that TRPC1, TRPC3 TRPC4 and

TRPC7 were present in cardiomyocytes but not TRPC5 and TRPC6, while the efficacy of the anti-

TRPC6 antibody had been controlled on brain tissue. B: Western blots revealed the two TRPC3 and

TRPC7 proteins in isolated rat cardiomyocytes. C: the TRPC3 antibody also immunoprecipitated

TRPC7. The origin of the band at 171 kD was not investigated.

Fig. 8. Inhibition of the ATP-induced current by the anti-TRPC3 antibody. Pooled data of the

amplitude of the ATP-induced inward currents recorded within 5 min after the giga-seal formation in rat

ventricular cardiomyocytes under whole-cell patch-clamp. The anti-TRPC3 antibody (Ab-C3) added to

the pipette solution (1:200 dilution) significantly reduced IATP. Antibody-induced inhibition was

prevented by further adding the TRPC3-antigenic peptide (Ab-C3+ Pep, 1:200) to the pipette solution.

Adding the anti-TRPC6 antibody (Ab-C6) to the pipette solution did not significantly affect the ATP-

induced current. Number of cells in bars, * P < 0.05.

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11. Drury AN, and Szent-Gyorgyi A. The physiological activity of adenine compounds with especial reference to their action upon the mammalian heart. J Physiol 68: 213-237, 1929. 12. Dutta AK, Sabirov RZ, Uramoto H, and Okada Y. Role of ATP-conductive anion channel in ATP release from neonatal rat cardiomyocytes in ischaemic or hypoxic conditions. J Physiol 559: 799-812, 2004. 13. Fauconnier J, Lanner JT, Sultan A, Zhang SJ, Katz A, Bruton JD, and Westerblad H. Insulin potentiates TRPC3-mediated cation currents in normal but not in insulin-resistant mouse cardiomyocytes. Cardiovasc Res 73: 376-385, 2007. 14. Fredholm BB, AP IJ, Jacobson KA, Klotz KN, and Linden J. International Union of Pharmacology. XXV. Nomenclature and classification of adenosine receptors. Pharmacol Rev 53: 527-552, 2001. 15. Goel M, Sinkins WG, and Schilling WP. Selective association of TRPC channel subunits in rat brain synaptosomes. J Biol Chem 277: 48303-48310, 2002. 16. Hofmann T, Schaefer M, Schultz G, and Gudermann T. Subunit composition of mammalian transient receptor potential channels in living cells. Proc Natl Acad Sci U S A 99: 7461-7466, 2002. 17. Hurst RS, Zhu X, Boulay G, Birnbaumer L, and Stefani E. Ionic currents underlying HTRP3 mediated agonist-dependent Ca2+ influx in stably transfected HEK293 cells. FEBS Lett 422: 333-338, 1998. 18. Inoue R, Jensen LJ, Shi J, Morita H, Nishida M, Honda A, and Ito Y. Transient receptor potential channels in cardiovascular function and disease. Circ Res 99: 119-131, 2006. 19. Inoue R, Okada T, Onoue H, Hara Y, Shimizu S, Naitoh S, Ito Y, and Mori Y. The transient receptor potential protein homologue TRP6 is the essential component of vascular alpha(1)-adrenoceptor-activated Ca(2+)-permeable cation channel. Circ Res 88: 325-332, 2001. 20. Ju YK, Chu Y, Chaulet H, Lai D, Gervasio OL, Graham RM, Cannell MB, and Allen DG. Store-operated Ca2+ influx and expression of TRPC genes in mouse sinoatrial node. Circ Res 100: 1605-1614, 2007. 21. Kuzmin AI, Lakomkin VL, Kapelko VI, and Vassort G. Interstitial ATP level and degradation in control and postmyocardial infarcted rats. Am J Physiol 275: C766-771, 1998. 22. Lievremont JP, Bird GS, and Putney JW, Jr. Canonical transient receptor potential TRPC7 can function as both a receptor- and store-operated channel in HEK-293 cells. Am J Physiol Cell Physiol 287: C1709-1716, 2004. 23. Maruyama Y, Nakanishi Y, Walsh EJ, Wilson DP, Welsh DG, and Cole WC. Heteromultimeric TRPC6-TRPC7 channels contribute to arginine vasopressin-induced cation current of A7r5 vascular smooth muscle cells. Circ Res 98: 1520-1527, 2006. 24. Montell C, Birnbaumer L, and Flockerzi V. The TRP channels, a remarkably functional family. Cell 108: 595-598, 2002. 25. Nilius B, Owsianik G, Voets T, and Peters JA. Transient receptor potential cation channels in disease. Physiol Rev 87: 165-217, 2007. 26. Okada T, Inoue R, Yamazaki K, Maeda A, Kurosaki T, Yamakuni T, Tanaka I, Shimizu S, Ikenaka K, Imoto K, and Mori Y. Molecular and functional characterization of a novel mouse transient receptor potential protein homologue TRP7. Ca(2+)-permeable cation channel that is constitutively activated and enhanced by stimulation of G protein-coupled receptor. J Biol Chem 274: 27359-27370, 1999. 27. Onohara N, Nishida M, Inoue R, Kobayashi H, Sumimoto H, Sato Y, Mori Y, Nagao T, and Kurose H. TRPC3 and TRPC6 are essential for angiotensin II-induced cardiac hypertrophy. Embo J 25: 5305-5316, 2006. 28. Owsianik G, D'Hoedt D, Voets T, and Nilius B. Structure-function relationship of the TRP channel superfamily. Rev Physiol Biochem Pharmacol 156: 61-90, 2006. 29. Peppiatt-Wildman CM, Albert AP, Saleh SN, and Large WA. Endothelin-1 activates a Ca2+-permeable cation channel with TRPC3 and TRPC7 properties in rabbit coronary artery myocytes. J Physiol 580: 755-764, 2007. 30. Puceat M, Clement O, Scamps F, and Vassort G. Extracellular ATP-induced acidification leads to cytosolic calcium transient rise in single rat cardiac myocytes. Biochem J 274 ( Pt 1): 55-62, 1991. 31. Scamps F, and Vassort G. Mechanism of extracellular ATP-induced depolarization in rat isolated ventricular cardiomyocytes. Pflugers Arch 417: 309-316, 1990. 32. Scamps F, and Vassort G. Pharmacological profile of the ATP-mediated increase in L-type calcium current amplitude and activation of a non-specific cationic current in rat ventricular cells. Br J Pharmacol 113: 982-986, 1994.

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33. Shen JB, Pappano AJ, and Liang BT. Extracellular ATP-stimulated current in wild-type and P2X4 receptor transgenic mouse ventricular myocytes: implications for a cardiac physiologic role of P2X4 receptors. Faseb J 20: 277-284, 2006. 34. Shi J, Mori E, Mori Y, Mori M, Li J, Ito Y, and Inoue R. Multiple regulation by calcium of murine homologues of transient receptor potential proteins TRPC6 and TRPC7 expressed in HEK293 cells. J Physiol 561: 415-432, 2004. 35. Shoda M, Hagiwara N, Kasanuki H, and Hosoda S. ATP-activated cationic current in rabbit sino-atrial node cells. J Mol Cell Cardiol 29: 689-695, 1997. 36. Sim JA, Chaumont S, Jo J, Ulmann L, Young MT, Cho K, Buell G, North RA, and Rassendren F. Altered hippocampal synaptic potentiation in P2X4 knock-out mice. J Neurosci 26: 9006-9009, 2006. 37. Sinkins WG, Goel M, Estacion M, and Schilling WP. Association of immunophilins with mammalian TRPC channels. J Biol Chem 279: 34521-34529, 2004. 38. Surprenant A, Rassendren F, Kawashima E, North RA, and Buell G. The cytolytic P2Z receptor for extracellular ATP identified as a P2X receptor (P2X7). Science 272: 735-738, 1996. 39. Szabo G, Szentandrassy N, Biro T, Toth BI, Czifra G, Magyar J, Banyasz T, Varro A, Kovacs L, and Nanasi PP. Asymmetrical distribution of ion channels in canine and human left-ventricular wall: epicardium versus midmyocardium. Pflugers Arch 450: 307-316, 2005. 40. Vassort G. Adenosine 5'-triphosphate: a P2-purinergic agonist in the myocardium. Physiol Rev 81: 767-806, 2001. 41. Venkatachalam K, and Montell C. TRP Channels. Annu Rev Biochem 76: 387-417, 2007. 42. Wihlborg AK, Balogh J, Wang L, Borna C, Dou Y, Joshi BV, Lazarowski E, Jacobson KA, Arner A, and Erlinge D. Positive inotropic effects by uridine triphosphate (UTP) and uridine diphosphate (UDP) via P2Y2 and P2Y6 receptors on cardiomyocytes and release of UTP in man during myocardial infarction. Circ Res 98: 970-976, 2006. 43. Wildman SS, Unwin RJ, and King BF. Extended pharmacological profiles of rat P2Y2 and rat P2Y4 receptors and their sensitivity to extracellular H+ and Zn2+ ions. Br J Pharmacol 140: 1177-1186, 2003. 44. Zagranichnaya TK, Wu X, and Villereal ML. Endogenous TRPC1, TRPC3, and TRPC7 proteins combine to form native store-operated channels in HEK-293 cells. J Biol Chem 280: 29559-29569, 2005. 45. Zhu X, Jiang M, and Birnbaumer L. Receptor-activated Ca2+ influx via human Trp3 stably expressed in human embryonic kidney (HEK)293 cells. Evidence for a non-capacitative Ca2+ entry. J Biol Chem 273: 133-142, 1998. 46. Zitt C, Obukhov AG, Strubing C, Zobel A, Kalkbrenner F, Luckhoff A, and Schultz G. Expression of TRPC3 in Chinese hamster ovary cells results in calcium-activated cation currents not related to store depletion. J Cell Biol 138: 1333-1341, 1997.

ATP/UTP (mM)

0

1

2

Cur

rent

(pA

/pF)

0.003 310.30.03

Rat, ATPRat, UTPPMI-rat; ATP

0

1

2

3

mice dogKO-mice

human

ATP 0.03 mM

ATP 3 mMC

Fig. 1

B

Cur

rent

(pA

/pF)

0

-1

-2

Mg2+ 1.8 Mg2+ 0 mM

ATP 1 mM ATP 1 mM

3 min

A

1

0.03 0.3 1 3 mM; Mg2+ 0 mM

1 min

ATP

0

-2

-1

Fig. 2

A0

-1

ATP 1; Mg2+ 0

ATP 1; Mg2+ 0

ATP 3; Mg2+ 3.5

3 minCur

rent

(pA

/pF)

Cur

rent

(pA

/pF)

B

0

0.5

1.0

1.5EC50ATP = 558 µMEC50ATP4- = 58 µM

ATP (mM)0.1 10.30.03

0.09 2.7 9.2 29 120 802

3

ATP4- (µM)

Propranolol

AACOCF3

LY294002

U-73122

Genistein0.0

0.2

0.4

0.6

Cur

rent

(pA

/pF) 4

45

5

10

154

*4

4 4

Fig. 3

C -80 mV -40 mV 0 mV0

-3

-2

-1

Cur

rent

(pA

/pF)

A-100 mV

+50 -100 - 50 (mV)

0.5

-0.5

-1.0

-1.5 (pA

/pF)

+ 50 mV -80 mV

500 ms

Fig. 4

Rel

ativ

e cu

rrent

D150

100

50

0

La3+

Gd3+

CPAFK506

FFA

SKF963657 6 55 4 10

** * * *

*

B

Cur

rent

(pA

/pF)

-4

-2

02 (mM)

0.6 0.60.1Ca2+

= 2 2

ATP1 mM

2 min

Ca2+ = 2 mMCa2+ = 0.6 mMCa2+ = 0.1 mM

B

0.0

0.1

0.2

0.3

Control

Na 2ATP

U-73122

15

19HP=-80 mV

16

Rel

ativ

e m

ean

curre

ntam

plitu

de

(mV)-80 -40 -20

Current (pA

)

-2

-114 pS

23 pS

C

Fig. 5

200 s

200 s

-80 mVU-73122

10 s

A

1 pA

Control

Na2ATP 200 s

200 s

10 s

25 ms

2 pA

∆F3

40/3

80

Control Ca2+ 0 + Ba2+ 2 + ATP 1 mM

0.3

0.5

0.4

B

5 min

C

0.01

∆F3

40/3

80 /

min

0 10 64 4

5* *

*

ATP UDP +U73122 +FK506UTP

Fig. 6

A

Fluo

resc

ence

(A.U

.)

ATP 30 µM + ATP 30 µMMn

0

1

340

380

2 min

Blot : TRPC7 TRPC3

B

Fig. 7

MyocytesBrain- RT - RT+ RT + RT

TRPC5

TRPC6

TRPC4

TRPC7

TRPC3

TRPC1

A120100

80

TRPC3 TRPC7

IP : TRPC3

120100

80

220

96 kDa

171 kDa

C

*

ControlAb-C3

Ab-C3 + PepAb-C6

0.5

0

0.2

11 9 8 7Cur

rent

(pA

/pF)

Fig. 8