Selective Association of TRPC Channel Subunits in Rat Brain Synaptosomes

TRPC genes encode a ubiquitous family of ion channel proteins responsible for Ca2+ influx following stimulation of G-protein-coupled membrane receptors linked to phospholipase C. These channels may be localized to large multimeric signaling complexes via association with PDZ-containing scaffolding proteins. Based on sequence homology, the TRPC channel family can be divided into two major subgroups: TRPC1, -C4, and -C5 and TRPC3, -C6, and -C7. Although TRPC channels are thought to be tetramers, the actual subunit composition remains unknown. To determine subunit arrangement, individual TRPC channel pairs were heterologously expressed in Sf9 insect cells and immunoprecipitated using affinity-purified rabbit polyclonal antibodies specific for each channel subtype. Reciprocal co-immunoprecipitations showed that TRPC1, -C4, and -C5 co-associate and that TRPC3, -C6, and -C7 co-associate but that cross-association between the two major subgroups does not occur. Additionally, the interaction between each TRPC channel and the PDZ-containing protein, INAD (protein responsible for theinactivation-no-after-potentialDrosophila mutant), was examined. TRPC1, -C4, and -C5 co-immunoprecipitated with INAD, whereas TRPC3, -C6, and -C7 did not. To define channel subunit interactions in vivo, immunoprecipitations were performed from isolated rat brain synaptosomal preparations. The results revealed that TRPC1, -C4, and -C5 co-associate and that TRPC3, -C6, and -C7 co-associate in both cortex and cerebellum but that cross-association between the two major subgroups does not occur. These results demonstrate that TRPC channels are present in nerve terminals and provide the first direct evidence for selective assembly of channel subunits in vivo. TRPC genes encode a ubiquitous family of ion channel proteins responsible for Ca2+ influx following stimulation of G-protein-coupled membrane receptors linked to phospholipase C. These channels may be localized to large multimeric signaling complexes via association with PDZ-containing scaffolding proteins. Based on sequence homology, the TRPC channel family can be divided into two major subgroups: TRPC1, -C4, and -C5 and TRPC3, -C6, and -C7. Although TRPC channels are thought to be tetramers, the actual subunit composition remains unknown. To determine subunit arrangement, individual TRPC channel pairs were heterologously expressed in Sf9 insect cells and immunoprecipitated using affinity-purified rabbit polyclonal antibodies specific for each channel subtype. Reciprocal co-immunoprecipitations showed that TRPC1, -C4, and -C5 co-associate and that TRPC3, -C6, and -C7 co-associate but that cross-association between the two major subgroups does not occur. Additionally, the interaction between each TRPC channel and the PDZ-containing protein, INAD (protein responsible for theinactivation-no-after-potentialDrosophila mutant), was examined. TRPC1, -C4, and -C5 co-immunoprecipitated with INAD, whereas TRPC3, -C6, and -C7 did not. To define channel subunit interactions in vivo, immunoprecipitations were performed from isolated rat brain synaptosomal preparations. The results revealed that TRPC1, -C4, and -C5 co-associate and that TRPC3, -C6, and -C7 co-associate in both cortex and cerebellum but that cross-association between the two major subgroups does not occur. These results demonstrate that TRPC channels are present in nerve terminals and provide the first direct evidence for selective assembly of channel subunits in vivo. TRP 1The abbreviations used are: TRP, transient receptor potential; NHERF, Na+-H+exchange regulatory factor; PLC, phospholipase C; IP, immunoprecipitation. genes, originally identified as critical components of phototransduction inDrosophila, encode a ubiquitous and heterogeneous family of ion channel proteins that appear to play a fundamental role in cell signaling, cell growth, and cell death (recently reviewed in Refs.1Montell C. Science's STKE. 2001; : RE1Google Scholar, 2Clapham D.E. Runnels L.W. Strübing C. Nat. Rev. Neurosci. 2001; 2: 387-396Google Scholar, 3Harteneck C. Plant T.D. Schultz G. Trends Neurosci. 2001; 23: 159-166Google Scholar). In the last 7 years, 21 mammalian homologs have been discovered. There are currently seven mammalian TRP siblings designated TRPC1–TRPC7 that exhibit 32–47% overall identity at the amino acid level compared with the Drosophila isoforms. Based on amino acid sequence homologies, the TRPC family can be into divided into two major subgroups: TRPC1, -C4, and -C5 and TRPC3, -C6, and -C7. TRPC2, which is a pseudogene in humans (4Wes P.D. Chevesich J. Jeromin A. Rosenberg C. Stetten G. Montell C. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 9652-9656Google Scholar), is related but is clearly distinct from the two major subgroups. The primary TRPC channels, like theDrosophila versions, appear to be regulated by phospholipase C (PLC)-dependent mechanisms and thus are thought to be intimately involved in receptor-mediated Ca2+ signaling. In many cell types, a specific receptor-activated channel appears to be regulated by the level of Ca2+ within the inositol 1,4,5-trisphosphate-sensitive internal Ca2+ store (5Putney Jr., J.W. McKay R.R. Bioessays. 1999; 21: 38-46Google Scholar, 6Putney Jr., J.W. Broad L.M. Braun F.J. Lievremont J.-P. Bird G.S. J. Cell Sci. 2001; 114: 2223-2229Google Scholar). These so-called store-operated channels are responsible for the Ca2+ release-activated current,ICRAC (7Parekh A.B. Penner R. Physiol. Rev. 1997; 77: 901-930Google Scholar). There is evidence from heterologous expression studies (8Zitt C. Zobel A. Obukhov A.G. Harteneck C. Kalkbrenner F. Lückhoff A. Schultz G. Neuron. 1996; 16: 1189-1196Google Scholar, 9Warnat J. Philipp S. Zimmer S. Flockerzi V. Cavalié A. J. Physiol. (Lond.). 1999; 518: 631-638Google Scholar, 10Philipp S. Cavalié A. Freichel M. Wissenbach U. Zimmer S. Trost C. Marquart A. Murakami M. Flockerzi V. EMBO J. 1996; 15: 6166-6171Google Scholar, 11Philipp S. Hambrecht J. Braslavski L. Schroth G. Freichel M. Murakami M. Cavalie A. Flockerzi V. EMBO J. 1998; 17: 4274-4282Google Scholar, 12Vazquez G. Lievremont J.-P. Bird G.S.J. Putney Jr., J.W. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 11777-11782Google Scholar), antisense experiments (13Wu X. Babnigg G. Villereal M.L. Am. J. Physiol. 2000; 278: C526-C536Google Scholar, 14Liu X. Wang W. Singh B.B. Lockwich T. Jadlowiec J. O'Connell B. Wellner R. Zhu M.X. Ambudkar I.S. J. Biol. Chem. 2000; 275: 3403-3411Google Scholar, 15Brough G.H. Wu S. Cioffi D. Moore T.M. Li M. Dean N. Stevens T. FASEB J. 2001; 15: 1727-1738Google Scholar, 16Philipp S. Trost C. Warnat J. Rautmann J. Himmerkus N. Schroth G. Kretz O. Nastainczy W. Cavalié A. Hoth M. Flockerzi V. J. Biol. Chem. 2000; 275: 23965-23972Google Scholar), genetic disruption (17Freichel M. Suh S.H. Pfeifer A. Schweig U. Trost C. Weibgerber P. Biel M. Philipp S. Freise D. Droogmans G. Hofmann F. Flockerzi V. Nilius B. Nat. Cell Biol. 2001; 3: 121-127Google Scholar, 18Mori Y. Wakamori M. Miyakawa T. Hermosura M. Hara Y. Nishida M. Hirose K. Mizushima A. Kurosaki M. Mori E. Gotoh K. Okada T. Fleig A. Penner R. Iino M. Kurosaki T. J. Exp. Med. 2002; 195: 1-10Google Scholar), and adenovirus-mediated in vivooverexpression (19Singh B.B. Zheng C. Liu X. Lockwich T. Liao D. Zhu M.X. Birnbaumer L. Ambudkar I.S. FASEB J. 2001; 15: 1652-1654Google Scholar) that mammalian TRPC channel subunits are components of store-operated channels, although results in the literature are often conflicting. There is, however, general agreement that TRPC3 and TRPC6, and perhaps TRPC7, comprise channels that can be regulated by diacylglycerol (20Hofmann T. Obukhov A.G. Schaefer M. Harteneck C. Gudermann T. Schultz G. Nature. 1999; 397: 259-263Google Scholar, 21Okada T. Inoue R. Yamazaki K. Maeda A. Kurosaki T. Yamakuni T. Tanaka I. Shimizu S. Ikenaka K. Imoto K. Mori Y. J. Biol. Chem. 1999; 274: 27359-27370Google Scholar). Based on the known structure of K+, Na+, and Ca2+ channels, it is assumed that TRPC channels are composed of four subunits, and there is growing evidence to suggest heteromultimeric channel assembly (22Xu X.Z.S. Li H.S. Guggino W.B. Montell C. Cell. 1997; 89: 1155-1164Google Scholar, 23Lintschinger B. Balzer-Geldsetzer M. Baskaran T. Graier W.F. Romanin C. Zhu M.X. Groschner K. J. Biol. Chem. 2002; 275: 27799-27805Google Scholar, 24Hofmann T. Schaefer M. Schultz G. Gudermann T. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 7461-7466Google Scholar, 25Strubing C. Krapivinsky G. Krapivinsky L. Clapham D.E. Neuron. 2001; 29: 645-655Google Scholar). However, the channel composition in vivo and the rules governing subunit assembly remain to be determined. Likewise, how these tetrameric channels are localized in the plasmalemma is unknown. Selective localization to specific domains of the membrane appears to play an important role in signal transduction (26Delmas P. Wanaverbecq N. Abogadie F.C. Mistry M. Brown D.A. Neuron. 2002; 34: 209-220Google Scholar). In this regard, studies inDrosophila photoreceptor cells have shown that TRP channels are held in a signaling complex (i.e. a signalplex) by a scaffolding protein called INAD (1Montell C. Science's STKE. 2001; : RE1Google Scholar). INAD contains five tandem PDZ domains that serve as protein binding modules mediating the clustering of proteins involved in the Drosophila phototransduction cascade (e.g. TRP, TRPL, PLC, protein kinase C, and calmodulin). Likewise, PDZ-containing proteins may provide the scaffolding necessary for TRPC channel signalplex formation and localization in mammalian cells. Immunoprecipitation experiments in heterologous expression systems and rat brain have shown that the first PDZ domain of the Na+-H+ exchanger regulatory factor (NHERF) binds TRPC4, TRPC5, PLCβ1, and PLCβ2 (27Tang Y. Tang J. Chen Z. Trost C. Flockerzi V. Li M. Ramesh V. Zhu M.X. J. Biol. Chem. 2000; 275: 37559-37564Google Scholar). Interestingly, neither NHERF nor INAD appear to bind TRPC3 (27Tang Y. Tang J. Chen Z. Trost C. Flockerzi V. Li M. Ramesh V. Zhu M.X. J. Biol. Chem. 2000; 275: 37559-37564Google Scholar, 28Xu X.S. Choudhury A. Li X. Montell C. J. Cell Biol. 1998; 142: 545-555Google Scholar), suggesting that only certain TRPC channel subunits may associate with PDZ-containing proteins and consequently contribute to the signalplex in mammalian cells. In order to determine the subunit arrangement of native TRPC channels, we isolated and affinity-purified rabbit polyclonal antibodies directed against amino acid sequences specific for each channel subtype. Reciprocal co-immunoprecipitations of individual TRPC channel pairs heterologously expressed in Sf9 insect cells using recombinant baculovirus revealed that TRPC1, -C4, and -C5 co-associate and that TRPC3, -C6, and -C7 co-associate but that cross-association between the two major subgroups does not occur. Additionally, we examined the interaction between each TRPC channel and the PDZ-containing protein, INAD. TRPC1, -C4, and -C5 co-immunoprecipitated with INAD, whereas TRPC3, -C6, and -C7 did not. In order to define channel subunit interactions in vivo, synaptosomal preparations were isolated from rat cerebral cortex and cerebellum. Again the immunoprecipitation experiments revealed that TRPC1, -C4, and -C5 co-associate and that TRPC3, -C6, and -C7 co-associate in both cortex and cerebellum but that cross-association between the two major subgroups does not occur. These results not only demonstrate that TRPC channels are present in nerve terminals, but they provide the first direct evidence for selective assembly of channel subunits in vivo. Selective subunit assembly and selective interaction with PDZ-containing proteins may underlie differences in the activation mechanism for the various TRPC channels and help to explain conflicting results with regard to their putative role as store-operated channels. Spodoptera frugiperda (Sf9) cells were obtained from ATCC and cultured as previously described (29Hu Y. Rajan L. Schilling W.P. Am. J. Physiol. 1994; 266: C1736-C1743Google Scholar,30O'Reilly D.R. Miller L.K. Luckow V.A. Baculovirus Expression Vectors: A Laboratory Manual. W. H. Freeman and Co., New York1992Google Scholar) using Grace's insect medium supplemented with 2% lactalbumin hydrolysate, 2% yeastolate solution, 2 mml-glutamine, 10% heat-inactivated fetal bovine serum, and 1% penicillin/streptomycin/neomycin solution (Invitrogen). Peptide sequences specific to each human TRPC protein (Table I) were selected based on specificity, potential immunogenicity, and similarity across species lines. Additionally, peptide sequences were selected that lacked potential phosphorylation sites, which might render antibodies ineffective for in vivo immunoprecipitation and immunohistochemistry. Because each sequence chosen had one the were with a at the for to proteins. acid was between the and the peptide sequence to provide of the peptide on the of which was used as the protein for rabbit Peptide the of and acid and peptide to and to were performed by and were performed by and Laboratory sequences used to human polyclonal in a The from of rabbit was isolated using a of rabbit to with of was a with The was with the of the was was with and as in of The of each was and the the of protein were was by 2 of to a The were and the was on a for at The was with the of the was was with as described and the protein were The were by using a The was with and the was to a of an of was and the was at of affinity-purified rabbit antibodies in from to the primary TRPC antibodies were used at a to a of the antibodies were used at The for the TRPC channel proteins were from the following human and TRPC3, Montell and TRPC5, Mori for and Zhu and TRPC6, and Birnbaumer The for bovine was using from cultured bovine cells. The INAD and antibodies were from The the various mammalian TRPC channel proteins and INAD were into baculovirus using were using the as described in the by the were and to a The was at Sf9 cells in Grace's medium were into for an of was of was and the cells were at in a cells were used at were isolated from rat cerebral cortex and cerebellum as described by Scholar). were as described following of the synaptosomal were at a protein of with recombinant Sf9 cells were to at for and at a of in and The cell was on using a on a of The cell was for with a between The cell was to at for at The was and the were at The were in at a protein of and at Sf9 cells with baculovirus were in of at for The protein from Sf9 cells synaptosomal were at for at to and membrane of were by with protein for at were with the antibodies for were by with protein at for were four with in and for Cell and were by of protein of and to membrane for in were with the primary and following with by The results from at the experiments in Sf9 this at were in against peptide sequences specific to each human TRPC channel protein (Table The sequences chosen were had only one two amino acid differences compared with the and bovine of the sequences are in the and are to be In each was for to the TRPC channel protein expressed in Sf9 insect cells using recombinant The proteins examined and their were as human and TRPC3 bovine and and Sf9 cells were and membrane were isolated as described and proteins were by and to using each of the affinity-purified the of and the results showed that each the TRPC channel protein, did not membrane proteins expressed in Sf9 did not baculovirus and with of the TRPC channel proteins in the channel protein, but an at a was often in Likewise, the channel protein, but an was at the These were in Sf9 cells each of the TRPC proteins and in Sf9 cells with an baculovirus the human receptor these an Sf9 cell membrane protein a baculovirus examined the of each to the protein from Sf9 cell 2 in was to the TRPC channel were immunoprecipitated using the the TRPC1, whereas the may an Sf9 cell The Sf9 is for the of proteins of protein used at of individual subunits are into a in Sf9 cells with individual recombinant 1997; Scholar, D. P. H. I. I. 2001; Scholar, J. Biol. Chem. 1994; Scholar, P. J. Biol. Chem. 2002; Scholar). of this to determine the TRPC channel proteins TRPC channel was in channel pairs with the of the channel Reciprocal experiments were performed for each The results that TRPC1, TRPC4, and from Sf9 cell expressed in the Likewise, TRPC3, TRPC6, and In TRPC1, TRPC4, and did not with TRPC3, TRPC6, the immunoprecipitations for each channel are not shown in but were These results suggest that there is association between the of the two major TRPC channel subgroups but that there is cross-association between TRPC6, and from Sf9 cell Sf9 cells were with recombinant baculovirus for expression of individual TRPC channel TRPC3 and and TRPC3 and a using the proteins from cell proteins immunoprecipitated with the channel TRPC4, and not from Sf9 cell with TRPC3, TRPC6, Sf9 cells were with recombinant baculovirus for expression of individual TRPC channel with TRPC3 with TRPC3 with TRPC3 a using the proteins from cell proteins immunoprecipitated with the channel studies have shown that and TRPC5, but not TRPC3, with NHERF (27Tang Y. Tang J. Chen Z. Trost C. Flockerzi V. Li M. Ramesh V. Zhu M.X. J. Biol. Chem. 2000; 275: 37559-37564Google Scholar). TRP and with INAD but not with TRPC3 X.S. Choudhury A. Li X. Montell C. J. Cell Biol. 1998; 142: 545-555Google Scholar). these results suggest that there may be interaction of with PDZ To to this we INAD in Sf9 cells with the individual TRPC channel proteins. in INAD co-immunoprecipitated with TRPC1, TRPC4, and but not with TRPC3, TRPC6, To determine the of interaction is of native TRPC channels as they in their signaling we performed immunoprecipitations from isolated nerve obtained from rat cerebral cortex and cerebellum. were isolated from the of a as previously described Scholar). In TRPC channel proteins were only in from obtained at the were not present at the is with of TRPC channel proteins with the of each to the channel protein from synaptosomal the of TRPC4, each of the antibodies immunoprecipitated a protein of from both cortex and although was to in cortex were to in cortex a that was in immunoprecipitations from Sf9 cells in from rat brain C. Krapivinsky G. Krapivinsky L. Clapham D.E. Neuron. 2001; 29: 645-655Google Scholar, Y. Tang J. Chen Z. Trost C. Flockerzi V. Li M. Ramesh V. Zhu M.X. J. Biol. Chem. 2000; 275: 37559-37564Google Scholar). In were These may of the TRPC proteins in performed experiments from cortex and synaptosomal in co-immunoprecipitated with to co-immunoprecipitated with and but neither nor immunoprecipitated with These results suggest that the on native is not to the the results suggest that TRPC1, TRPC4, and co-associate in rat cerebral The results were obtained in synaptosomal preparations isolated from cerebellum in TRPC3, TRPC6, and co-immunoprecipitated from rat synaptosomal but did not with TRPC1, TRPC4, and the immunoprecipitations for each channel are not shown in but were to shown in 7 and were obtained in synaptosomal preparations isolated from cortex These results suggest that there is association between the of the two major TRPC channel subgroups in vivo but that there is cross-association between TRPC4, and from rat cortex from synaptosomal preparations isolated from rat cortex were immunoprecipitated and to a using the proteins from synaptosomal proteins immunoprecipitated with the channel TRPC6, and from rat from synaptosomal preparations isolated from rat cerebellum were immunoprecipitated and to a using the proteins from synaptosomal proteins immunoprecipitated with the channel TRPC4, and not from rat cerebellum with TRPC3, TRPC6, from synaptosomal preparations isolated from rat cerebellum were immunoprecipitated and to a using the proteins from synaptosomal proteins immunoprecipitated with the channel In order to determine the subunit composition and to a of the mammalian TRPC channel specific antibodies are In the present we rabbit polyclonal antibodies against amino acid sequences specific for each TRPC channel protein expressed in Sf9 insect cells using recombinant this we that the affinity-purified TRPC antibodies were specific and selective and the TRPC channel proteins with each TRPC was of the TRPC protein from Sf9 cell suggesting that the specific by the antibodies was in each the of TRPC4, each TRPC was to the TRPC channel protein from rat brain synaptosomal suggesting that the is in the native channel However, we were to from using the did not the of protein in the nerve terminals, was immunoprecipitated with and The with of the is that the for is not in the native channel amino of may be by TRPC subunits present in the heteromultimeric channel may be by protein present in the Based on amino acid sequence the TRPC channel family can be into two major subgroups: TRPC1, TRPC4, and and TRPC3, TRPC6, and of evidence suggest that of each to channel but that cross-association between subgroups does not occur. only of each following in expression of individual subunit of the immunoprecipitations were performed in a association of the individual subunits is not on the used for Likewise, co-immunoprecipitations of channel between the two major subgroups were the interactions within subgroups were specific and to as only of each in association was between subgroups. these experiments were performed in a and in synaptosomal preparations isolated from both cortex and cerebellum. the results suggest that within each the TRPC proteins heteromultimeric channels both in and in vivo, but the actual subunit of native channels remains unknown. studies that and TRPC3 subunits (22Xu X.Z.S. Li H.S. Guggino W.B. Montell C. Cell. 1997; 89: 1155-1164Google Scholar) the of and TRPC3 heterologously in and B. Balzer-Geldsetzer M. Baskaran T. Graier W.F. Romanin C. Zhu M.X. Groschner K. J. Biol. Chem. 2002; 275: 27799-27805Google Scholar) that membrane with were following of and TRPC3 in cells. However, neither interactions of native TRPC C. Krapivinsky G. Krapivinsky L. Clapham D.E. Neuron. 2001; 29: 645-655Google Scholar) that channels were following heterologous expression of and in cells and that co-immunoprecipitated with from brain and with from rat a of and to heterologously expressed TRPC channel Hofmann T. Schaefer M. Schultz G. Gudermann T. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 7461-7466Google Scholar) that TRPC1, TRPC4, and and that TRPC3, TRPC6, and but that there was interaction between the two major subgroups. The in results of the present using a expression with channel proteins and the in vivo results obtained in rat brain clearly this subunit of of the TRPC proteins the may these are expressed in rat they not be in and may exhibit a interaction Likewise, with to be is that Drosophila TRP and are held in a large signalplex by a PDZ-containing scaffolding protein called INAD (1Montell C. Science's STKE. 2001; : RE1Google Scholar). signalplex appears to be for signal and both TRP and INAD are the interaction is genetic B. Neuron. 1995; Scholar, J. Montell C. Neuron. 1997; Scholar). signalplex may be necessary for signal transduction and localization in mammalian but there is direct evidence for TRPC signalplex A of mammalian PDZ-containing proteins with to INAD have been These human S. Flockerzi V. 1997; Scholar), PDZ-containing protein C. K. A. H. 1998; Scholar), and the protein H. Nature. 1997; Scholar). interaction with TRPC channels, however, not been and have been shown to with NHERF from brain and from of cells heterologously the proteins (27Tang Y. Tang J. Chen Z. Trost C. Flockerzi V. Li M. Ramesh V. Zhu M.X. J. Biol. Chem. 2000; 275: 37559-37564Google Scholar), but the of this interaction remain unknown. However, neither NHERF nor INAD bind to TRPC3, suggesting that the interaction of PDZ domains with TRPC channel proteins may be The results of the present that TRPC1, TRPC4, and with INAD, whereas TRPC3, TRPC6, and not. that TRPC1, TRPC4, and may heteromultimeric channels that are to specific via interaction with PDZ-containing whereas TRPC3, TRPC6, and may In this regard, P. Wanaverbecq N. Abogadie F.C. Mistry M. Brown D.A. Neuron. 2002; 34: 209-220Google Scholar) showed that in store-operated channels localized to a specific that a receptor and an inositol these showed that channels are not localized to specific domains but are on the cell In activation of expressed in cells is of Ca2+ store C. Krapivinsky G. Krapivinsky L. Clapham D.E. Neuron. 2001; 29: 645-655Google Scholar). the mechanism of TRPC channel activation may on subunit localization to specific and the of proteins present in the There is in the literature TRPC protein in the of rat brain showed that is present in the cell of the and of the cortex and C. Krapivinsky G. Krapivinsky L. Clapham D.E. Neuron. 2001; 29: 645-655Google Scholar). In primary cultured by was in the cell and but was not in identified by the of In the present of the appear to be present in isolated synaptosomal preparations from rat cerebral cortex and suggesting that native TRPC channels are present in nerve that the TRPC channel proteins are expressed in various of rat C. P. P. and D. L. in be to determine the association selective localization to specific nerve nerve terminals both and of these channels. for help with The was by with