ATPUTP activate cation-permeable channels with TRPC37 properties in rat cardiomyocytes

TRANSLATIONAL PHYSIOLOGY
ATP/UTP activate cation-permeable channels with TRPC3/7properties
in rat cardiomyocytes
Julio Alvarez,1,2Alain Coulombe,3Olivier Cazorla,1Mehmet Ugur,1Jean-Michel Rauzier,1Janos Magyar,4 Eve-Lyne Mathieu,5Guylain Boulay,5Rafael Souto,2Patrice Bideaux,1Guillermo Salazar,1
Franc¸ois Rassendren,6Alain Lacampagne,1Je´re´my Fauconnier,1and Guy Vassort1
1Institut National de la Sante´et de la Recherche Me´dicale(INSERM),Unite´637,Universite´Montpellier1,Unite´de
Formation et de Recherche de Me´decine,Centre Hospitalier Universitaire(CHU)Arnaud de Villeneuve,Montpellier,France;
2Laboratorio de Electrofisiologı´a,Instituto de Cardiologı´a,Havana,Cuba;3INSERM,Unite´Mixte de Recherche(UMR)
S621,Universite´Pierre et Marie Curie,CHU Pitie´-Salpeˆtrie`re,Paris,France;4Department of Physiology,University of
Debrecen,Debrecen,Hungary;5Pharmacology,Faculty of Medicine,University of Sherbrooke,Fleurimont,Quebec,Canada;
and6Department of Pharmacology,Institut de Ge´nomique Fonctionnelle,Centre National de la Recherche Scientifique UMR
5203,INSERM Unite´661,Universite´Montpellier I and Universite´Montpellier II,Montpellier Cedex,France
Submitted7February2008;accepted infinal form16May2008
Alvarez J,Coulombe A,Cazorla O,Ugur M,Rauzier JM,Magyar J, Mathieu EL,Boulay G,Souto R,Bideaux P,Salazar G,Rassendren F, Lacampagne A,Fauconnier J,Vassort G.ATP/UTP activate cation-per-meable channels with TRPC3/7properties in rat cardiomyocytes.Am J Physiol Heart Circ Physiol295:H21–H28,2008.First published May23, 2008;doi:10.1152/ajpheart.00135.2008.—Extracellular purines and pyrimi-dines 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 iso-lated from various mammalian species,ATP and UTP elicited a sustained, nonselective cationic current,I ATP.UDP was ineffective,whereas2Ј(3Ј)-O-(4-benzoylbenzoyl)-ATP was active,suggesting that P2Y2receptors are involved.I ATP resulted from the binding of ATP4Ϫto P2Y2purinoceptors.
I ATP was maintained after ATP removal in the presence of guanosine 5Ј-[␥-thio]triphosphate and was inhibited by U-73122,a PLC 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 of14and23pS were easily distinguished.Similarly,in fura-2-loaded cardiomyocytes,Mn2ϩquench-ing and Ba2ϩinflux were significant only in the presence of ATP or UTP. Adult rat ventricular cardiomyocytes expressed transient receptor poten-tial channel TRPC1,-3,-4,and-7mRNA and the TRPC3and TRPC7 proteins that coimmunoprecipitated.Finally,the anti-TRPC3antibody added to the patch pipette solution inhibited I ATP.In conclusion,activa-tion of P2Y2receptors,via a G protein and stimulation of PLC␤,induces the opening of heteromeric TRPC3/7channels,leading to a sustained, nonspecific 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,poten-tially deleterious role of TRPC channel activation.
purinergic receptor;signal transduction;infarction;arrhythmia
A HIGH-ENERGY PHOSPHATE DONOR,ATP has been extensively studied since the early description of a role for extracellular purines by Drury and Szent-Gyo¨rgyi in1929(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 condi-tions(40).In particular,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 pharmaco-logical agents.
Extracellular ATP activates both the ionotropic(ligand gated)receptors of the P2X receptor family and the metabo-tropic(G protein coupled)receptors of the P2Y receptor family (40).The P2Y family is divided into two structurally distinct subfamilies.Thefirst is composed of P2Y1,P2Y2,P2Y4,P2Y6, and P2Y11receptors,all coupled to G q,which promotes PLC activation and diacylglycerol(DAG)production.The others, P2Y12,P2Y13,and P2Y14,are coupled to G i,inhibiting ade-nylate cyclase(6,14).Among thefirst subfamily,P2Y2,P2Y4, and P2Y6also can 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 Mg2ϩ(8,31,32).Furthermore,during prolonged ATP application,in the presence or absence of Mg2ϩ,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 channels(TRPCs)werefirst described in the phototransduction system of Drosophila mela-nogaster.Mammalian homologs encode channel proteins that have six transmembrane domains and assemble into heterotet-
Address for reprint requests and other correspondence:G.Vassort,INSERM U-637,Physiopathologie cardiovasculaire,CHU Arnaud de Villeneuve,F-34295 Montpellier,France(e-mail:guy.vassort@inserm.fr).
The costs of publication of this article were defrayed in part by the payment of page charges.The article must therefore be hereby marked“advertisement”in accordance with18U.S.C.Section1734solely to indicate this fact.
Am J Physiol Heart Circ Physiol295:H21–H28,2008.
First published May23,2008;doi:10.1152/ajpheart.00135.2008.
ramers(9,28,41).TRPCs are widely distributed in mamma-lian tissues and are involved in several cardiovascular func-tions and diseases(18,25).Like P2X purinoceptors,most TRPCs are nonselective to cations and act to shift the mem-brane potential to around0mV,thus depolarizing cells from resting potential and allowing Ca2ϩinflux and cell automatic-ity.The TRPC subfamily is composed of seven members, TRPC1–7,with the TRPC3,TRPC6,and TRPC7subgroup being directly activated by DAG(24).TRPC3-and TRPC7-expressing cells have been demonstrated to have both consti-tutively activated and ATP-enhanced inward currents that al-low Ca2ϩinflux(17,26).Recently,TRPC6and TRPC6/7have been identified as an essential part of the␣1-adrenoceptor-activated cation currents in smooth muscle cells(19),whereas in heart,TRPC3and TRPC6proteins are essential for angio-tensin II-induced hypertrophy(7,27),and TRPC3is necessary for the potentiated insulin-induced current(13).
In the present work at the cellular level,we have shown that ATP and UTP activate purinergic P2Y2receptors.These re-ceptors,via a G protein-dependent activation of PLC␤,trigger a fast-activating,sustained,nonselective cationic current through TRPC3/7channels.
MATERIALS AND METHODS
Experimental models and chemicals.Experiments were performed on cardiomyocytes isolated from adult male Wistar rats or as other-wise specified.Rats were killed by an intravenous injection of pen-tobarbital(100mg/kg).The investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the U.S. National Institutes of Health(NIH Publication No.85-23,revised 1996).The protocol was approved by the Animal Research Commit-tee of INSERM.Cardiomyocytes were also isolated from postmyo-cardial infarcted(PMI)rats(1)and from control and transgenic mice deficient for the P2X1,P2X4,or both P2X1-P2X4purinergic receptors (36)using a similar procedure,as well as from control dog and human according to Szabo et al.(39).All materials were purchased from Sigma-Aldrich except SKF-96365(Calbiochem),U-73122(Biomol), and fura-2AM(Molecular Probes,Eugene,OR).Anti-TRPC(anti-TRPC3and anti-TRPC6)antibodies raised against putative intracel-lular epitopes were obtained from Alomone Laboratories(Jerusalem, Israel),and the anti-TRPC7antibody,a generous gift from Prof.W.P. Schilling(Case Western Reserve University School of Medicine, Cleveland,OH),was made against the sequence843EKFGKNLNK-DHLRVN857and demonstrated to recognize no other TRPCs(15).
Electrophysiological experiments.Rat ventricular myocytes were enzymatically dissociated using a method previously described(4), kept in physiological solution containing(mM)117NaCl,5.4KCl,1 CaCl2,1.0MgCl2,10glucose,and10HEPES,pH7.4at22–24°C, and used within6–8h.Currents were recorded using the whole cell variant of the patch-clamp method at22–24°C.Kϩcurrents were blocked by Csϩ.Compared with the physiological solution,the standard extracellular solution contained20CsCl,instead of KCl,and 2CaCl2.The pipette(intracellular)solution contained(mM)130 CsCl,0.4Na2GTP,5Na2ATP,5Na2-creatine phosphate,11EGTA, 4.7CaCl2(free Ca2ϩ,108nM),and10HEPES,with pH adjusted to 7.2with 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 six microcapillaries(250-␮m inner diameter, Tygon microbore tubing;Norton Performance Plastics,Wayne,NJ) through which the different extracellular solutions were perfused by gravity at a rate of0.1ml/min.Solution changes were accomplished within1s.Alterations of the current at a holding potential ofϪ80mV were analyzed every4s.Currents were scaled to cell capacitance.
Single-channel recordings were performed with the classic 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 code7052;WPI,Sarasota,FL)using a horizontal puller(DMZ-Universal Puller;Zeitz Instruments,Munich, Germany)andfire-polished before use.The pipette resistance was 5–10M⍀.The currents were recorded using a patch-clamp amplifier (Axopatch200B;Axon Instruments,Foster City,CA)andfiltered through an eight-pole Bessel low-passfilter920LPF(Frequency Devices,Ottawa,IL)at a setting of1kHz(Ϫ3dB point).Data were digitized at5kHz with a Digidata1200(Axon Instruments)using Acquis1software(G.Sadoc,Centre National de la Recherche Scien-tifique,Gif/Yvette,France).Single-channel activity was recorded only when the seal resistance wasՆ10G⍀.All experiments were con-ducted at room temperature(20–24°C).Elementary conductances were determined as previously reported(10).Channel activity(mean patch current)was calculated by integrating currentflow during the channel openings and dividing the integral by the total sampling time. The Kϩ-rich medium in which cells were maintained before use contained(mM)70L-glutamic acid monopotassium salt,25KCl,10 KH2PO4,3MgCl2,0.5EGTA,10HEPES,20glucose,and10taurine, and pH was adjusted to7.4with KOH.The superfusion solution contained(mM)135NaCl,4KCl,2MgCl2,2CaCl2,10HEPES,and 20glucose,and pH was adjusted to7.4with NaOH.The pipette solution contained(mM)135NaCl,4KCl,2CaCl2,10HEPES,and 20glucose,and pH was adjusted to7.4with NaOH.In some experiments,Na2ATP(100␮M)was added to the pipette solution.
Measurements of changes in intracellular Ca2ϩconcentration.Rat ventricular cardiomyocytes bathed in the physiological solution were loaded with fura-2AM(2.5␮M)for30min at35°C and then allowed to attach on a coverslip.Fluorescence images(2–4rod-shaped cells in afield)were recorded and analyzed with a video image analysis system(MetaMorph6.0/6.1;Universal Imaging).The fura-2fluores-cence image at an emission wavelength of510nm(bandwidth20nm) was followed at22–24°C by excitation of fura-2alternatively at340 and380nm(bandwidth11nm)to obtain a340/380ratio on a pixel/pixel basis.Ca2ϩwas removed(Ca2ϩ-free solution),and later Ba2ϩwas added(2mM,Ba2ϩsolution).
Biochemical analyses.For RT-PCR,total RNA was extracted from myocytes or brain using TRIzol(Invitrogen)according to the manufac-turer’s specifications and reverse transcribed into cDNA using random hexamer(Amersham Pharmacia Biotech).An aliquot of thefirst-strand cDNA was used as a template for PCR,and rat TRPCs were amplified using the following primer sets:TRPC1,forward,5Ј-CAAGATTTTGG-GAAATTTCTAG-3Ј,and reverse,5Ј-TTTATCCTCATGATTTGC-TAT-3Ј;TRPC3,forward,5Ј-TGACTTCTGTTGTGCTCAAATATG-3Ј,and reverse,5Ј-CCTTCTGAAGTCTTCTCCTCCTGC-3Ј;TRPC4, forward,5Ј-TCTGCAGATATCTCTGGGAAGAATGC-3Ј,and reverse, 5Ј-AAGCTTTGTTCGAGCAAATTTCC-3Ј;TRPC5,forward,5Ј-ATC-TACTGCCTAGTACTACTGGCT-3Ј,and reverse,5Ј-CAGCATGGTCG-GCAATGAGCTG-3Ј;TRPC6,forward,5Ј-TCACTTGGAAGAACAGT-GAAAGA-3Ј,and reverse,5Ј-CATCCTCAATTTCCTGGAATGAAC-3Ј; and TRPC7,forward,5Ј-ACCTTCACAGACTACCCCAAAC-3Ј,and re-verse,5Ј-GCCAAATATGGACCAAAACAAGG-3Ј.The resulting PCR product was analyzed using ethidium bromide-agarose gel electrophore-sis and cloned into the pCR2.1-TOPO plasmid vector(Invitrogen)for sequencing analysis.
For Western blotting,adult rat cardiomyocytes isolated from left ventricles were quick-frozen and then homogenized.Proteins were heated for5min at60°C in SDS sample buffer(2%SDS,8M urea, 0.08M DTT,0.05M Tris,1mM EDTA,1mM EGTA,0.5mM PMSF,10␮M E64,and40␮M leupeptin).Proteins were separated by 7.5%SDS-PAGE,followed by transfer to0.45-␮m polyvinylidene diflouride membranes using standard techniques.The membranes were blocked with3%bovine serum albumin(BSA)in0.1%Tween
H22ATP/UTP ACTIVATE TRPC3/7CHANNELS IN RAT CARDIOMYOCYTES
20-TBS(TBS-T).Membranes were labeled overnight with primary antibodies(anti-TRPC3and anti-TRPC7,1:200)in0.1%BSA in TBS-T and washed with TBS-T before being labeled with1:5,000 horseradish peroxidase-conjugated anti-rabbit antibody.Immunode-tection was revealed with West Pico chemiluminescent substrate (Pierce Biotechnology).Quantification of signals was performed by densitometry using an imaging system(Kodak Image Station2000R).
For the immunoprecipitation experiments,all reactions were per-formed during tumbling at4°C.Cell protein extracts(1mg)were precleared for1h with protein A-Sepharose CL-4B beads(Pharma-cia)and then incubated for1h with5␮l of antibody.Antibody-protein complexes were captured by the addition of protein A-Sepharose and incubated for1h to facilitate binding.Immunoprecipi-tated complexes were eluted from the beads using SDS sample buffer before SDS-PAGE and immunoblotting.Blots were visualized as described above.
Statistical analysis.Values are meansϮSE of n cells.Statistical analysis was carried out using paired(comparing effects of agents on same cell)or unpaired(comparing effects between cells)Student’s t-tests with the level of significance set at PϽ0.05.
RESULTS
P2Y2purinergic receptor mediates the ATP/UTP effect in isolated cardiomyocytes.The mechanisms by which ATP trig-gers cardiac tissue automaticity,namely,the nature of the ATP-induced sustained inward current I ATP and its signal transduction pathway,were investigated at the cellular level. The present study was mostly conducted in Mg2ϩ-free extra-cellular solutions,taking advantage of the fact that I ATP did not require the presence of Mg2ϩfor its activation.This allowed us to eliminate the initial transient surge of inward current(31). As shown in Fig.1A,in the presence of Mg2ϩ,application of 1mM 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
Mg2ϩ,only a sustained ATP-induced inward current,I ATP,was seen.Furthermore,I ATP was significantly increased in the Mg2ϩ-free solution.I ATP activated and deactivated within a minute on ATP application and withdrawal.I ATP amplitude increased stepwise with cumulative ATP concentrations within the range from30␮M to3mM(Fig.1B).Under our experi-mental conditions that omitted Mg2ϩ,I ATP could be elicited by ATP and UTP with similar concentration dependency and amplitude range in adult ventricular cardiomyocytes isolated from control and transgenic mice deficient for the P2X1,P2X4, or both P2X1-P2X4purinergic receptors and from PMI rats,as well as from control dog and human(Fig.1C).
Under our experimental conditions,2Ј(3Ј)-O-(benzoylben-zoyl)ATP(BzATP)and adenosine-5Ј-[␥-thio]triphosphate (ATP␥S),both applied at100␮M,were slightly less effective (70%of the ATP effect,nϾ5cells).Furthermore,whenever used at30␮M or1mM,the ATP analogs adenylyl imido-diphosphate,adenosine5Ј-(␤,␥-imido)triphosphate,and␣,␤-methylene ATP(see also Fig.6in Ref.32),as well as ADP,did not affect the holding current.UDP was also ineffective in triggering I ATP.I ATP exhibited a specific pharmacology that excluded it from being carried by any of the P2X receptors, including P2X7(38).First,BzATP,described to be more potent than ATP to activate P2X7receptor,induced a slightly weaker current.Second,the strong P2X inhibitors pyridoxal phosphate-6-azo(benzene-2Ј,4Ј-disulfonic acid),oxidized ATP, and brilliant blue did not significantly affect I ATP.However,suramin,a relatively selective P2Y1-P2Y2antagonist,added at 100␮M,reduced by45%the sustained current induced by1 mM ATP(not shown;nϾ4).Moreover,in cells patched with a pipette that contained a solution with500␮M guanosine 5Ј-[␥-thio]triphosphate(GTP␥S),I ATP 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 P2Y2subtype receptors (40,43).
To elucidate the nature of the P2Y2ligand and ATP2Ϫor ATP4Ϫ(and UTP2Ϫor UTP4Ϫ),we compared the effects of two external solutions with similar calculated free ATP4Ϫand Ca2ϩactivities but varying ATP and MgCl2concentrations. Except for the initial surge of transient current related to the presence of Mg2ϩ,I ATP had similar amplitude under these two experimental conditions,indicating that ATP4Ϫwas the P2Y2-specific agonist(Fig.2A).At a constant300␮M free external Ca2ϩconcentration([Ca2ϩ]o),the EC50for the ATP4Ϫeffect determined in various ATP-containing but Mg2ϩ-free solutions was58␮M(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,but not by its
inactive Fig.1.ATP and UTP activate a sustained inward current that is mediated by P2Y receptors.A:currents elicited at a holding potential(HP)ofϪ80mV on application of1mM ATP in the presence or absence of Mg2ϩon a rat ventricular cardiomyocyte.The fast transient current required the presence of Mg2ϩand was elicited only on fast ATP application.B:inward sustained currents(I ATP)elicited at a HP ofϪ80mV by applying increasing concentra-tions of ATP.C:concentration-effect relationships of current density elicited by ATP or UTP in the absence of Mg2ϩon ventricular cardiomyocytes isolated from control(nՆ9)and infarcted rats(PMI rat,nՆ6).The inset shows current densities induced by2ATP concentrations in control(nՆ4)and transgenic mice deficient for the P2X1,P2X4,or the P2X1-P2X4purinoceptors (KO mice,nՆ3for each single and double KO,pooled)and in dog(nՆ5) and human(nϭ4)ventricular myocytes.Values are meansϮSE;n is the number of cardiomyocytes from at least2hearts.
H23
ATP/UTP ACTIVATE TRPC3/7CHANNELS IN RAT CARDIOMYOCYTES
analog,U-73433,or by propranolol and arachidonyltrifluorom-ethyl ketone,inhibitors of PLD and PLA 2cascades,respec-tively,to produce DAG (2).Furthermore,I ATP was modified by neither LY-294,002and wortmannin nor by genistein,respec-tively PLC ␥,phosphatidylinositol 3-kinase,and tyrosine ki-nase inhibitors (Fig.3).Together,these data indicate that PLC ␤was stimulated by the P2Y 2receptors through a G protein to produce DAG.
Characteristics of the ATP/UTP-induced inward current in rat cardiomyocytes.The I ATP -voltage relationship,recorded during a negative voltage ramp from ϩ50to Ϫ100mV,exhibited a weak voltage dependence and showed a current reversal potential near 0mV,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 ϩby N -methyl-D -glucamine only slightly reduced (6mV)the reversal potential of the ATP-induced current (n ϭ3).
To investigate the effect of various [Ca 2ϩ]o and avoid changing the active ATP 4Ϫconcentration,we used a GTP ␥S-containing solution in the pipette and applied ATP for a short period that was,however,sufficient to activate the then main-tained I ATP .At that point,reducing Ca 2ϩin the perfusing solution markedly enhanced I ATP .This effect was reversible and voltage independent with a twofold increase in chord conductance when [Ca 2ϩ]o was reduced 20-fold and without significant effect on the reversal potential (Fig.4,B and C ).This indicates that the channel was inhibited by external Ca 2ϩdespite Ca 2ϩbeing allowed to pass through.Furthermore,cyclopiazonic acid (CPA)and FK-506,reported to increase [Ca 2ϩ]i ,significantly reduced I ATP (Fig.4D ).To further char-acterize the channel properties,we used various pharmacolog-ical compounds,namely,La 3ϩ,Gd 3ϩ,and the imidazole de-rivative SKF-96365,which were reported to inhibit Ca 2ϩ-permeable TRPCs,including the ATP-activated TRPC7(3,26).Likewise,these compounds significantly reduced the ATP-induced current in rat ventricular cardiomyocytes.Flufe-namic acid,a common inhibitor of expressed homomeric TRPCs,except TRPC6(19)(but see Ref.5),slightly increased I ATP (Fig.4D ).In addition,several compounds known to alter various currents in cardiac tissues,including lidocaine
(100
Fig. 2.Free ATP 4Ϫis the agonist at the purinergic receptor.A :typical recording of the I ATP in 2solutions that contained both 2mM Ca 2ϩand either 1mM ATP and 0mM Mg 2ϩor 3mM ATP and 3.5mM Mg 2ϩ,leading to similar estimated free ATP 4Ϫand Ca 2ϩactivities (150and 136␮M and 1.14and 1.18mM,respectively).Data are representative of 5similar experiments.B :dose-response curve of I ATP amplitude elicited by various ATP concentra-tions in a Mg 2ϩ-free,300␮M Ca 2ϩsolution (n ϭ6).The apparent half-effective ATP concentration (EC 50ATP )was 558␮M,corresponding to a calculated EC 50ATP 4Ϫof 58␮M.HP,Ϫ80
mV.
Fig.3.ATP-induced current requires the activation of PLC ␤.Incubating the cardiomyocytes with the PLD inhibitor propranolol (200␮M),the PLA 2inhibitor arachidonyltrifluoromethyl ketone (AACOCF 3;50␮M,incubated 10–20min),and the PLC ␥inhibitor LY-294,002(100␮M,incubated 30min),as well as with the broad-spectrum tyrosine kinase inhibitor genistein (20␮g/ml,incubated 30min at 37°C),had no effect on the inward current elicited at Ϫ80mV on the application of 300␮M ATP in the absence of Mg 2ϩ.I ATP was prevented only in the presence of the PLC ␤inhibitor U-73122(10␮M,incubated 10–15min).Filled bars represent experimental values,and open bars represent respective control cells;the number of cells is indicated above each bar.*P Ͻ
0.05.
Fig.4.Characteristics of the ATP-induced current.A :current-voltage rela-tionship established after the application of 300␮M ATP in the absence of Mg 2ϩduring a ramp potential as indicated at top .B :with 500␮M guanosine 5Ј-[␥-thio]triphosphate (GTP ␥S)in the pipette solution,the brief application of ATP induced a current that was not reversible after ATP removal.Decreasing extracellular Ca 2ϩthen significantly increased I ATP amplitude.HP,Ϫ80mV.C :mean sustained inward currents elicited under the conditions in B .Mean estimated chord conductances are 3.7Ϯ0.8,5.7Ϯ1.2,and 7.3Ϯ1.2nS at 2,0.6,and 0.1mM extracellular Ca 2ϩconcentrations,respectively (n ϭ5).D :the sustained ATP-induced current in Mg 2ϩ-free solution shared pharmacological properties with the expressed transient receptor potential channel TRPC7.Cyclopiazonic acid (CPA;30␮M,incubated 10min)and FK-506(25␮M,incubated 10–30min),both thought to increase intracellular Ca 2ϩconcentra-tion,inhibited the 300␮M ATP-induced 3ϩ(100␮M),Gd 2ϩ(100␮M),and SKF-96375(25␮M)induced a marked reversible inhibition of I ATP ,whereas flufenamic acid (FFA;100␮M)significantly potentiated the sustained current.Values are presented as percentages of controls.The number of cells is indicated within bars for each experimental condition.*P Ͻ0.05.
H24
ATP/UTP ACTIVATE TRPC3/7CHANNELS IN RAT CARDIOMYOCYTES
␮M),amiodarone (10–100␮M),quinidine (10–100␮M),and glibenclamide (50␮M),as well as isoproterenol (1␮M),did not significantly affect I ATP (n Ն4).
Under control conditions in cell-attached patch clamp,the patch membrane continuously held at Ϫ80mV demonstrated very rare single-channel openings.However,when the pipette contained 100␮M ATP,numerous openings were recorded (Fig.5).The current reversed near 0mV and showed no rectification.The two most frequently observed current levels exhibited conductances of 14and 23pS.In line with the above-reported signal transduction cascade,channel activity was markedly reduced when the cell was bathed in the pres-ence of U-73122.
Further properties of the sustained cationic current were determined by microspectrofluorescence analysis.The applica-tion of 30␮M ATP,in the absence of Mg 2ϩ,was sufficient to trigger an influx of Mn 2ϩ,as indicated by a marked quenching of the fura-2emissions following excitations at 340-and 380-nm wavelengths,whereas the signals varied in the oppo-site direction on ATP application in the control conditions as a consequence of Ca 2ϩinflux and ATP-induced intracellular Ca 2ϩrelease (8,30).Quenching was only very weak under basal conditions in the presence of Mn 2ϩbefore ATP applica-
tion (Fig.6A ).We also used Ba 2ϩas a surrogate for Ca 2ϩto estimate cation influx.Adding Ba 2ϩto the Mg 2ϩ-free,Ca 2ϩ-free solution did not affect basal cell fluorescence.The further ATP application induced a significant Ba 2ϩinflux (Fig.6B ).Ba 2ϩinflux rate was similar in the presence of UTP,whereas UDP was inefficient to elicit Ba 2ϩinflux.The ATP-induced Ba 2ϩinflux was for the most part inhibited by U-73122and reduced by one-half in the presence of 30␮M FK-506.
TRPC3/7channel proteins carry the ATP/UTP-induced in-ward current.The presence of various TRPC mRNAs was checked in isolated adult rat ventricular cardiomyocytes.The mRNAs of TRPC1,TRPC3,TRPC4,and TRPC7,but not TRPC5and TRPC6,were detected.Western blots further revealed the presence of TRPC3and TRPC7proteins having apparent molecular mass around 90–95kDa.Furthermore,it was possible to immunoprecipitate TRPC7with the anti-TRPC3antibody,suggesting that both proteins contribute to heteromeric TRPC3/7channels (Fig.7).A band whose
nature
Fig. 5.Single-channel characteristics of the ATP-induced current.A :no opening was observed in a patch from a control rat cardiomyocyte,whereas frequent openings were seen on another cell when the patch pipette contained 100␮M Na 2ATP.Downward deflections of the current trace represent in-wardly directed membrane currents at Ϫ80mV.Note that bathing the cell in the presence of 10␮M U-73122prevented channel openings.B :mean ATP-induced current in cell-attached patches on rat cardiomyocytes in control solution or in the presence of U-73122.The number of cells is indicated above each bar.C :mean single-channel current amplitudes as a function of mem-brane potential for the 2most frequently observed low levels of current,determined from at least 14membrane patches.The straight lines represent least squares fits of the
data.
Fig.6.Fluorescence analysis of the ATP-induced current.A :original record-ings in a rat ventricular cardiomyocyte loaded with fura-2.Changes in fluorescence induced by 30␮M ATP were in the opposite direction after excitation at either 340or 380nm,whereas there was a marked quenching effect after excitation at both wavelengths when the same ATP concentration was applied in the presence of 1mM Mn 2ϩ(3similar experiments).A.U.,arbitrary units.B :original tracings of the fluorescence ratio (⌬F 340/380)in 3rat ventricular cardiomyocytes sequentially submitted to Ca 2ϩ-free,2mM Ba 2ϩ-containing solution.A significant slow increase in Ba 2ϩfluorescence was observed only after 1mM ATP application.C :pooled data of the maximal rate of increase in Ba 2ϩfluorescence induced by ATP,UTP,and UDP all at 1mM or by 1mM ATP on cells incubated with 10␮M U-73122or 25␮M FK-506.The number of cells is indicated within bars for each experimental condition.*P Ͻ0.05.
H25
ATP/UTP ACTIVATE TRPC3/7CHANNELS IN RAT CARDIOMYOCYTES。