TRPC3 and TRPC4 Associate to Form a Redox-sensitive Cation Channel

Abstract

Canonical transient receptor potential proteins (TRPC) have been proposed to form homo- or heteromeric cation channels in a variety of tissues, including the vascular endothelium. Assembly of TRPC multimers is incompletely understood. In particular, heteromeric assembly of distantly related TRPC isoforms is still a controversial issue. Because we have previously suggested TRPC proteins as the basis of the redox-activated cation conductance of porcine aortic endothelial cells (PAECs), we set out to analyze the TRPC subunit composition of endogenous endothelial TRPC channels and report here on a redox-sensitive TRPC3-TRPC4 channel complex. The ability of TRPC3 and TRPC4 proteins to associate and to form a cation-conducting pore complex was supported by four lines of evidence as follows: 1) Co-immunoprecipitation experiments in PAECs and in HEK293 cells demonstrated the association of TRPC3 and TRPC4 in the same complex. 2) Fluorescence resonance energy transfer analysis demonstrated TRPC3-TRPC4 association, involving close proximity between the N terminus of TRPC4 and the C terminus of TRPC3 subunits. 3) Co-expression of TRPC3 and TRPC4 in HEK293 cells generated a channel that displayed distinct biophysical and regulatory properties. 4) Expression of dominant-negative TRPC4 proteins suppressed TRPC3-related channel activity in the HEK293 expression system and in native endothelial cells. Specifically, an extracellularly hemagglutinin (HA)-tagged TRPC4 mutant, which is sensitive to blockage by anti-HA-antibody, was found to transfer anti-HA sensitivity to both TRPC3-related currents in the HEK293 expression system and the redox-sensitive cation conductance of PAECs. We propose TRPC3 and TRPC4 as subunits of native endothelial cation channels that are governed by the cellular redox state. Canonical transient receptor potential proteins (TRPC) have been proposed to form homo- or heteromeric cation channels in a variety of tissues, including the vascular endothelium. Assembly of TRPC multimers is incompletely understood. In particular, heteromeric assembly of distantly related TRPC isoforms is still a controversial issue. Because we have previously suggested TRPC proteins as the basis of the redox-activated cation conductance of porcine aortic endothelial cells (PAECs), we set out to analyze the TRPC subunit composition of endogenous endothelial TRPC channels and report here on a redox-sensitive TRPC3-TRPC4 channel complex. The ability of TRPC3 and TRPC4 proteins to associate and to form a cation-conducting pore complex was supported by four lines of evidence as follows: 1) Co-immunoprecipitation experiments in PAECs and in HEK293 cells demonstrated the association of TRPC3 and TRPC4 in the same complex. 2) Fluorescence resonance energy transfer analysis demonstrated TRPC3-TRPC4 association, involving close proximity between the N terminus of TRPC4 and the C terminus of TRPC3 subunits. 3) Co-expression of TRPC3 and TRPC4 in HEK293 cells generated a channel that displayed distinct biophysical and regulatory properties. 4) Expression of dominant-negative TRPC4 proteins suppressed TRPC3-related channel activity in the HEK293 expression system and in native endothelial cells. Specifically, an extracellularly hemagglutinin (HA)-tagged TRPC4 mutant, which is sensitive to blockage by anti-HA-antibody, was found to transfer anti-HA sensitivity to both TRPC3-related currents in the HEK293 expression system and the redox-sensitive cation conductance of PAECs. We propose TRPC3 and TRPC4 as subunits of native endothelial cation channels that are governed by the cellular redox state. The TRPC 3The abbreviations used are: TRPC, transient receptor potential protein; PAEC, porcine aortic endothelial cell; FRET, fluorescence resonance energy transfer; HA, hemagglutinin; YFP, yellow fluorescent protein; CFP, cyan fluorescent protein; GFP, green fluorescent protein; PBS, phosphate-buffered saline; DPBS, Dulbecco's PBS; CCH, carbachol; OAG, 1-oleoyl-2-acetyl-sn-glycerol; t-BHP, tert-butylhydroperoxide. subfamily consists of seven isoforms of which several are typically co-expressed in most cell types studied so far, including vascular endothelial cells (1Chang A.S. Chang S.M. Garcia R.L. Schilling W.P. FEBS Lett. 1997; 415: 335-340Crossref PubMed Scopus (52) Google Scholar, 2Groschner K. Hingel S. Lintschinger B. Balzer M. Romanin C. Zhu X. Schreibmayer W. FEBS Lett. 1998; 437: 101-106Crossref PubMed Scopus (142) Google Scholar, 3Moore T.M. Brough G.H. Babal P. Kelly J.J. Li M. Stevens T. Am. J. Physiol. 1998; 275: L574-L582Crossref PubMed Google Scholar, 4Tiruppathi C. Freichel M. Vogel S.M. Paria B.C. Mehta D. Flockerzi V. Malik A.B. Circ. Res. 2002; 91: 70-76Crossref PubMed Scopus (332) Google Scholar, 5Kohler R. Brakemeier S. Kuhn M. Degenhardt C. Buhr H. Pries A. Hoyer J. Cardiovasc Res. 2001; 51: 160-168Crossref PubMed Scopus (72) Google Scholar). These TRPC proteins assemble to form multimeric pore structures, which enable cation transport across the plasma membrane (6Vazquez G. Wedel B.J. Aziz O. Trebak M. Putney Jr., J.W. Biochim. Biophys. Acta. 2004; 1742: 21-36Crossref PubMed Scopus (273) Google Scholar). Multimerization of TRPC species has emerged as a complex issue, because both homo- and heteromeric assemblies appear possible and up to three isoforms may contribute to formation of native pore complexes (7Strubing C. Krapivinsky G. Krapivinsky L. Clapham D.E. J. Biol. Chem. 2003; 278: 39014-39019Abstract Full Text Full Text PDF PubMed Scopus (347) Google Scholar). TRPC channel activation was found to be triggered by a variety of stimuli, most prominently phospholipase C-derived signals, including classic Ca2+ store depletion as well as mechanical forces and redox processes (8Hofmann T. Obukhov A.G. Schaefer M. Harteneck C. Gudermann T. Schultz G. Nature. 1999; 397: 259-263Crossref PubMed Scopus (1255) Google Scholar, 9Trebak M. Bird G.S. McKay R.R. Putney Jr., J.W. J. Biol. Chem. 2002; 277: 21617-21623Abstract Full Text Full Text PDF PubMed Scopus (221) Google Scholar, 10Zhang Z. Tang J. Tikunova S. Johnson J.D. Chen Z. Qin N. Dietrich A. Stefani E. Birnbaumer L. Zhu M.X. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 3168-3173Crossref PubMed Scopus (208) Google Scholar, 11Kiselyov K. Xu X. Mozhayeva G. Kuo T. Pessah I. Mignery G. Zhu X. Birnbaumer L. Muallem S. Nature. 1998; 396: 478-482Crossref PubMed Scopus (561) Google Scholar, 12Boulay G. Brown D.M. Qin N. Jiang M. Dietrich A. Zhu M.X. Chen Z. Birnbaumer M. Mikoshiba K. Birnbaumer L. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 14955-14960Crossref PubMed Scopus (348) Google Scholar, 13Zhu X. Jiang M. Birnbaumer L. J. Biol. Chem. 1998; 273: 133-142Abstract Full Text Full Text PDF PubMed Scopus (307) Google Scholar, 14Balzer M. Lintschinger B. Groschner K. Cardiovasc. Res. 1999; 42: 543-549Crossref PubMed Scopus (132) Google Scholar, 15Groschner K. Rosker C. Lukas M. Novartis Found. Symp. 2004; 258 (discussion 231-225, 263-266): 222-230Crossref PubMed Google Scholar). We have previously demonstrated that TRPC3 is involved in a cation conductance that is activated in response to oxidative stress in porcine aortic endothelial cells (PAECs) (14Balzer M. Lintschinger B. Groschner K. Cardiovasc. Res. 1999; 42: 543-549Crossref PubMed Scopus (132) Google Scholar). TRPC channels may sense redox stress due to oxidation-induced changes in the membrane lipid composition that has been recognized as an important regulator of TRPC channel function (16Lockwich T.P. Liu X. Singh B.B. Jadlowiec J. Weiland S. Ambudkar I.S. J. Biol. Chem. 2000; 275: 11934-11942Abstract Full Text Full Text PDF PubMed Scopus (359) Google Scholar). Accordingly, we identified cholesterol oxidase-induced modification of membrane lipid composition and lipid raft structure, as an effective trigger for activation of TRPC3 channels in HEK293 cells (15Groschner K. Rosker C. Lukas M. Novartis Found. Symp. 2004; 258 (discussion 231-225, 263-266): 222-230Crossref PubMed Google Scholar). Moreover, a TRPC isoform may exist as a homo- and heteromeric channel complex in a given cell type, and multimeric assembly of TRPC complexes may depend on cellular localization and on the availability of specific interacting protein partners (17Schaefer M. Pflugers Arch. 2005; 451: 35-42Crossref PubMed Scopus (122) Google Scholar). Co-immunoprecipitation and FRET microscopy studies suggested that TRPC3 may form not only homomeric channels but also heteromultimeric pore structures with its close relatives, TRPC6 und TRPC7 (18Hofmann T. Schaefer M. Schultz G. Gudermann T. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 7461-7466Crossref PubMed Scopus (629) Google Scholar). Although these investigations excluded heteromers that consist of distant relatives of the TRPC family, other studies provided evidence for heteromerization of TRPC3 with its more distant relative TRPC1 (19Lintschinger B. Balzer-Geldsetzer M. Baskaran T. Graier W.F. Romanin C. Zhu M.X. Groschner K. J. Biol. Chem. 2000; 275: 27799-27805Abstract Full Text Full Text PDF PubMed Scopus (263) Google Scholar, 20Liu X. Bandyopadhyay B.C. Singh B.B. Groschner K. Ambudkar I.S. J. Biol. Chem. 2005; 280: 21600-21606Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar) and of TRPC1 with TRPC5 or TRPC4 (7Strubing C. Krapivinsky G. Krapivinsky L. Clapham D.E. J. Biol. Chem. 2003; 278: 39014-39019Abstract Full Text Full Text PDF PubMed Scopus (347) Google Scholar, 21Strubing C. Krapivinsky G. Krapivinsky L. Clapham D.E. Neuron. 2001; 29: 645-655Abstract Full Text Full Text PDF PubMed Scopus (636) Google Scholar). Moreover, even the presence of a heteromer consisting of TRPC1, TRPC3, and TRPC5 has been proposed (7Strubing C. Krapivinsky G. Krapivinsky L. Clapham D.E. J. Biol. Chem. 2003; 278: 39014-39019Abstract Full Text Full Text PDF PubMed Scopus (347) Google Scholar). For the vascular endothelium the exact subunit composition of native TRPC channels is so far elusive. Here we report evidence for the existence of an endothelial cation channel that represents a TRPC3/C4 heteromer and is sensitive to oxidative modification of plasma membrane cholesterol. Cell Culture and Transfections—Endothelial cells were isolated from porcine aorta as described previously (14Balzer M. Lintschinger B. Groschner K. Cardiovasc. Res. 1999; 42: 543-549Crossref PubMed Scopus (132) Google Scholar) and cultured in Dulbecco's modified Eagle's medium (Sigma-Aldrich) containing 10% heat-inactivated fetal calf serum, 100 units/ml penicillin, 0.1 mg/ml streptomycin, and 1.25 μg/ml amphotericin B. Only the first passage of cells was used for experiments. Cells were grown to ∼80% confluency in either 100-mm Petri dishes (6 × 106 cells/dish), for biotinylation and immunoblotting experiments, or in 6-well plates (∼106 cells/well) for measurement of membrane currents. Human embryonic kidney (HEK293) cells stably expressing C-terminal HA-tagged human TRPC3 or the wild-type mouse TRPC4α, designated as T3-9 or T4 - 60 cells (22Zhang Z. Tang Y. Zhu M.X. Biochem. J. 2001; 354: 717-725Crossref PubMed Google Scholar), respectively, were established and maintained as previously described (13Zhu X. Jiang M. Birnbaumer L. J. Biol. Chem. 1998; 273: 133-142Abstract Full Text Full Text PDF PubMed Scopus (307) Google Scholar). An extracellularly HA-tagged TRPC3 (exoHA-TRPC3) construct was made by inserting a sequence encoding YPYDVPD before the codons for Y524A525 of human TRPC3, thus creating a complete HA epitope YPYDVPDYA in the putative second extracellular loop of the channel subunit. Similarly, an exoHA-TRPC4α construct was created by inserting codons for PYDVPDYA after that of Tyr-458 of the wild-type TRPC4α. The N-terminal fragment N292-TRPC4, including two ankyrin-like repeats and the coiled-coil region of the mouse TRPC4β isoform, was obtained by internal restriction site RsaI and the multicloning site KpnI. fragment was in for and in for both and FRET T3-9 cells were with or and with to the experiments were with wild-type HEK293 which were cultured in the of PAECs were with TRPC species and as a to the For FRET and FRET experiments, and of TRPC proteins were wild-type HEK293 cells or in cells stably expressing TRPC3 or TRPC4 or of were three with containing and on for with mg/ml in The biotinylation was by the cells three with containing 100 Cells were with PBS, from the dishes in PBS, and The were in of 60 and the and for on The cell were and × for to cell proteins were isolated by cell to 60 of in a of of The were The were by for × and the were three with The proteins were from the in 60 of 60 for and to and cell from PAECs and T3-9 with obtained as described were and × for to cell proteins were by of with of protein (Sigma-Aldrich) and for were with of either TRPC3, or HA of protein was to the complex for The were three with in and to for from cell and were by and to were with containing and with TRPC4 TRPC3 in or for with PBS, were with a were the system and a with an of were from and a of either cell or channel were either with a containing for cell experiments or and for experiments. In cell cells were with a containing either Ca2+ or a including In channel experiments, the and The of was to Cells were with either 100 or cholesterol For experiments cells were with a in were a channel currents were a of and with from to or to potential were by were Fluorescence HEK293 cells grown on for were to an extracellular to the used in experiments was used for FRET and was to a system was to an in with a a cyan set and a yellow was used to and to the were The FRET be due to from channel to For the FRET was after the to - - The relative fluorescence between the and were obtained from experiments only or was For the cellular in cells was after are as were for a in PAECs and in T3-9 was previously found to contribute to the oxidative conductance of PAECs (14Balzer M. Lintschinger B. Groschner K. Cardiovasc. Res. 1999; 42: 543-549Crossref PubMed Scopus (132) Google Scholar). a to analyze the of TRPC3 as a redox we of PAECs and HEK293 cells stably expressing TRPC3 to oxidative redox stress was by with cholesterol in and both cell types displayed a in the membrane conductance in response to cholesterol to and of and were in PAECs and T3-9 The ability of TRPC3 to a redox-sensitive cation conductance supported the of TRPC3 as a of the native redox-sensitive cation of the redox sensitivity for wild-type HEK293 cells and T3-9 both the membrane and cholesterol to redox is in The that cells displayed an membrane conductance that was in wild-type that cation channels by TRPC3 in HEK293 which are sensitive to oxidative the redox-activated membrane displayed a to which is not in with the of classic phospholipase TRPC3 conductance in HEK293 cells. TRPC3 and TRPC4 in PAECs to a analyze the composition of redox-sensitive TRPC channels in we biotinylation and experiments to for plasma TRPC species and for possible In immunoblotting experiments of TRPC1, TRPC3, and TRPC4 a of TRPC6 was in not in biotinylation experiments only TRPC3 and TRPC4 were as plasma TRPC species The presence of both TRPC3 and TRPC4 in the plasma membrane of PAECs to a possible heteromeric assembly of these TRPC species by experiments. in TRPC4 was in and These the existence of a TRPC3-TRPC4 a potential redox-sensitive channel complex in PAECs. of TRPC3 and TRPC4 in the HEK293 Expression a to the ability of TRPC3 and TRPC4 proteins to we experiments with T3-9 cells that were to in of the HA-tagged TRPC3 from T3-9 anti-HA the for the Similarly, from T3-9 cells to TRPC4 TRPC4 not the to TRPC4 in the T4 - 60 cell which TRPC3 expression for of the were in both HEK293 wild-type and T3-9 cells and that proteins with a to that of TRPC4 are not with these in the HEK293 system In an to and analyze TRPC3-TRPC4 association, we used expression of and proteins of TRPC3 and TRPC4 proteins to FRET analysis of Because a which for TRPC3-TRPC4 not FRET C-terminal proteins (17Schaefer M. Pflugers Arch. 2005; 451: 35-42Crossref PubMed Scopus (122) Google Scholar), we are the of FRET microscopy in cells that proteins in which the were to the N and C of the respectively, as in the the We a FRET that was in the plasma membrane region of the cells Moreover, FRET experiments were to for the ability of TRPC4 to the homomeric TRPC3 in of and TRPC3 in FRET, which was suppressed TRPC4 was in to the TRPC3 FRET partners and These of TRPC3 and TRPC4 proteins in the HEK293 expression The and TRPC3 in the HEK293 Expression by TRPC4 of heteromeric that of are to the channel function generated by the other TRPC We set out to the of TRPC4 on TRPC3 currents. were to for sensitivity of TRPC3 to by TRPC4 we have previously found that N-terminal of proteins are effective as K. Hingel S. Lintschinger B. Balzer M. Romanin C. Zhu X. Schreibmayer W. FEBS Lett. 1998; 437: 101-106Crossref PubMed Scopus (142) Google Scholar, 14Balzer M. Lintschinger B. Groschner K. Cardiovasc. Res. 1999; 42: 543-549Crossref PubMed Scopus (132) Google Scholar), we co-expressed TRPC3 with an N-terminal fragment of Expression of N-terminal TRPC4 fragment suppressed the currents typically in the HEK293 expression system In a second we co-expressed TRPC3 with an which by not the conductance by construct a sensitivity of the TRPC3 channel activity to by anti-HA in TRPC3-related currents in T3-9 cells were in cells with anti-HA we of anti-HA sensitivity of a TRPC3-related conductance by of an construct the ability of the TRPC4 construct to form a pore complex with Similarly, expression of generated a anti-HA sensitivity of the TRPC3 currents activated by cholesterol as in and expression of the TRPC4 construct not the cholesterol oxidase-induced currents in T3-9 cells but generated sensitivity of conductance to by anti-HA The cholesterol oxidase-induced was by anti-HA that the TRPC4 construct is the redox-sensitive TRPC3-related pore complex. in redox-activated currents in T3-9 cells were to anti-HA The of PAECs by TRPC3 and TRPC4 of both TRPC3 and TRPC4 in the cholesterol conductance of PAECs was by experiments in to either or in anti-HA of PAECs to either TRPC species suppressed the redox membrane to by in cholesterol which were with Expression of TRPC species by not the redox-activated conductance but generated anti-HA sensitivity to that in T3-9 cells These the of TRPC3/C4 heteromerization in native endothelial cells and that TRPC3/C4 heteromeric cation channel as a for the cellular redox state. of existence of redox-sensitive pore complexes of TRPC3 and TRPC4 subunits was the channel the activated by in and HEK293 cells as well as in native endothelial cells close to and the of the channels was complex in three cell as well as The channel in T3-9 cells a conductance of 3) channel was also activated in response to of the cells with not In T3-9 cells to TRPC4 in to TRPC3, the conductance activated by cholesterol was is of that from a cell TRPC3-TRPC4 a channel of conductance not the conductance of the channel in cells was close to that for TRPC4 channels M. N. N. Schultz G. J. Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus Google we regulatory of of cells with which has been to TRPC3 but not TRPC4 channels (8Hofmann T. Obukhov A.G. Schaefer M. Harteneck C. Gudermann T. Schultz G. Nature. 1999; 397: 259-263Crossref PubMed Scopus (1255) Google Scholar), a in the activity of the channel in cells that cells channel activity that is from that of HEK293 cells expressing TRPC3 or TRPC4 The cation channels in cholesterol PAECs a conductance that was and that for TRPC3 of channel typically in These from channel that of TRPC3 and TRPC4 in HEK293 cells a pore structure, from homomeric TRPC3 or TRPC4 of heteromeric TRPC3-TRPC4 channels in the HEK293 expression to the native redox-sensitive cation conductance of endothelial cells. the we evidence for heteromeric assembly of TRPC3 and TRPC4 and that TRPC3/C4 heteromers are in PAECs to as redox-sensitive cation the previously proposed of TRPC3 in the redox-sensitive membrane conductance of PAECs (14Balzer M. Lintschinger B. Groschner K. Cardiovasc. Res. 1999; 42: 543-549Crossref PubMed Scopus (132) Google Scholar, 15Groschner K. Rosker C. Lukas M. Novartis Found. Symp. 2004; 258 (discussion 231-225, 263-266): 222-230Crossref PubMed Google Scholar). redox sensitivity of membrane cholesterol was as an that for oxidative of membrane oxidative stress with of cellular function as with of (15Groschner K. Rosker C. Lukas M. Novartis Found. Symp. 2004; 258 (discussion 231-225, 263-266): 222-230Crossref PubMed Google Scholar). of cells with cholesterol was found to a cation conductance in both native endothelial cells and HEK293 cells The membrane by cholesterol were to obtained with the t-BHP, which was used in studies the of redox-sensitive membrane Schilling W.P. J. Biol. Chem. Full Text PDF PubMed Google Scholar). activated cation in both native endothelial cells (PAECs) as well as in cells. These currents were but not in of biophysical because were more in native endothelial cells. Moreover, the currents not typically phospholipase C activation in HEK293 cells C. A. Lukas M. P. Zhu M.X. Romanin C. Groschner K. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). was to either from of redox-sensitive in PAECs or from a composition of the redox-sensitive pore both and cholesterol activated a cation conductance in cells expressing TRPC3 but not in wild-type HEK293 the potential of TRPC3 as a of redox-sensitive cation The previously proposed of TRPC3 as a redox in PAECs (14Balzer M. Lintschinger B. Groschner K. Cardiovasc. Res. 1999; 42: 543-549Crossref PubMed Scopus (132) Google Scholar) was in on the expression of TRPC3 in these native endothelial cells. In the we TRPC3 expression the protein and demonstrated that TRPC species is to the plasma membrane of these endothelial cells. native TRPC channels are to we for plasma membrane expression of potential heteromerization partners of only a related protein that has so far not been identified as heteromerization of TRPC3 (18Hofmann T. Schaefer M. Schultz G. Gudermann T. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 7461-7466Crossref PubMed Scopus (629) Google Scholar, W.P. M. Novartis Symp. 2004; 258 (discussion Google Scholar), was in the plasma membrane of PAECs. (7Strubing C. Krapivinsky G. Krapivinsky L. Clapham D.E. J. Biol. Chem. 2003; 278: 39014-39019Abstract Full Text Full Text PDF PubMed Scopus (347) Google Scholar) demonstrated the of the formation of heteromeric channel structures of TRPC3 and TRPC4 TRPC1 was as the channel subunit. experiments in PAECs that endogenous TRPC3 and TRPC4 are to associate in the membrane of PAECs. with the of (7Strubing C. Krapivinsky G. Krapivinsky L. Clapham D.E. J. Biol. Chem. 2003; 278: 39014-39019Abstract Full Text Full Text PDF PubMed Scopus (347) Google Scholar), we found TRPC1 expression and evidence for a between TRPC3 and plasma membrane expression of TRPC1 was not in biotinylation experiments. the of TRPC1 in the formation of heteromeric TRPC channels in PAECs to be Because TRPC3 and TRPC4 have been excluded by two studies with and FRET experiments (18Hofmann T. Schaefer M. Schultz G. Gudermann T. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 7461-7466Crossref PubMed Scopus (629) Google Scholar, W.P. M. Novartis Symp. 2004; 258 (discussion Google Scholar), we several to for the existence of pore TRPC3/C4 The association of TRPC3 with TRPC4 proteins in HEK293 cells was by experiments and FRET we energy transfer between and TRPC3 and TRPC4 proteins the fluorescence were to the and C-terminal of the TRPC4 and TRPC3 were not fluorescence proteins were to only the N or C of the These a of localization of the for the of TRPC3/C4 association, which may of the obtained with FRET a close proximity of N and C the TRPC3-TRPC4 TRPC3-TRPC4 was by the ability of wild-type TRPC4 to the between the and of TRPC3 and TRPC4 in of formation of a distinct pore was in channel experiments, which demonstrated that of TRPC3 and TRPC4 to cation channel activity of properties. The channel was found sensitive to activation by OAG, a cation conductance from the so far described TRPC3 and TRPC4 generated by of channel proteins M. N. N. Schultz G. J. Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus Google Scholar, K. Mignery Zhu M.X. Muallem S. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). the existence of TRPC3-TRPC4 pore complexes we the of TRPC4 on membrane currents in HEK293 cells and in PAECs. demonstrated that both an N-terminal fragment of TRPC4 as well as an TRPC4 is to TRPC3-related currents in T3-9 cells and PAECs. we demonstrated that TRPC containing a HA epitope in the second extracellular a on is in that the presence of an with anti-HA of the TRPC4 construct with TRPC3 in HEK293 cells the currents in native PAECs Moreover, the were in these cells by anti-HA that TRPC4 is the TRPC3 channel In with the of TRPC3 and TRPC4 association a redox-sensitive channel complex in we found that either expression of or was effective in of the dominant-negative of the redox-sensitive in PAECs. of the cholesterol cation channels in PAECs conductance and between the endothelial redox-sensitive channel and the channel in HEK293 cells. The basis of the in channel is is to the existence of TRPC3-TRPC4 heteromers with a which may the between native cells and the expression we in of a cell a conductance of with the an that be in of of a second important to that the redox-sensitive channel in porcine aortic endothelium from a previously redox-sensitive cation conductance in endothelial cells of calf aorta J. Physiol. PubMed Scopus Google Scholar), of endothelial cation In we the first evidence for cation channels of both TRPC3 and TRPC4 subunits. We that a TRPC3-TRPC4 heteromeric channel is in porcine aortic endothelial cells and represents a possible for changes in the endothelium may be as a potential for We T. and M. for TRPC3 and TRPC4 as well as and for