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The Membrane Topology of Human Transient Receptor Potential 3 as Inferred from Glycosylation-scanning Mutagenesis and Epitope Immunocytochemistry

1998; Elsevier BV; Volume: 273; Issue: 15 Linguagem: Inglês

10.1074/jbc.273.15.8675

1083-351X

Brigitte Vannier, Xi Zhu, Darren L. Brown, Lutz Birnbaumer,

Cardiac electrophysiology and arrhythmias

Transient receptor potential (Trp) proteins form ion channels implicated in the calcium entry observed after stimulation of the phospholipase C pathway. Kyte-Doolittle analysis of the amino acid sequence of Trp proteins identifies seven hydrophobic regions (H1–H7) with potential of forming transmembrane segments. A limited sequence similarity to voltage-gated calcium channel α1 subunits lead to the prediction of six transmembrane (TM) segments flanked by intracellular N and C termini and a putative pore region between TM5 and TM6. However, experimental evidence supporting this model is missing. Using human Trp 3 to test Trp topology, we now confirm the intracellular nature of the termini by immunocytochemistry. We also demonstrate presence of a unique glycosylation site in position 418, which defines one extracellular loop between H2 and H3. After removal of this site and insertion of ten separate glycosylation sites, we defined two additional extracellular loops between H4 and H5, and H6 and H7. This demonstrated the existence of six transmembrane segments formed of H2–H7. Thus, the first hydrophobic region of Trp rather than being a transmembrane segment is intracellular and available for protein-protein interactions. A site placed in the center of the putative pore region was glycosylated, suggesting that this region may have been luminal and was reinserted into the membrane at a late stage of channel assembly. Transient receptor potential (Trp) proteins form ion channels implicated in the calcium entry observed after stimulation of the phospholipase C pathway. Kyte-Doolittle analysis of the amino acid sequence of Trp proteins identifies seven hydrophobic regions (H1–H7) with potential of forming transmembrane segments. A limited sequence similarity to voltage-gated calcium channel α1 subunits lead to the prediction of six transmembrane (TM) segments flanked by intracellular N and C termini and a putative pore region between TM5 and TM6. However, experimental evidence supporting this model is missing. Using human Trp 3 to test Trp topology, we now confirm the intracellular nature of the termini by immunocytochemistry. We also demonstrate presence of a unique glycosylation site in position 418, which defines one extracellular loop between H2 and H3. After removal of this site and insertion of ten separate glycosylation sites, we defined two additional extracellular loops between H4 and H5, and H6 and H7. This demonstrated the existence of six transmembrane segments formed of H2–H7. Thus, the first hydrophobic region of Trp rather than being a transmembrane segment is intracellular and available for protein-protein interactions. A site placed in the center of the putative pore region was glycosylated, suggesting that this region may have been luminal and was reinserted into the membrane at a late stage of channel assembly. Activation of a Gq protein-coupled receptor leads to the production of inositol 1,4,5-trisphosphate (IP3) 1The abbreviations used are: IP3, inositol 1,4,5- trisphosphate; H1–H7, hydrophobic regions of Trp proteins; HA, hemagglutinin antigen; RIPA, radioimmunoprecipitation assay buffer; PCR, polymerase chain reaction; PAGE, polyacrylamide gel electrophoresis; TM, transmembrane; Trp, transient receptor potential; hTrp3, human Trp 3; PNGase F, peptide N-glycosidase F; Endo H, endoglycosidase H. 1The abbreviations used are: IP3, inositol 1,4,5- trisphosphate; H1–H7, hydrophobic regions of Trp proteins; HA, hemagglutinin antigen; RIPA, radioimmunoprecipitation assay buffer; PCR, polymerase chain reaction; PAGE, polyacrylamide gel electrophoresis; TM, transmembrane; Trp, transient receptor potential; hTrp3, human Trp 3; PNGase F, peptide N-glycosidase F; Endo H, endoglycosidase H. via phospholipase C and subsequently to a biphasic increase of intracellular Ca2+ concentration. The first phase is due to the release of Ca2+ from intracellular stores. The second depends on extracellular Ca2+ that regulates cellular effector systems and replenish the stores. We refer to this form of Ca2+ entry as capacitative calcium entry, a term originally coined by Putney (1Putney Jr., J.W. J. Exp. Biol. 1988; 139: 135-150PubMed Google Scholar, 2Putney Jr., J.W. Bird G. St J. Trends Endocrinol. Metab. 1994; 5: 256-260Abstract Full Text PDF PubMed Scopus (32) Google Scholar). Recently, cDNAs coding for Trp proteins, a family of mammalian proteins homologous to Drosophila Trp and Trp-like have been cloned (3Montell C. Rubin G.M. Neuron. 1989; 2: 1313-1323Abstract Full Text PDF PubMed Scopus (851) Google Scholar, 4Phillips A.M. Bull A. Kelly L.E. Neuron. 1992; 8: 631-642Abstract Full Text PDF PubMed Scopus (384) Google Scholar) and shown to encode ion channels that participate in capacitative calcium entry (5Vaca L. Sinkins W.G. Hu Y. Kunze D. Schilling W.P. Am. J. Physiol. 1994; 267: C1501-C1505Crossref PubMed Google Scholar, 6Zhu X. Jiang M. Peyton M.J. Boulay G. Hurst R. Stefani E. Birnbaumer L. Cell. 1996; 85: 661-671Abstract Full Text Full Text PDF PubMed Scopus (596) Google Scholar).Although the functional aspects of mammalian and DrosophilaTrp proteins have been studied (5Vaca L. Sinkins W.G. Hu Y. Kunze D. Schilling W.P. Am. J. Physiol. 1994; 267: C1501-C1505Crossref PubMed Google Scholar, 6Zhu X. Jiang M. Peyton M.J. Boulay G. Hurst R. Stefani E. Birnbaumer L. Cell. 1996; 85: 661-671Abstract Full Text Full Text PDF PubMed Scopus (596) Google Scholar, 7Harteneck C. Obukhov A.G. Zobel A. Kalkbrenner F. Schultz G. FEBS Lett. 1995; 358: 297-300Crossref PubMed Scopus (76) Google Scholar, 8Philipp S. Cavalie 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), their transmembrane topology has not been addressed by methods other than computer-based predictions of hydrophobicity with the recognition that they exhibit a limited sequence similarity to portions of voltage-gated Ca2+. Hydrophobicity analysis shows the existence of seven regions (H1–H7) able to traverse the plasma membrane. We found similarity to Ca2+ channels in regions H6 and H7 and the intervening segment that is thought to contribute to the pore structure (4Phillips A.M. Bull A. Kelly L.E. Neuron. 1992; 8: 631-642Abstract Full Text PDF PubMed Scopus (384) Google Scholar).The present study addresses the transmembrane topology of one of the Trp proteins, human transient receptor potential 3 (hTrp3), as seen after transient expression in COS cells. hTrp3 is predicted to be a protein of 848 amino acids with the seven hydrophobic regions mentioned above and six endogenous NX(S/T) consensus glycosylation (6Zhu X. Jiang M. Peyton M.J. Boulay G. Hurst R. Stefani E. Birnbaumer L. Cell. 1996; 85: 661-671Abstract Full Text Full Text PDF PubMed Scopus (596) Google Scholar). Previous studies from our laboratory in which hTrp3, tagged at the C terminus with the hemagglutinin antigen (HA) epitope, was immunoprecipitated from extracts of metabolically labeled HEK cells and analyzed by SDS-PAGE and autoradiography, showed that hTrp3 migrates as a doublet of ∼97–100 kDa (9Birnbaumer L. Zhu X. Jiang M. Boulay G. Peyton M. Vannier B. Brown D. Platano D. Sadeghi H. Stefani E. Birnbaumer M. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 15195-15202Crossref PubMed Scopus (354) Google Scholar). Digestion with peptide N glycosidase F (PNGase F) and endoglycosidase H (Endo H) indicated that the upper band of the doublet corresponded to a mature, Endo H-insensitive and endoglycosidase F-sensitive form of hTrp3, whereas the lower band was an immature Endo H-sensitive form (9Birnbaumer L. Zhu X. Jiang M. Boulay G. Peyton M. Vannier B. Brown D. Platano D. Sadeghi H. Stefani E. Birnbaumer M. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 15195-15202Crossref PubMed Scopus (354) Google Scholar). A similar experiment in COS cells, showed only the Endo H sensitive form (9Birnbaumer L. Zhu X. Jiang M. Boulay G. Peyton M. Vannier B. Brown D. Platano D. Sadeghi H. Stefani E. Birnbaumer M. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 15195-15202Crossref PubMed Scopus (354) Google Scholar).These initial results showed that at least one of the six putative sites present in the hTrp3 protein is available to the glycosylation machinery of COS and HEK cells. Below, we identify the location of the glycosylated site. After sequential introduction of consensus glycosylation sites into a Trp from which the endogenous glycosylated site had been removed by site-directed mutagenesis, we then show which of the hydrophobic regions span the membrane and thus form transmembrane segments (TMs). Glycosylation scanning mutagenesis has been used to elucidate topologies of several proteins, including the cystic fibrosis transmembrane conductance regulator (10Chang X.B. Hou Y.X. Jensen T.J. Riordan J.R. J. Biol. Chem. 1994; 269: 18572-18575Abstract Full Text PDF PubMed Google Scholar) and a potassium channel, ROMK1 (11Schwalbe R.A. Wang Z. Bianchi L. Brown A.M. J. Biol. Chem. 1996; 271: 24201-24206Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). Localization of N and C termini on the cytoplasmic side of the membrane allowed us to assign the direction in which the transmembrane segments span the membrane.DISCUSSIONThe results obtained in this study through glycosylation mutants indicate that Trp proteins indeed have six transmembrane segments (Fig. 4), of which the last two are connected by a large loop. Although data to this effect are not yet available, the likelihood of it being the pore is predicated on the sequence homology between TM5–TM6 loops of Trps and the S5 → S6 loops of voltage-gated Ca2+ channels (cf. Fig. 5 of Ref.4Phillips A.M. Bull A. Kelly L.E. Neuron. 1992; 8: 631-642Abstract Full Text PDF PubMed Scopus (384) Google Scholar).Ours are not the first experiments showing glycosylation of a pore region, as the pore region of the ROMK1 K+ channel has been shown to be susceptible to glycosylation when suitably mutated (11Schwalbe R.A. Wang Z. Bianchi L. Brown A.M. J. Biol. Chem. 1996; 271: 24201-24206Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). Once assembled, the pore regions are presumed to be intramembranous. Their susceptibility to glycosylation indicates that the "pores" at one point in time were luminal and suggests that their insertion into the lipid bilayer is a late event in the maturation of the channel. Further experiments in which single amino acids of the putative pore formed by the H6 to H7 linker are changed followed by determination of changes in ion selectivity will be needed to confirm that the linker indeed contributes to the formation of the pore of the channels formed by Trp proteins. The present study identifies on an experimental basis which of the seven hydrophobic domains of Trp proteins traverse the plasma membrane in which direction. Contrary to previous assumptions, the H1 domain is not a TM segment, whereas the much shorter H3 is a TM segment.The delineation of six transmembrane regions formed of hydrophobic regions 2, 3, 4, 5, 6, and 7 clearly confirms at the topological level the relatedness of Trp channels to other channels formed of units that traverse the membrane six times. In addition by analogy, the data suggest that Trp channels should be tetrameric in nature, as are voltage-gated K+ channels, Ca2+, and Na+ channels, which are concatenated tetramers (18Catterall W.A. Science. 1988; 242: 50-61Crossref PubMed Scopus (943) Google Scholar).In a recent work the Drosophila Trp and Trp-like have been shown by co-immunoprecipitation to form heteromultimers as well as homomultimers (19Xu X.Z. Li H.S. Guggino W.B. Montell C. Cell. 1997; 89: 1155-1164Abstract Full Text Full Text PDF PubMed Scopus (279) Google Scholar). The production of chimeras between Trp or Trp-like and the Shaker B channel demonstrated that these interactions not only occur in intact cells but may have a functional significance inDrosophila. Formation of heteromultimers between Trp3 and Trp1 was also shown in this study, but no functional data were presented. Although Trp3 and Trp1 are both expressed in the brain, we do not know if hTrp1 and hTrp3 are expressed in the same cells. Further studies will be needed not only to demonstrate that functional multimers of Trp are formed in mammalian cells but also to determine the composition and stoichiometry of these complexes. Activation of a Gq protein-coupled receptor leads to the production of inositol 1,4,5-trisphosphate (IP3) 1The abbreviations used are: IP3, inositol 1,4,5- trisphosphate; H1–H7, hydrophobic regions of Trp proteins; HA, hemagglutinin antigen; RIPA, radioimmunoprecipitation assay buffer; PCR, polymerase chain reaction; PAGE, polyacrylamide gel electrophoresis; TM, transmembrane; Trp, transient receptor potential; hTrp3, human Trp 3; PNGase F, peptide N-glycosidase F; Endo H, endoglycosidase H. 1The abbreviations used are: IP3, inositol 1,4,5- trisphosphate; H1–H7, hydrophobic regions of Trp proteins; HA, hemagglutinin antigen; RIPA, radioimmunoprecipitation assay buffer; PCR, polymerase chain reaction; PAGE, polyacrylamide gel electrophoresis; TM, transmembrane; Trp, transient receptor potential; hTrp3, human Trp 3; PNGase F, peptide N-glycosidase F; Endo H, endoglycosidase H. via phospholipase C and subsequently to a biphasic increase of intracellular Ca2+ concentration. The first phase is due to the release of Ca2+ from intracellular stores. The second depends on extracellular Ca2+ that regulates cellular effector systems and replenish the stores. We refer to this form of Ca2+ entry as capacitative calcium entry, a term originally coined by Putney (1Putney Jr., J.W. J. Exp. Biol. 1988; 139: 135-150PubMed Google Scholar, 2Putney Jr., J.W. Bird G. St J. Trends Endocrinol. Metab. 1994; 5: 256-260Abstract Full Text PDF PubMed Scopus (32) Google Scholar). Recently, cDNAs coding for Trp proteins, a family of mammalian proteins homologous to Drosophila Trp and Trp-like have been cloned (3Montell C. Rubin G.M. Neuron. 1989; 2: 1313-1323Abstract Full Text PDF PubMed Scopus (851) Google Scholar, 4Phillips A.M. Bull A. Kelly L.E. Neuron. 1992; 8: 631-642Abstract Full Text PDF PubMed Scopus (384) Google Scholar) and shown to encode ion channels that participate in capacitative calcium entry (5Vaca L. Sinkins W.G. Hu Y. Kunze D. Schilling W.P. Am. J. Physiol. 1994; 267: C1501-C1505Crossref PubMed Google Scholar, 6Zhu X. Jiang M. Peyton M.J. Boulay G. Hurst R. Stefani E. Birnbaumer L. Cell. 1996; 85: 661-671Abstract Full Text Full Text PDF PubMed Scopus (596) Google Scholar). Although the functional aspects of mammalian and DrosophilaTrp proteins have been studied (5Vaca L. Sinkins W.G. Hu Y. Kunze D. Schilling W.P. Am. J. Physiol. 1994; 267: C1501-C1505Crossref PubMed Google Scholar, 6Zhu X. Jiang M. Peyton M.J. Boulay G. Hurst R. Stefani E. Birnbaumer L. Cell. 1996; 85: 661-671Abstract Full Text Full Text PDF PubMed Scopus (596) Google Scholar, 7Harteneck C. Obukhov A.G. Zobel A. Kalkbrenner F. Schultz G. FEBS Lett. 1995; 358: 297-300Crossref PubMed Scopus (76) Google Scholar, 8Philipp S. Cavalie 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), their transmembrane topology has not been addressed by methods other than computer-based predictions of hydrophobicity with the recognition that they exhibit a limited sequence similarity to portions of voltage-gated Ca2+. Hydrophobicity analysis shows the existence of seven regions (H1–H7) able to traverse the plasma membrane. We found similarity to Ca2+ channels in regions H6 and H7 and the intervening segment that is thought to contribute to the pore structure (4Phillips A.M. Bull A. Kelly L.E. Neuron. 1992; 8: 631-642Abstract Full Text PDF PubMed Scopus (384) Google Scholar). The present study addresses the transmembrane topology of one of the Trp proteins, human transient receptor potential 3 (hTrp3), as seen after transient expression in COS cells. hTrp3 is predicted to be a protein of 848 amino acids with the seven hydrophobic regions mentioned above and six endogenous NX(S/T) consensus glycosylation (6Zhu X. Jiang M. Peyton M.J. Boulay G. Hurst R. Stefani E. Birnbaumer L. Cell. 1996; 85: 661-671Abstract Full Text Full Text PDF PubMed Scopus (596) Google Scholar). Previous studies from our laboratory in which hTrp3, tagged at the C terminus with the hemagglutinin antigen (HA) epitope, was immunoprecipitated from extracts of metabolically labeled HEK cells and analyzed by SDS-PAGE and autoradiography, showed that hTrp3 migrates as a doublet of ∼97–100 kDa (9Birnbaumer L. Zhu X. Jiang M. Boulay G. Peyton M. Vannier B. Brown D. Platano D. Sadeghi H. Stefani E. Birnbaumer M. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 15195-15202Crossref PubMed Scopus (354) Google Scholar). Digestion with peptide N glycosidase F (PNGase F) and endoglycosidase H (Endo H) indicated that the upper band of the doublet corresponded to a mature, Endo H-insensitive and endoglycosidase F-sensitive form of hTrp3, whereas the lower band was an immature Endo H-sensitive form (9Birnbaumer L. Zhu X. Jiang M. Boulay G. Peyton M. Vannier B. Brown D. Platano D. Sadeghi H. Stefani E. Birnbaumer M. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 15195-15202Crossref PubMed Scopus (354) Google Scholar). A similar experiment in COS cells, showed only the Endo H sensitive form (9Birnbaumer L. Zhu X. Jiang M. Boulay G. Peyton M. Vannier B. Brown D. Platano D. Sadeghi H. Stefani E. Birnbaumer M. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 15195-15202Crossref PubMed Scopus (354) Google Scholar). These initial results showed that at least one of the six putative sites present in the hTrp3 protein is available to the glycosylation machinery of COS and HEK cells. Below, we identify the location of the glycosylated site. After sequential introduction of consensus glycosylation sites into a Trp from which the endogenous glycosylated site had been removed by site-directed mutagenesis, we then show which of the hydrophobic regions span the membrane and thus form transmembrane segments (TMs). Glycosylation scanning mutagenesis has been used to elucidate topologies of several proteins, including the cystic fibrosis transmembrane conductance regulator (10Chang X.B. Hou Y.X. Jensen T.J. Riordan J.R. J. Biol. Chem. 1994; 269: 18572-18575Abstract Full Text PDF PubMed Google Scholar) and a potassium channel, ROMK1 (11Schwalbe R.A. Wang Z. Bianchi L. Brown A.M. J. Biol. Chem. 1996; 271: 24201-24206Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). Localization of N and C termini on the cytoplasmic side of the membrane allowed us to assign the direction in which the transmembrane segments span the membrane. DISCUSSIONThe results obtained in this study through glycosylation mutants indicate that Trp proteins indeed have six transmembrane segments (Fig. 4), of which the last two are connected by a large loop. Although data to this effect are not yet available, the likelihood of it being the pore is predicated on the sequence homology between TM5–TM6 loops of Trps and the S5 → S6 loops of voltage-gated Ca2+ channels (cf. Fig. 5 of Ref.4Phillips A.M. Bull A. Kelly L.E. Neuron. 1992; 8: 631-642Abstract Full Text PDF PubMed Scopus (384) Google Scholar).Ours are not the first experiments showing glycosylation of a pore region, as the pore region of the ROMK1 K+ channel has been shown to be susceptible to glycosylation when suitably mutated (11Schwalbe R.A. Wang Z. Bianchi L. Brown A.M. J. Biol. Chem. 1996; 271: 24201-24206Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). Once assembled, the pore regions are presumed to be intramembranous. Their susceptibility to glycosylation indicates that the "pores" at one point in time were luminal and suggests that their insertion into the lipid bilayer is a late event in the maturation of the channel. Further experiments in which single amino acids of the putative pore formed by the H6 to H7 linker are changed followed by determination of changes in ion selectivity will be needed to confirm that the linker indeed contributes to the formation of the pore of the channels formed by Trp proteins. The present study identifies on an experimental basis which of the seven hydrophobic domains of Trp proteins traverse the plasma membrane in which direction. Contrary to previous assumptions, the H1 domain is not a TM segment, whereas the much shorter H3 is a TM segment.The delineation of six transmembrane regions formed of hydrophobic regions 2, 3, 4, 5, 6, and 7 clearly confirms at the topological level the relatedness of Trp channels to other channels formed of units that traverse the membrane six times. In addition by analogy, the data suggest that Trp channels should be tetrameric in nature, as are voltage-gated K+ channels, Ca2+, and Na+ channels, which are concatenated tetramers (18Catterall W.A. Science. 1988; 242: 50-61Crossref PubMed Scopus (943) Google Scholar).In a recent work the Drosophila Trp and Trp-like have been shown by co-immunoprecipitation to form heteromultimers as well as homomultimers (19Xu X.Z. Li H.S. Guggino W.B. Montell C. Cell. 1997; 89: 1155-1164Abstract Full Text Full Text PDF PubMed Scopus (279) Google Scholar). The production of chimeras between Trp or Trp-like and the Shaker B channel demonstrated that these interactions not only occur in intact cells but may have a functional significance inDrosophila. Formation of heteromultimers between Trp3 and Trp1 was also shown in this study, but no functional data were presented. Although Trp3 and Trp1 are both expressed in the brain, we do not know if hTrp1 and hTrp3 are expressed in the same cells. Further studies will be needed not only to demonstrate that functional multimers of Trp are formed in mammalian cells but also to determine the composition and stoichiometry of these complexes. The results obtained in this study through glycosylation mutants indicate that Trp proteins indeed have six transmembrane segments (Fig. 4), of which the last two are connected by a large loop. Although data to this effect are not yet available, the likelihood of it being the pore is predicated on the sequence homology between TM5–TM6 loops of Trps and the S5 → S6 loops of voltage-gated Ca2+ channels (cf. Fig. 5 of Ref.4Phillips A.M. Bull A. Kelly L.E. Neuron. 1992; 8: 631-642Abstract Full Text PDF PubMed Scopus (384) Google Scholar). Ours are not the first experiments showing glycosylation of a pore region, as the pore region of the ROMK1 K+ channel has been shown to be susceptible to glycosylation when suitably mutated (11Schwalbe R.A. Wang Z. Bianchi L. Brown A.M. J. Biol. Chem. 1996; 271: 24201-24206Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). Once assembled, the pore regions are presumed to be intramembranous. Their susceptibility to glycosylation indicates that the "pores" at one point in time were luminal and suggests that their insertion into the lipid bilayer is a late event in the maturation of the channel. Further experiments in which single amino acids of the putative pore formed by the H6 to H7 linker are changed followed by determination of changes in ion selectivity will be needed to confirm that the linker indeed contributes to the formation of the pore of the channels formed by Trp proteins. The present study identifies on an experimental basis which of the seven hydrophobic domains of Trp proteins traverse the plasma membrane in which direction. Contrary to previous assumptions, the H1 domain is not a TM segment, whereas the much shorter H3 is a TM segment. The delineation of six transmembrane regions formed of hydrophobic regions 2, 3, 4, 5, 6, and 7 clearly confirms at the topological level the relatedness of Trp channels to other channels formed of units that traverse the membrane six times. In addition by analogy, the data suggest that Trp channels should be tetrameric in nature, as are voltage-gated K+ channels, Ca2+, and Na+ channels, which are concatenated tetramers (18Catterall W.A. Science. 1988; 242: 50-61Crossref PubMed Scopus (943) Google Scholar). In a recent work the Drosophila Trp and Trp-like have been shown by co-immunoprecipitation to form heteromultimers as well as homomultimers (19Xu X.Z. Li H.S. Guggino W.B. Montell C. Cell. 1997; 89: 1155-1164Abstract Full Text Full Text PDF PubMed Scopus (279) Google Scholar). The production of chimeras between Trp or Trp-like and the Shaker B channel demonstrated that these interactions not only occur in intact cells but may have a functional significance inDrosophila. Formation of heteromultimers between Trp3 and Trp1 was also shown in this study, but no functional data were presented. Although Trp3 and Trp1 are both expressed in the brain, we do not know if hTrp1 and hTrp3 are expressed in the same cells. Further studies will be needed not only to demonstrate that functional multimers of Trp are formed in mammalian cells but also to determine the composition and stoichiometry of these complexes.