The Phospholipid-binding Protein SESTD1 Is a Novel Regulator of the Transient Receptor Potential Channels TRPC4 and TRPC5

Abstract

TRPC4 and TRPC5 are two closely related members of the mammalian transient receptor potential cation channel family that have been implicated in important physiological functions, such as growth cone guidance and smooth muscle contraction. To further unravel the role of TRPC4 and TRPC5 in these processes in vivo, detailed information about the molecular composition of native channel complexes and their association with cellular signaling networks is needed. We therefore searched a human aortic cDNA library for novel TRPC4-interacting proteins using a modified yeast two-hybrid assay. This screen identified SESTD1, a previously uncharacterized protein containing a lipid-binding SEC14-like domain as well as spectrin-type cytoskeleton interaction domains. SESTD1 was found to associate with TRPC4 and TRPC5 via the channel's calmodulin- and inositol 1,4,5-trisphosphate receptor-binding domain. In functional studies, we demonstrate that SESTD1 binds several phospholipid species in vitro and is essential for efficient receptor-mediated activation of TRPC5. Notably, phospholipid binding to SESTD1 was Ca2+-dependent. Because TRPC4 and -5 conduct Ca2+, SESTD1-channel signaling may be bidirectional and also couple TRPC activity to lipid signaling through SESTD1. The modulation of TRPC channel function by specific lipid-binding proteins, such as SESTD1, adds another facet to the complex regulation of these channels complementary to the previously described effects of direct channel-phospholipid interaction. TRPC4 and TRPC5 are two closely related members of the mammalian transient receptor potential cation channel family that have been implicated in important physiological functions, such as growth cone guidance and smooth muscle contraction. To further unravel the role of TRPC4 and TRPC5 in these processes in vivo, detailed information about the molecular composition of native channel complexes and their association with cellular signaling networks is needed. We therefore searched a human aortic cDNA library for novel TRPC4-interacting proteins using a modified yeast two-hybrid assay. This screen identified SESTD1, a previously uncharacterized protein containing a lipid-binding SEC14-like domain as well as spectrin-type cytoskeleton interaction domains. SESTD1 was found to associate with TRPC4 and TRPC5 via the channel's calmodulin- and inositol 1,4,5-trisphosphate receptor-binding domain. In functional studies, we demonstrate that SESTD1 binds several phospholipid species in vitro and is essential for efficient receptor-mediated activation of TRPC5. Notably, phospholipid binding to SESTD1 was Ca2+-dependent. Because TRPC4 and -5 conduct Ca2+, SESTD1-channel signaling may be bidirectional and also couple TRPC activity to lipid signaling through SESTD1. The modulation of TRPC channel function by specific lipid-binding proteins, such as SESTD1, adds another facet to the complex regulation of these channels complementary to the previously described effects of direct channel-phospholipid interaction. IntroductionSeven homologous channel proteins belong to the TRPC 3The abbreviations used are: TRPCtransient receptor potential canonicalmTRPCmurine TRPCGSTglutathione S-transferaseCIRB domaincalmodulin- and inositol 1,4,5-trisphosphate receptor-binding domainsiRNAsmall interfering RNAGFPgreen fluorescent proteinBisTris2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diolGAPDHglyceraldehyde-3-phosphate dehydrogenaseYFPyellow fluorescent proteinHAhemagglutinin. family of nonselective cation channels. Within the TRPC family, TRPC4 and TRPC5 represent a structurally distinct subgroup that is characterized by its ability to form homo- and heteromultimeric channels with each other as well as with TRPC1 (1Hofmann T. Schaefer M. Schultz G. Gudermann T. Proc. Natl. Acad. Sci. U.S.A. 2002; 99: 7461-7466Crossref PubMed Scopus (625) Google Scholar).Moreover, TRPC4 and TRPC5 are functionally set apart from other TRPCs by their unique activation mechanism that is dependent on phospholipase C activity but does not involve the phospholipid hydrolysis product diacylglycerol (2Schaefer M. Plant T.D. Obukhov A.G. Hofmann T. Gudermann T. Schultz G. J. Biol. Chem. 2000; 275: 17517-17526Abstract Full Text Full Text PDF PubMed Scopus (357) Google Scholar). Further signaling pathways, including Ca2+ store depletion and diverse lipid-channel interactions, were also shown to control channel function (3Trebak M. Lemonnier L. DeHaven W.I. Wedel B.J. Bird G.S. Putney Jr., J.W. Pflugers Arch. 2009; 457: 757-769Crossref PubMed Scopus (89) Google Scholar, 4Flemming P.K. Dedman A.M. Xu S.Z. Li J. Zeng F. Naylor J. Benham C.D. Bateson A.N. Muraki K. Beech D.J. J. Biol. Chem. 2006; 281: 4977-4982Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar, 5Philipp S. Cavalié A. Freichel M. Wissenbach U. Zimmer S. Trost C. Marquart A. Murakami M. Flockerzi V. EMBO J. 1996; 15: 6166-6171Crossref PubMed Scopus (257) Google Scholar, 6Philipp S. Hambrecht J. Braslavski L. Schroth G. Freichel M. Murakami M. Cavalié A. Flockerzi V. EMBO J. 1998; 17: 4274-4282Crossref PubMed Scopus (271) Google Scholar, 7Zeng F. Xu S.Z. Jackson P.K. McHugh D. Kumar B. Fountain S.J. Beech D.J. J. Physiol. 2004; 559: 739-750Crossref PubMed Scopus (114) Google Scholar). Nevertheless, the exact principles that govern TRPC4 and -5 gating have not been finally clarified and probably depend on the cellular context.Studies in knock-out mice showed that TRPC4 plays an important role in vascular physiology. TRPC4−/− mice have markedly reduced store- and receptor-induced endothelial Ca2+ entry and impaired endothelium-dependent vasorelaxation (8Freichel M. Suh S.H. Pfeifer A. Schweig U. Trost C. Weissgerber P. Biel M. Philipp S. Freise D. Droogmans G. Hofmann F. Flockerzi V. Nilius B. Nat. Cell Biol. 2001; 3: 121-127Crossref PubMed Scopus (506) Google Scholar). Moreover, TRPC4 knock-out strongly reduced acetylcholine-activated non-selective cation currents in visceral smooth muscle cells that are involved in the regulation of gastric motility (9Lee K.P. Jun J.Y. Chang I.Y. Suh S.H. So I. Kim K.W. Mol. Cells. 2005; 20: 435-441PubMed Google Scholar). Similarly, functional expression of TRPC5 has been found in several vascular tissues, including human saphenous vein (10Xu S.Z. Muraki K. Zeng F. Li J. Sukumar P. Shah S. Dedman A.M. Flemming P.K. McHugh D. Naylor J. Cheong A. Bateson A.N. Munsch C.M. Porter K.E. Beech D.J. Circ. Res. 2006; 98: 1381-1389Crossref PubMed Scopus (137) Google Scholar) and rabbit arteriolar smooth muscle cells (11Xu S.Z. Boulay G. Flemming R. Beech D.J. Am. J. Physiol. Heart Circ. Physiol. 2006; 291: H2653-H2659Crossref PubMed Scopus (81) Google Scholar). In addition, both TRPC4 and TRPC5 are strongly expressed in neuronal tissue and have been assigned functions in neurotransmitter release (12Munsch T. Freichel M. Flockerzi V. Pape H.C. Proc. Natl. Acad. Sci. U.S.A. 2003; 100: 16065-16070Crossref PubMed Scopus (98) Google Scholar) and regulation of neurite growth, respectively (13Greka A. Navarro B. Oancea E. A. Nat. 2003; PubMed Scopus Google Scholar, McHugh D. M. Zeng F. Xu S.Z. R. Beech D.J. J. Physiol. 2006; PubMed Scopus Google Scholar). TRPC5 has also been found to control in mice A. Li J. Kim U. S. E. S.J. A. Jr., 2009; Full Text Full Text PDF PubMed Scopus Google their physiological the molecular composition of TRPC4 and -5 channel complexes is in expression that TRPC4 and TRPC5 form functionally diverse channel complexes dependent on the with other TRPC in TRPC1 C. G. L. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). In to several other proteins with TRPC4 and -5 and and signaling of the channel the Ca2+ was to with both and of receptor-mediated activation Zeng S. Nat. Cell Biol. PubMed Scopus Google Scholar). a direct interaction with the inositol 1,4,5-trisphosphate receptor has been to signaling with channel activity J. S. L. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google a association with the several of a with the of TRPC4 to was shown to and of the channel J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). interaction with proteins, such as protein and was for activation of TRPC4 in endothelial cells and in cells L. B. M. J. Cell Sci. 2002; PubMed Google Scholar, S. M. T. Circ. Res. 2005; PubMed Scopus Google Scholar). TRPC5 was found in a screen to with several M. A. M. Pflugers Arch. 2005; PubMed Scopus Google Scholar). of TRPC5 in neuronal cells was via binding to the protein (13Greka A. Navarro B. Oancea E. A. Nat. 2003; PubMed Scopus Google that TRPC4 and -5 as well as other are in signaling complexes C. Sci. 2005; Google to be the proteins to channel Moreover, the proteins that and TRPCs in diverse are to on the of of TRPC4 and -5 channels in vascular SESTD1, a previously uncharacterized lipid-binding protein strongly expressed in was found to with both TRPC proteins and TRPC5 currents in a of SESTD1 is involved in phospholipid regulation of TRPC channels in and to function further to novel TRPC4-interacting proteins, we a human aortic cDNA library using the The TRPC4 yeast two-hybrid was by a to the C of in Because TRPC channels are to of a B. Proc. Natl. Acad. Sci. U.S.A. 2001; 98: PubMed Scopus Google Scholar, T. Kim T. PubMed Scopus Google Scholar) was the C and the binding domain. The of the and was The TRPC5 was the by the of of control the C of the channels were by To the SESTD1 interaction on the were of on were for from were and the human SESTD1 cDNA The of the yeast two-hybrid interaction was by of the SESTD1 with the control as for expression of proteins in were by SESTD1 cDNA for the domain in proteins were expressed in proteins were from using and used for of of cells with M. Plant T.D. Schultz G. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google T. S. M. A. T. K. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar) were with of proteins and for were with and by for and SESTD1 were using and cells were with C. G. L. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google and SESTD1 as a cells were with and with were with were with protein protein with proteins were with and by and protein of was with the protein in were with and on was in were to using and a the to the To the proteins were with The was with and with and were using the of human from and of were and of each was cDNA using and in of for of human SESTD1 and the were using and the mammalian expressed were The was with The SESTD1 was with was with of cDNA in a in was in with a with as a of and used were and was on the using were as activation for by of of the of was to the expression of as expression is the the of SESTD1 and SESTD1 is to be expressed the is and is and Cell were in a cells were in with and cells the human receptor A. J.W. J. D.J. PubMed Scopus Google Scholar) were in modified with with and was for and of human aortic smooth muscle smooth muscle aortic endothelial endothelial cells and the were to the were in growth using to the cells were with control with a specific SESTD1 of an of human SESTD1 was by of and were as for functional with to a with TRPC5 we cells that the human receptor A. J.W. J. D.J. PubMed Scopus Google Scholar). was from C. G. L. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar) by with and The was by were functionally characterized by Ca2+ and and was for further currents were with the A. E. B. Pflugers Arch. PubMed Scopus Google Scholar). with of were from using a and with on were with and were using an were with an and were and were by from to were and with of was and by were not were of cells to an on were with and with in were and with was with an and using a cells were in and to an were in with and to cells were with were in a was and and The was using were and Ca2+ were from the the with were were with with Ca2+ with Ca2+ and Ca2+ were using on the was by human SESTD1 in The protein was expressed and as described was from with and were with protein in the were with and with by was and the were with a rabbit the of human SESTD1 was by used were and rabbit and rabbit and used were and rabbit and human SESTD1 and were from and were from was from was from and was from other were from are expressed as of was with were as further and proteins of TRPC4 channels in smooth we a human aortic cDNA library for interaction with the channel's C in Because TRPC channels are probably Schultz G. Schaefer M. Cell 2003; PubMed Scopus Google we a modified containing a domain B. Proc. Natl. Acad. Sci. U.S.A. 2001; 98: PubMed Scopus Google Scholar, T. Kim T. PubMed Scopus Google Scholar) to of the channel In several proteins, including the previously described were identified as TRPC4 binding that of the potential TRPC4-interacting proteins was SESTD1, a protein containing a SEC14-like lipid binding domain as well as two and interaction with the the of TRPC4 activity on phospholipid signaling and its to SESTD1 a for a yeast two-hybrid with of the TRPC4 showed that binding to SESTD1 was by a This is in TRPC5 and with the previously described calmodulin- and inositol 1,4,5-trisphosphate receptor-binding domain of We that TRPC5 also with SESTD1. To binding of SESTD1 to TRPC4 and were were also to the TRPC binding of SESTD1. proteins were that of the identified of SESTD1 and The shown in demonstrate that the domain of SESTD1 to and the as well as TRPC5. In interaction of the channels with be to the SEC14-like domain not be in the protein was to E. a yeast two-hybrid using the domain not binding to binding to was SESTD1 via its domain with the of TRPC4 and of the SESTD1 domain to TRPC4 and TRPC5. proteins of the of SESTD1 were used to from of cells with the were and with and of the was as expression was used to of the the of the interaction in we of SESTD1 with and TRPC5 cells were with SESTD1, and the protein was on using an a direct an protein in with the molecular of SESTD1 with channel SESTD1 be identified in TRPC4 and -5 its association with the TRPC channel with and of cells and cells with were on using and a SESTD1. an protein in that protein is SESTD1. also an protein in cells that we is native SESTD1 also In addition, the an of and the from cells with SESTD1 and SESTD1 are of and the from cells with SESTD1 and SESTD1 functional of the channel we SESTD1 and TRPC4 and -5 are in To we an expression of SESTD1. we found that SESTD1 is expressed in human and cells to The of SESTD1 was in with expression about of that of the SESTD1 expression in and is also expressed in two in TRPC4 and -5 previously been (8Freichel M. Suh S.H. Pfeifer A. Schweig U. Trost C. Weissgerber P. Biel M. Philipp S. Freise D. Droogmans G. Hofmann F. Flockerzi V. Nilius B. Nat. Cell Biol. 2001; 3: 121-127Crossref PubMed Scopus (506) Google Scholar, T. S. M. A. T. K. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar, R. Xu S.Z. Beech D.J. J. 2003; PubMed Scopus Google Scholar, A. C. Benham C.D. Res. Mol. Res. 2002; PubMed Scopus Google Scholar). The of SESTD1 in several human vascular and in was on the protein by we were to currents in cells from these tissues, the of native SESTD1 and TRPC protein expression of SESTD1 SESTD1 expression was in human and using were to the expression of the shown are of Cell are to the and cells used were cells aortic smooth muscle cells and human vein endothelial cells of SESTD1 in and human from of the smooth muscle human aortic endothelial cells endothelial cells smooth muscle cells and aortic smooth muscle cells were by and SESTD1 was on using SESTD1 we a protein of the of SESTD1 in cells that these cells SESTD1 we to a for further In to the and the receptor cells showed currents from currents through channels that to Ca2+ with the was to the of SESTD1 on TRPC5. in of specific SESTD1 reduced SESTD1 protein by about expression of an protein also by and expression of the were Moreover, on the expression of the of TRPC5 currents and Ca2+ entry in of cells by The and receptor nonselective cation currents with for TRPC5. of in cells were using a Ca2+ release from in cells was by of are of Ca2+ release from in to of was in are of of SESTD1 expression by specific SESTD1 of cells with control a of a specific SESTD1 were with and SESTD1 protein by with with cells with expression of an protein by as well as expression were of and Ca2+ that Ca2+ was reduced in cells with with with cells of the of Ca2+ release was showed a of the in SESTD1 Ca2+ as from the the were also in SESTD1 In the SESTD1 the the to with in the and in the control control of activity is reduced in cells with SESTD1 of in cells with SESTD1 control Ca2+ release from was in by of Ca2+ was in are of with a of Ca2+ in SESTD1 cells of Ca2+ release as well as the of the of Ca2+ were reduced by SESTD1 The the from SESTD1 cells to in the and in the control respectively control of was the Ca2+ release in was This that the receptor signaling of TRPC5 were not by the J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) have shown that TRPC4 with of the protein via a to the binding identified in is that SESTD1 a regulation mechanism and that its as of TRPC4 and -5 to the Nevertheless, SESTD1 another distinct the that has been implicated in phospholipid in yeast for protein in mammalian cells K. L. T. PubMed Scopus Google Scholar). that domain of SESTD1 is involved in of the channel proteins that channel function by the lipid was important to the of the SESTD1 domain. To we the ability of SESTD1 to using an assay. that SESTD1 and binding to including and was binding to was and was strongly reduced in containing a Ca2+ for is that Ca2+ through TRPC4 -5 phospholipid binding of SESTD1. the interaction in a a functional of the binding of SESTD1 to containing were with in containing proteins were with and using and we identified SESTD1 as a novel and SESTD1 to a of mammalian proteins that a lipid binding domain. of the domain is on the protein that is essential for phospholipid and function in yeast S. L. Li S. 2005; PubMed Scopus Google Scholar, P. R. D. 2006; PubMed Scopus Google Scholar). the function of in mammalian phospholipid is and functional information was for of SESTD1 of two was for binding to TRPC4 and TRPC5. The binding on was to the C interaction has with the interaction of TRPC4 with and an association for and of TRPC4 J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). and binding to TRPC4 was by of a of the SESTD1 binding is that both binding are in In the channel the binding identified in may the further described by J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) for Moreover, of as well as SESTD1 to impaired channel is that both proteins are of a has been that as well as the protein TRPC4 in a complex that is for efficient of the channel to the association with other signaling J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, S. M. T. Circ. Res. 2005; PubMed Scopus Google Scholar). SESTD1 may as another to protein and TRPC4 and TRPC5 to the cytoskeleton through its the of for of TRPC5 the S. A. Nat. Cell Biol. 2004; PubMed Scopus Google Scholar) and the that SESTD1 binds another is that SESTD1 the protein and of TRPC4 and of the channel proteins, SESTD1 may have direct effects on TRPC In has been that of from the to TRPC4 activation J. S. L. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). Because the SESTD1 binding is to the of by SESTD1 may be for activation of TRPC4 and we not the mechanism by SESTD1 TRPC5 currents in be to the and to of TRPC channel important of is that the of SESTD1 with is Ca2+-dependent. of TRPC4 and -5 Ca2+ binding of SESTD1 to The of on TRPC4 and -5 function is but a of the channels for be with both the of for TRPC5 activity (3Trebak M. Lemonnier L. DeHaven W.I. Wedel B.J. Bird G.S. Putney Jr., J.W. Pflugers Arch. 2009; 457: 757-769Crossref PubMed Scopus (89) Google Scholar) and the role of in TRPC5 S. A. Nat. Cell Biol. 2004; PubMed Scopus Google its SESTD1 is to have other functions apart from TRPC channel In we in by a of the protein in SESTD1 Moreover, an interaction of SESTD1 with has been found to with TRPC4 in endothelial cells and to TRPC4 activity in a A. M. M. D. C. K. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). have to be further is an that SESTD1 is to and its complex functional novel TRPC proteins in vascular and of vascular channel complexes an important the of these channels have not been finally The the function and for endothelial TRPC channels is by that Ca2+ entry in endothelial cells is by channels TRPC4 TRPC1 M. M. Circ. Res. PubMed Scopus Google Scholar). Nevertheless, is knock-out an of TRPC4 in endothelial Ca2+ signaling (8Freichel M. Suh S.H. Pfeifer A. Schweig U. Trost C. Weissgerber P. Biel M. Philipp S. Freise D. Droogmans G. Hofmann F. Flockerzi V. Nilius B. Nat. Cell Biol. 2001; 3: 121-127Crossref PubMed Scopus (506) Google Scholar). to be for the of specific TRPC channel that may have in the of on its association with the C of TRPC4 in we SESTD1 from an aortic cDNA we expression of SESTD1 in human endothelial and smooth muscle cells by Because we not TRPC4 TRPC5 proteins in the using be for functional on native proteins to that both SESTD1 and TRPC4 TRPC5. is to the physiological of the the represent a further the complex that govern the function of TRPC4 and -5 in IntroductionSeven homologous channel proteins belong to the TRPC 3The abbreviations used are: TRPCtransient receptor potential canonicalmTRPCmurine TRPCGSTglutathione S-transferaseCIRB domaincalmodulin- and inositol 1,4,5-trisphosphate receptor-binding domainsiRNAsmall interfering RNAGFPgreen fluorescent proteinBisTris2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diolGAPDHglyceraldehyde-3-phosphate dehydrogenaseYFPyellow fluorescent proteinHAhemagglutinin. family of nonselective cation channels. Within the TRPC family, TRPC4 and TRPC5 represent a structurally distinct subgroup that is characterized by its ability to form homo- and heteromultimeric channels with each other as well as with TRPC1 (1Hofmann T. Schaefer M. Schultz G. Gudermann T. Proc. Natl. Acad. Sci. U.S.A. 2002; 99: 7461-7466Crossref PubMed Scopus (625) Google Scholar).Moreover, TRPC4 and TRPC5 are functionally set apart from other TRPCs by their unique activation mechanism that is dependent on phospholipase C activity but does not involve the phospholipid hydrolysis product diacylglycerol (2Schaefer M. Plant T.D. Obukhov A.G. Hofmann T. Gudermann T. Schultz G. J. Biol. Chem. 2000; 275: 17517-17526Abstract Full Text Full Text PDF PubMed Scopus (357) Google Scholar). Further signaling pathways, including Ca2+ store depletion and diverse lipid-channel interactions, were also shown to control channel function (3Trebak M. Lemonnier L. DeHaven W.I. Wedel B.J. Bird G.S. Putney Jr., J.W. Pflugers Arch. 2009; 457: 757-769Crossref PubMed Scopus (89) Google Scholar, 4Flemming P.K. Dedman A.M. Xu S.Z. Li J. Zeng F. Naylor J. Benham C.D. Bateson A.N. Muraki K. Beech D.J. J. Biol. Chem. 2006; 281: 4977-4982Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar, 5Philipp S. Cavalié A. Freichel M. Wissenbach U. Zimmer S. Trost C. Marquart A. Murakami M. Flockerzi V. EMBO J. 1996; 15: 6166-6171Crossref PubMed Scopus (257) Google Scholar, 6Philipp S. Hambrecht J. Braslavski L. Schroth G. Freichel M. Murakami M. Cavalié A. Flockerzi V. EMBO J. 1998; 17: 4274-4282Crossref PubMed Scopus (271) Google Scholar, 7Zeng F. Xu S.Z. Jackson P.K. McHugh D. Kumar B. Fountain S.J. Beech D.J. J. Physiol. 2004; 559: 739-750Crossref PubMed Scopus (114) Google Scholar). Nevertheless, the exact principles that govern TRPC4 and -5 gating have not been finally clarified and probably depend on the cellular context.Studies in knock-out mice showed that TRPC4 plays an important role in vascular physiology. TRPC4−/− mice have markedly reduced store- and receptor-induced endothelial Ca2+ entry and impaired endothelium-dependent vasorelaxation (8Freichel M. Suh S.H. Pfeifer A. Schweig U. Trost C. Weissgerber P. Biel M. Philipp S. Freise D. Droogmans G. Hofmann F. Flockerzi V. Nilius B. Nat. Cell Biol. 2001; 3: 121-127Crossref PubMed Scopus (506) Google Scholar). Moreover, TRPC4 knock-out strongly reduced acetylcholine-activated non-selective cation currents in visceral smooth muscle cells that are involved in the regulation of gastric motility (9Lee K.P. Jun J.Y. Chang I.Y. Suh S.H. So I. Kim K.W. Mol. Cells. 2005; 20: 435-441PubMed Google Scholar). Similarly, functional expression of TRPC5 has been found in several vascular tissues, including human saphenous vein (10Xu S.Z. Muraki K. Zeng F. Li J. Sukumar P. Shah S. Dedman A.M. Flemming P.K. McHugh D. Naylor J. Cheong A. Bateson A.N. Munsch C.M. Porter K.E. Beech D.J. Circ. Res. 2006; 98: 1381-1389Crossref PubMed Scopus (137) Google Scholar) and rabbit arteriolar smooth muscle cells (11Xu S.Z. Boulay G. Flemming R. Beech D.J. Am. J. Physiol. Heart Circ. Physiol. 2006; 291: H2653-H2659Crossref PubMed Scopus (81) Google Scholar). In addition, both TRPC4 and TRPC5 are strongly expressed in neuronal tissue and have been assigned functions in neurotransmitter release (12Munsch T. Freichel M. Flockerzi V. Pape H.C. Proc. Natl. Acad. Sci. U.S.A. 2003; 100: 16065-16070Crossref PubMed Scopus (98) Google Scholar) and regulation of neurite growth, respectively (13Greka A. Navarro B. Oancea E. A. Nat. 2003; PubMed Scopus Google Scholar, McHugh D. M. Zeng F. Xu S.Z. R. Beech D.J. J. Physiol. 2006; PubMed Scopus Google Scholar). TRPC5 has also been found to control in mice A. Li J. Kim U. S. E. S.J. A. Jr., 2009; Full Text Full Text PDF PubMed Scopus Google their physiological the molecular composition of TRPC4 and -5 channel complexes is in expression that TRPC4 and TRPC5 form functionally diverse channel complexes dependent on the with other TRPC in TRPC1 C. G. L. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). In to several other proteins with TRPC4 and -5 and and signaling of the channel the Ca2+ was to with both and of receptor-mediated activation Zeng S. Nat. Cell Biol. PubMed Scopus Google Scholar). a direct interaction with the inositol 1,4,5-trisphosphate receptor has been to signaling with channel activity J. S. L. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google a association with the several of a with the of TRPC4 to was shown to and of the channel J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). interaction with proteins, such as protein and was for activation of TRPC4 in endothelial cells and in cells L. B. M. J. Cell Sci. 2002; PubMed Google Scholar, S. M. T. Circ. Res. 2005; PubMed Scopus Google Scholar). TRPC5 was found in a screen to with several M. A. M. Pflugers Arch. 2005; PubMed Scopus Google Scholar). of TRPC5 in neuronal cells was via binding to the protein (13Greka A. Navarro B. Oancea E. A. Nat. 2003; PubMed Scopus Google that TRPC4 and -5 as well as other are in signaling complexes C. Sci. 2005; Google to be the proteins to channel Moreover, the proteins that and TRPCs in diverse are to on the of of TRPC4 and -5 channels in vascular SESTD1, a previously uncharacterized lipid-binding protein strongly expressed in was found to with both TRPC proteins and TRPC5 currents in a of SESTD1 is involved in phospholipid regulation of TRPC channels in and to function further