Regulation of Canonical Transient Receptor Potential (TRPC) Channel Function by Diacylglycerol and Protein Kinase C
2003; Elsevier BV; Volume: 278; Issue: 31 Linguagem: Inglês
10.1074/jbc.m302751200
ISSN1083-351X
AutoresKartik Venkatachalam, Fei Zheng, Donald L. Gill,
Tópico(s)Biochemical Analysis and Sensing Techniques
ResumoThe mechanism of receptor-induced activation of the ubiquitously expressed family of mammalian canonical transient receptor potential (TRPC) channels has been the focus of intense study. Primarily responding to phospholipase C (PLC)-coupled receptors, the channels are reported to receive modulatory input from diacylglycerol, endoplasmic reticulum inositol 1,4,5-trisphosphate receptors and Ca2+ stores. Analysis of TRPC5 channels transfected within DT40 B cells and deletion mutants thereof revealed efficient activation in response to PLC-β or PLC-γ activation, which was independent of inositol 1,4,5-trisphoshate receptors or the content of stores. In both HEK293 cells and DT40 cells, TRPC5 and TRPC3 channel responses to PLC activation were highly analogous, but only TRPC3 and not TRPC5 channels responded to the addition of the permeant diacylglycerol (DAG) analogue, 1-oleoyl-2-acetyl-sn-glycerol (OAG). However, OAG application or elevated endogenous DAG, resulting from either DAG lipase or DAG kinase inhibition, completely prevented TRPC5 or TRPC4 activation. This inhibitory action of DAG on TRPC5 and TRPC4 channels was clearly mediated by protein kinase C (PKC), in distinction to the stimulatory action of DAG on TRPC3, which is established to be PKC-independent. PKC activation totally blocked TRPC3 channel activation in response to OAG, and the activation was restored by PKC-blockade. PKC inhibition resulted in decreased TRPC3 channel deactivation. Store-operated Ca2+ entry in response to PLC-coupled receptor activation was substantially reduced by OAG or DAG-lipase inhibition in a PKC-dependent manner. However, store-operated Ca2+ entry in response to the pump blocker, thapsigargin, was unaffected by PKC. The results reveal that each TRPC subtype is strongly inhibited by DAG-induced PKC activation, reflecting a likely universal feedback control on TRPCs, and that DAG-mediated PKC-independent activation of TRPC channels is highly subtype-specific. The profound yet distinct control by PKC and DAG of the activation of TRPC channel subtypes is likely the basis of a spectrum of regulatory phenotypes of expressed TRPC channels. The mechanism of receptor-induced activation of the ubiquitously expressed family of mammalian canonical transient receptor potential (TRPC) channels has been the focus of intense study. Primarily responding to phospholipase C (PLC)-coupled receptors, the channels are reported to receive modulatory input from diacylglycerol, endoplasmic reticulum inositol 1,4,5-trisphosphate receptors and Ca2+ stores. Analysis of TRPC5 channels transfected within DT40 B cells and deletion mutants thereof revealed efficient activation in response to PLC-β or PLC-γ activation, which was independent of inositol 1,4,5-trisphoshate receptors or the content of stores. In both HEK293 cells and DT40 cells, TRPC5 and TRPC3 channel responses to PLC activation were highly analogous, but only TRPC3 and not TRPC5 channels responded to the addition of the permeant diacylglycerol (DAG) analogue, 1-oleoyl-2-acetyl-sn-glycerol (OAG). However, OAG application or elevated endogenous DAG, resulting from either DAG lipase or DAG kinase inhibition, completely prevented TRPC5 or TRPC4 activation. This inhibitory action of DAG on TRPC5 and TRPC4 channels was clearly mediated by protein kinase C (PKC), in distinction to the stimulatory action of DAG on TRPC3, which is established to be PKC-independent. PKC activation totally blocked TRPC3 channel activation in response to OAG, and the activation was restored by PKC-blockade. PKC inhibition resulted in decreased TRPC3 channel deactivation. Store-operated Ca2+ entry in response to PLC-coupled receptor activation was substantially reduced by OAG or DAG-lipase inhibition in a PKC-dependent manner. However, store-operated Ca2+ entry in response to the pump blocker, thapsigargin, was unaffected by PKC. The results reveal that each TRPC subtype is strongly inhibited by DAG-induced PKC activation, reflecting a likely universal feedback control on TRPCs, and that DAG-mediated PKC-independent activation of TRPC channels is highly subtype-specific. The profound yet distinct control by PKC and DAG of the activation of TRPC channel subtypes is likely the basis of a spectrum of regulatory phenotypes of expressed TRPC channels. The superfamily of TRP 1The abbreviations and trivial names used are: TRP, transient receptor potential; SOC, store-operated channel; TRPC, canonical TRP; PLC, phospholipase C; PKC, protein kinase C; InsP3, inositol 1,4,5-trisphosphate; InsP3R, InsP3 receptor; DAG, diacylglycerol; OAG, 1-oleoyl-2-acetyl-sn-glycerol; TG, thapsigargin; BCR, B-cell receptor; GPCR, G protein-coupled receptor; M5R, M5 muscarinic receptor; CCh, carbachol; SERCA, sarcoplasmic/endoplasmic reticulum Ca2+ ATPase; eYFP, enhanced yellow fluorescent protein; PMA, phorbol 12-myristate 13-acetate; R59949, 3-[2-[4-(bis-(4-fluorophenyl)methylene]piperidin-1-yl)ethyl]-2, 3-dihydro-2-thioxo-4(1H)-quin-azolinone. ion channels contains a large group of channels mediating an array of signal and sensory transduction pathways (1Montell C. Birnbaumer L. Flockerzi V. Cell. 2002; 108: 595-598Abstract Full Text Full Text PDF PubMed Scopus (736) Google Scholar, 2Montell C. 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Schultz G. Gudermann T. J. Mol. Med. 2000; 78: 14-25Crossref PubMed Scopus (117) Google Scholar, 6Venkatachalam K. van Rossum D.B. Patterson R.L. Ma H.T. Gill D.L. Nat. Cell Biol. 2002; 4: E263-E272Crossref PubMed Scopus (340) Google Scholar, 8Harteneck C. Plant T.D. Schultz G. Trends. Neurosci. 2000; 23: 159-166Abstract Full Text Full Text PDF PubMed Scopus (434) Google Scholar, 10Putney Jr., J.W. Broad L.M. Braun F.J. Lievremont J.P. Bird G.S. J. Cell Sci. 2001; 114: 2223-2229Crossref PubMed Google Scholar). In studies utilizing the triple InsP3R-deficient variant of the chicken B-cell line, DT40 (DT40 InsP 3 R –/–), we and others have determined that endogenous store-operated channels are observed to operate identically as in wild-type DT40 cells (DT40wt), indicating that the InsP3R is nonessential for endogenous store-operated channels (25Ma H.-T. Venkatachalam K. Li H.S. Montell C. Kurosaki T. Patterson R.L. Gill D.L. J. Biol. Chem. 2001; 276: 18888-18896Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar, 26Sugawara H. Kurosaki M. Takata M. Kurosaki T. EMBO J. 1997; 16: 3078-3088Crossref PubMed Scopus (377) Google Scholar, 27Broad L.M. Braun F.J. Lievremont J.P. Bird G.S. Kurosaki T. Putney Jr., J.W. J. Biol. Chem. 2001; 276: 15945-15952Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar). Moreover, we recently reported (28Venkatachalam K. Ma H.T. Ford D.L. Gill D.L. J. Biol. Chem. 2001; 276: 33980-33985Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar, 29Ma H.T. Venkatachalam K. Parys J.B. Gill D.L. J. Biol. Chem. 2002; 277: 6915-6922Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar) that TRPC3 channels expressed in the DT40 InsP 3 R –/– line can be activated in response to PLC-coupled receptors and function identically as TRPC3 channels expressed in DT40 wt cells. Our analyses reveal that TRPC3 channels are activated in response to PLC-coupled receptors and are mimicked by the application of exogenous DAG (28Venkatachalam K. Ma H.T. Ford D.L. Gill D.L. J. Biol. Chem. 2001; 276: 33980-33985Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar). Another report using the same cells revealed that the expressed TRPC3 channels can reflect input from stores and InsP3Rs (30Vazquez G. Lievremont J.P. St J. Bird G. Putney Jr., J.W. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 11777-11782Crossref PubMed Scopus (156) Google Scholar), and it appears that the conditions under which channels are expressed may alter their coupling phenotype (6Venkatachalam K. van Rossum D.B. Patterson R.L. Ma H.T. Gill D.L. Nat. Cell Biol. 2002; 4: E263-E272Crossref PubMed Scopus (340) Google Scholar). Nevertheless, under all conditions of expression, the TRPC3 channels are clearly activated by the application of DAG (28Venkatachalam K. Ma H.T. Ford D.L. Gill D.L. J. Biol. Chem. 2001; 276: 33980-33985Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar, 30Vazquez G. Lievremont J.P. St J. Bird G. Putney Jr., J.W. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 11777-11782Crossref PubMed Scopus (156) Google Scholar). Certainly, these results are consistent with the earlier report from Hofmann et al. (31Hofmann T. Obukhov A.G. Schaefer M. Harteneck C. Gudermann T. Schultz G. Nature. 1999; 397: 259-263Crossref PubMed Scopus (1285) Google Scholar) indicating that members of the closely related subgroup of TRPC3, TRPC6, and TRPC7 channels can each be activated in response to DAG through a mechanism independent of PKC. Other members of the TRPC channel family appear to behave differently. Thus, the subgroup represented by the closely related TRPC4 and TRPC5 channel proteins are reported to respond to store depletion (11Philipp S. Cavalié A. Freichel M. Wissenbach U. Zimmer S. Trost C. Marquart A. Murakami M. Flockerzi V. 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We therefore considered it important to investigate the role of store emptying and InsP3Rs in the activation of TRPC4 and TRPC5 channels utilizing the DT40 knockout cell lines and to assess how the activation of these channels in response to PLC-coupled receptors compares with the activation of TRPC3 channels. Our results indicate some important differences in the role of DAG as a mediator of TRPC channel activation and reveal that each TRPC subtype is strongly inhibited by DAG-induced PKC activation, reflecting a likely universal feedback control mechanism for TRPC channels. Culture of Cells—The DT40 chicken B-cell lines, wild-type (DT40-wt), triple InsP3R knockout (DT40 InsP 3 R –/–), and the PLC-γ2 knockout (DT40 PLCγ2 –/–) cells were cultured in RPMI 1640 (Invitrogen) supplemented with 10% fetal bovine serum, penicillin, streptomycin, and glutamine, as described previously (25Ma H.-T. Venkatachalam K. Li H.S. Montell C. Kurosaki T. Patterson R.L. Gill D.L. J. Biol. Chem. 2001; 276: 18888-18896Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar, 25Ma H.-T. Venkatachalam K. Li H.S. Montell C. Kurosaki T. Patterson R.L. Gill D.L. J. Biol. Chem. 2001; 276: 18888-18896Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar, 26Sugawara H. Kurosaki M. Takata M. Kurosaki T. EMBO J. 1997; 16: 3078-3088Crossref PubMed Scopus (377) Google Scholar, 29Ma H.T. Venkatachalam K. Parys J.B. Gill D.L. J. Biol. Chem. 2002; 277: 6915-6922Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar). HEK293 cells and T3-65 cells were cultured in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% fetal bovine serum, penicillin, streptomycin, and G-418 as described previously (20Ma H.-T. Patterson R.L. van Rossum D.B. Birnbaumer L. Mikoshiba K. Gill D.L. Science. 2000; 287: 1647-1651Crossref PubMed Scopus (534) Google Scholar) Transfection of Cells—All three DT40 cell types were cultured overnight in RPMI 1640 with 10% fetal bovine serum, harvested from plates by scraping, washed in reduced-serum Optimem (Invitrogen), and then resuspended in Optimem at a final concentration of 107 cells/ml. 12 μg of each of the plasmids to be transfected (containing either the human M5 muscarinic receptor, mouse TRPC5 channel, or human TRPC3 channel, each in the pcDNA3.1 vector) were taken with 5 μg of the marker DNA (eYFP) and added to 0.5-ml transfection cuvettes with an electrode gap of 0.4 cm followed by the addition of 0.5 ml of the cells in Optimem (107 cells/ml). After thorough mixing of cells and DNA, transfection was carried out using the Gene Pulser II electroporation system (Bio-Rad) at 350 mV, 960 microfarads, and infinite resistance. The cells were recovered in Optimem (no serum added) for 3 h and then resuspended in Optimem with 10% fetal bovine serum and applied to coverslips. Cells were allowed to attach for 3 h in the case of DT40 cells and overnight in the case of HEK293 cells before fura-2 measurements were undertaken. The overall efficiency of transfection (eYFP-positive cells) was 20–30% as detected during fluorescent imaging. The methods were similar to those described previously (28Venkatachalam K. Ma H.T. Ford D.L. Gill D.L. J. Biol. Chem. 2001; 276: 33980-33985Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar, 33Patterson R.L. van Rossum D.B. Gill D.L. Cell. 1999; 98: 487-499Abstract Full Text Full Text PDF PubMed Scopus (387) Google Scholar, 34Patterson R.L. van Rossum D.B. Ford D.L. Hurt K.J. Bae S.S. Suh P.G. Kurosaki T. Snyder S.H. Gill D.L. Cell. 2002; 111: 529-541Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar). Imaging of Intracellular Calcium in Single Transfected Cells—Cells grown on coverslips after transfection were placed in Hepes-buffered Krebs medium (107 mm NaCl, 6 mm KCl, 1.2 mm MgSO4, 1 mm CaCl2, 1.2 mm KH2PO4, 11.5 mm glucose, 0.1% bovine serum albumin, 20 mm Hepes-KOH, pH 7.4) and loaded with fura-2/acetoxymethylester (2 μm) for 25 min at 20 °C. Cells were washed, and dye was allowed to deesterify for a minimum of 15 min at 20 °C. Approximately 95% of the dye was confined to the cytoplasm as determined by the signal remaining after saponin permeabilization (35Waldron R.T. Short A.D. Gill D.L. J. Biol. Chem. 1995; 270: 11955-11961Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar, 36Short A.D. Klein M.G. Schneider M.F. Gill D.L. J. Biol. Chem. 1993; 268: 25887-25893Abstract Full Text PDF PubMed Google Scholar). Cells on coverslips were place in “cation-safe” medium free of sulfate and phosphate anions (107 mm NaCl, 7.2 mm KCl, 1.2 mm MgCl2, 11.5 mm glucose, 20 mm Hepes-NaOH, pH 7.2) in the absence or presence of 1 mm CaCl2, SrCl2, or BaCl2 as shown elsewhere (Ref. 28Venkatachalam K. Ma H.T. Ford D.L. Gill D.L. J. Biol. Chem. 2001; 276: 33980-33985Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar, figures therein). Ca2+ measurements in single transfected and groups of untransfected cells were made using an InCyt dual-wavelength fluorescence imaging system (Intracellular Imaging Inc.). Cotransfected eYFP served as the transfection marker and was detected at an excitation wavelength of 485 nm. Untransfected cells (not expressing eYFP) were identified from the same field and served as control cells. After cell identification, fluorescence emission at 505 nm was monitored with excitation at 340 and 380 nm; intracellular divalent cation measurements (either Ca2+, Sr2+, or Ba2+) are shown as 340/380 nm ratios obtained from groups of single untransfected and transfected cells. The details of these divalent cation measurements were described previously (20Ma H.-T. Patterson R.L. van Rossum D.B. Birnbaumer L. Mikoshiba K. Gill D.L. Science. 2000; 287: 1647-1651Crossref PubMed Scopus (534) Google Scholar, 25Ma H.-T. Venkatachalam K. Li H.S. Montell C. Kurosaki T. Patterson R.L. Gill D.L. J. Biol. Chem. 2001; 276: 18888-18896Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar, 25Ma H.-T. Venkatachalam K. Li H.S. Montell C. Kurosaki T. Patterson R.L. Gill D.L. J. Biol. Chem. 2001; 276: 18888-18896Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar, 28Venkatachalam K. Ma H.T. Ford D.L. Gill D.L. J. Biol. Chem. 2001; 276: 33980-33985Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar, 37van Rossum D.B. Patterson R.L. Ma H.-T. Gill D.L. J. Biol. Chem. 2000; 275: 28562-28568Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). Resting Ca2+ levels in all of the DT40 cell lines were similar, ∼100–130 nm. Resting Ca2+ levels in the HEK293 cells and T3-65 cells were 50–100 nm. All measurements shown are representative of a minimum of three and in most cases of a larger number of independent experiments. Materials and Miscellaneous Procedures—Plasmids obtained were as follows: human TRPC3 cDNA from Craig Montell (Johns Hopkins University), mouse TRPC5 from Michael Schaeffer (Freie Universitaet, Berlin), eYFP cDNA from Clontech, and human M5 musarinic receptor cDNA from L. Birnbaumer (UCLA). 1-Oleoyl-2-acetyl-sn-glyecrol (OAG), RHC-80267, R59949, and GF-109203X were from Calbiochem. EGTA, carbachol, and PMA were from Sigma. Thapsigargin (TG) was from LC Services (Woburn, MA). Fura-2/acetoxymethylester was from Molecular Probes, Eugene, OR. Anti-chicken IgM (M4 clone) was from Southern Biotechnology Associates, Birmingham, AL. The DT40 cell lines were kindly supplied by Dr. Tomohiro Kurosaki (Kyoto, Japan). The T3-65 clone was a kind gift from Lutz Birnbaumer (UCLA). The DT40 chicken B-cell line has been useful for evaluating the mechanisms by which store-operated channels and TRPC channels are activated. The high rate of homologous recombination in these cells facilitates targeted disruption of certain genes or groups of genes (26Sugawara H. Kurosaki M. Takata M. Kurosaki T. EMBO J. 1997; 16: 3078-3088Crossref PubMed Scopus (377) Google Scholar, 38Winding P. Berchtold M.W. J. Immunol. Methods. 2001; 249: 1-16Crossref PubMed Scopus (110) Google Scholar). Two such derivatives of these cells have been of particular use in assessing Ca2+ signaling mechanisms: the triple InsP3R-knockout variant DT40 cell line (DT40-InsP 3 R –/–), in which all three InsP3R genes have been eliminated (26Sugawara H. Kurosaki M. Takata M. Kurosaki T. EMBO J. 1997; 16: 3078-3088Crossref PubMed Scopus (377) Google Scholar), and the PLC-γ2-knockout variant (DT40-PLC-γ2 –/–), which is devoid of both PLC-γ1 and PLC-γ2 subtypes (34Patterson R.L. van Rossum D.B. Ford D.L. Hurt K.J. Bae S.S. Suh P.G. Kurosaki T. Snyder S.H. Gill D.L. Cell. 2002; 111: 529-541Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar, 39Takata M. Homma Y. Kurosaki T. J. Exp. Med. 1995; 182: 907-914Crossref PubMed Scopus (183) Google Scholar). These cell lines have allowed us to assess the roles of InsP3Rs and PLC-γ in the activation and maintenance of both endogenous SOCs and also of over-expressed TRPC3 channels (25Ma H.-T. Venkatachalam K. Li H.S. Montell C. Kurosaki T. Patterson R.L. Gill D.L. J. Biol. Chem. 2001; 276: 18888-18896Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar, 28Venkatachalam K. Ma H.T. Ford D.L. Gill D.L. J. Biol. Chem. 2001; 276: 33980-33985Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar, 29Ma H.T. Venkatachalam K. Parys J.B. Gill D.L. J. Biol. Chem. 2002; 277: 6915-6922Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar, 34Patterson R.L. van Rossum D.B. Ford D.L. Hurt K.J. Bae S.S. Suh P.G. Kurosaki T. Snyder S.H. Gill D.L. Cell. 2002; 111: 529-541Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar). To gain more information on the activation of TRPC channels, we investigated the role of InsP3Rs and PLC-γ on the activation of TRPC5 channels in DT40 cells. As in earlier studies (25Ma H.-T. Venkatachalam K. Li H.S. Montell C. Kurosaki T. Patterson R.L. Gill D.L. J. Biol. Chem. 2001; 276: 18888-18896Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar, 28Venkatachalam K. Ma H.T. Ford D.L. Gill D.L. J. Biol. Chem. 2001; 276: 33980-33985Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar, 29Ma H.T. Venkatachalam K. Parys J.B. Gill D.L. J. Biol. Chem. 2002; 277: 6915-6922Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar), we assessed the entry of Ba2+, which does not enter through endogenous SOCs in DT40 cells because of their high Ca2+ selectivity. DT40 cells were transiently cotransfected with TRPC5 together with eYFP to identify transfected cells as described previously (28Venkatachalam K. Ma H.T. Ford D.L. Gill D.L. J. Biol. Chem. 2001; 276: 33980-33985Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar). The activation of TRPC5 channels in response to B-cell receptor cross-linking induced by the addition of 3 μg/ml anti-IgM was assessed in fura-2-loaded wild-type B cells in the absence of extracellular Ca2+ (Fig. 1A). Stimulation of the BCR complex results in a cascade of non-receptor tyrosine phosphorylation events leading to activation of the PLC-γ2 enzyme, which cleaves phosphatidylinositol 4,5-bisphosphate to the products InsP3 and DAG (28Venkatachalam K. Ma H.T. Ford D.L. Gill D.L. J. Biol. Chem. 2001; 276: 33980-33985Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar). As shown in Fig. 1A, a substantial InsP3-mediated release of stored Ca2+ is observed, which slowly declines over a 6-min period as stores are depleted. As described previously (25Ma H.-T. Venkatachalam K. Li H.S. Montell C. Kurosaki T. Patterson R.L. Gill D.L. J. Biol. Chem. 2001; 276: 18888-18896Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar, 28Venkatachalam K. Ma H.T. Ford D.L. Gill D.L. J. Biol. Chem. 2001; 276: 33980-33985Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar), the subsequent addition of extracellular Ca2+ under this condition results in substantial store-operated Ca2+ entry in DT40 cells. However, the addition of Ba2+ does not result in entry (Fig. 1A), reflecting the high divalent cation specificity of the endogenous store-operated Ca2+ entry process in these cells. Indeed, in keeping with other cells of hematopoietic origin, recent evidence clearly identifies operation of the CRAC (Ca2+ release-activated Ca2+) channel in DT40 cells (29Ma H.T. Venkatachalam K. Parys J.B. Gill D.L. J. Biol. Chem. 2002; 277: 6915-6922Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar, 40Prakriya M. Lewis R.S. J. Physiol. 2001; 536: 3-19Crossref PubMed Scopus (437) Google Scholar), a channel with remarkable selectivity for Ca2+, that is virtually impermeable to other alkaline-earth cations including Sr2+ and Ba2+ (28Venkatachalam K. Ma H.T. Ford D.L. Gill D.L. J. Biol. Chem. 2001; 276: 33980-33985Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar, 41Parekh A.B. Penner R. Physiol. Rev. 1997; 77: 901-930Crossref PubMed Scopus (1295) Google Scholar). In contrast to wild-type cells, the transfected cells showed clear Ba2+ entry, reflecting the function of the exogenously expressed TRPC5 channel (Fig. 1A). Evidence has indicated that certain members of the TRPC channel family interact with and require the presence of InsP3Rs (6Venkatachalam K. van Rossum D.B. Patterson R.L. Ma H.T. Gill D.L. Nat. Cell Biol. 2002; 4: E263-E272Crossref PubMed Scopus (340) Google Scholar, 12Kiselyov K.I. Xu X. Mohayeva G. Kuo T. Pessah I.N. Mignery G.A. Zhu X. Birnbaumer L. Muallem S. 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