Journal of Molecular Neuroscience

, Volume 18, Issue 3, pp 169–178 | Cite as

Immunological characterization of 5-HT3 receptor transmembrane topology



The 5-hydroxytryptamine3 (5-HT3) receptor is a member of the Cys-loop family of ligand-gated ion channels. These receptors are pentamers with the greatest homology to nicotinic acetylcholine (nACh) receptors. The proposed topological organization of a 5-HT3 receptor subunit is based largely on hydropathy profiles and by homology to nACh receptors, and indicates a large N-terminal extracellular domain and four transmembrane regions. There is, however, little direct evidence for this model. We therefore investigated the topology of the 5-HT3A receptor subunit using a panel of 5-HT3 receptor-specific antisera that interact with defined regions of the receptor. An antiserum generated against a short peptide from the N-terminal domain of the 5-HT3A receptor subunit, pAb120, was shown to bind to 5-HT3 receptor-expressing cells with intact cell membranes, indicating that the N-terminal end of the subunit is extracellular. Two antisera generated against regions of the loop between predicted transmembrane regions three and four did not bind to cells with intact membranes. However on membrane permeabilization these antibodies both bound to the receptor in intracellular areas, thus indicating that the loop between transmembrane domains three and four is intracellular. These data therefore provide direct evidence for an extracellular N-terminal domain and an intracellular loop between the third and fourth transmembrane domains, thus supporting the conventional ligand-gated ion channel subunit topological model.

Index Entries

Serotonin 5-hydroxytryptamine ligand-gated ion channel 5-HT3 receptor immunochemistry 


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  1. Anand R., Bason L., Saedi M. S., Gerzanich V., Peng X., and Lindstrom J. (1993) Reporter epitopes: A novel approach to examine transmembrane topology of integral membrane proteins applied to the α1 subunit of the nicotinic acetylcholine receptor. Biochemistry 32, 9975–9984.PubMedCrossRefGoogle Scholar
  2. Boess F. G., Steward L. J., Steele J. A., et al. (1997) Analysis of the ligand binding site of the 5-HT3 receptor using site directed mutagenesis: importance of glutamate 106. Neuropharmacology 36, 637–647.PubMedCrossRefGoogle Scholar
  3. Brejc K., van Dijk W. J., Klaassen R. V., et al. (2001) Crystal structure of an ACh-binding protein reveals the ligand-binding domain of nicotinic receptors. Nature 411, 269–276.PubMedCrossRefGoogle Scholar
  4. Chavez R. A. and Hall Z. W. (1991) The transmembrane topology of the amino terminus of the α subunit of the nicotinic acetylcholine receptor. J. Biol. Chem. 266, 15532–15538.PubMedGoogle Scholar
  5. Chavez R. A. and Hall Z. W. (1992) Expression of fusion proteins of the nicotinic acetylcholine receptor from mammalian muscle identifies the membrane-spanning regions in the alpha and delta subunits. J. Cell Biol. 116, 385–393.PubMedCrossRefGoogle Scholar
  6. Conti-Fine B. M., Lei S., and McLane K. E. (1996) Antibodies as tools to study the structure of membrane proteins: the case of the nicotinic acetylcholine receptor. Annu. Rev. Biophys. Biomol. Struct. 25, 197–229.PubMedGoogle Scholar
  7. Corringer P. J., Le Novere N., and Changeux J.-P. (2000) Nicotinic receptors at the amino acid level. Ann. Rev. Pharmacol. Toxicol. 40, 431–458.CrossRefGoogle Scholar
  8. Criado M., Hochschwender S., Sarin V., Fox J. L., and Lindstrom J. (1985) Evidence for unpredicted transmembrane domains in acetylcholine receptor subunits. Proc. Natl. Acad. Sci. USA 82, 2004–2008.PubMedCrossRefGoogle Scholar
  9. Davies P. A., Pistis M., Hanna M. C., et al. (1999) The 5-HT3B subunit is a major determinant of serotonin-receptor function. Nature 397, 359–363.PubMedCrossRefGoogle Scholar
  10. Derkach V., Surprenant A., and North R. A. (1989) 5-HT3 receptors are membrane ion channels. Nature 339, 706–709.PubMedCrossRefGoogle Scholar
  11. Dwyer B. P. (1991) Topological dispositions of lysine α380 and lysine γ486 in the acetylcholine receptor from Torpedo californica. Biochemistry 30, 4105–4112.PubMedCrossRefGoogle Scholar
  12. Finer-Moore J. and Stroud R. M. (1984) Amphipathic analysis and possible formation of the ion channel in an acetylcholine receptor. Proc. Natl. Acad. Sci. USA 81, 155–159.PubMedCrossRefGoogle Scholar
  13. Gorne-Tschelnokow U., Strecker A., Kaduk C., Naumann D., and Hucho F. (1994) The transmembrane domains of the nicotinic acetylcholine receptor contain α-helical and β structures. EMBO J. 13, 338–341.PubMedGoogle Scholar
  14. Hargreaves A. C., Lummis C. C. R., and Taylor C. W. (1994) Ca2+ permeability of cloned and native 5-hydroxytryptamine type 3 receptors. Mol. Pharmacol. 46, 1120–1128.PubMedGoogle Scholar
  15. Hope A. G., Downie D. L., Sutherland L., Lambert J. L., Peter J. A., and Burchell B. (1993) Cloning and functional expression of an apparent splice variant of the murine 5-HT3 receptor Asubunit. Eur. J. Pharmacol. 254, 187–192.Google Scholar
  16. Karlin A. and Akabas M. H. (1995) Toward a structural basis for the function of nicotinic receptors and their cousins. Neuron 15, 1231–1244.PubMedCrossRefGoogle Scholar
  17. Leite J. F., Amoscato A. A., and Cascio M. (2000) Coupled proteolytic and mass spectrometry studies indicate a novel topology for the glycine receptor. J. Biol. Chem. 275(18), 13683–13689.PubMedCrossRefGoogle Scholar
  18. Lummis S. C. R. and Fletcher, E. J. (1996). Functional and binding studies of glycosylation site mutants of 5-HT3 receptors. Br. J. Pharmacol. 119, 291P.Google Scholar
  19. Maricq A. V., Peterson A. S., Brake A. J., Myers R. M., and Julius D. (1991) Primary structure and functional expression of the 5HT3 receptor, a serotonin gated ion channel. Science 254, 432–437.PubMedCrossRefGoogle Scholar
  20. Morales M., Battenberg E., Lecea L. D., and Bloom F. E. (1996) The type 3 serotonin receptor is expressed in a subpopulation of GABAergic neurons in the rat neocortex and hippocampus. Brain Res. 731, 199–202.PubMedCrossRefGoogle Scholar
  21. Moore C. R., Yates J. R. 3rd., Griffin P. R., et al. (1989) Proteolytic fragments of the nicotinic acetylcholine receptor identified by mass spectrometry: implications for receptor topography. Biochemistry 28, 9184–9191.PubMedCrossRefGoogle Scholar
  22. Moss S. J., Smart T. G., Blackstone C. D., and Huganir R. L. (1992) Functional modulation of GABAA receptors by cAMP-dependent protein phosphorylation. Science 257, 661–665.PubMedCrossRefGoogle Scholar
  23. Mukerji J., Haghighi A., and Seguela P. (1996) Immunological characterization and transmembrane topology of 5-hydroxytryptamine3 receptors by functional epitope tagging. J. Neurochem. 66, 1027–1032.PubMedCrossRefGoogle Scholar
  24. Noda M., Takahashi H., Tanabe T., et al. (1983) Structural homology of Torpedo californica acetylcholine subunits. Nature 302, 528–553.PubMedCrossRefGoogle Scholar
  25. Pedersen S. E. and Cohen J. B. (1990) d-Tubocurarine binding sites are located at α-γ and α-δ subunit interfaces of the nicotinic acetylcholine receptor. Proc. Natl. Acad. Sci. USA 87, 2785–2789.PubMedCrossRefGoogle Scholar
  26. Ratnam M. and Lindstrom J. (1984) Structural features of the nicotinic acetylcholine receptor revealed by antibodies to synthetic peptides. Biochem. Biophys. Res. Commun. 122, 1225–1233.PubMedCrossRefGoogle Scholar
  27. Ratnam M., Sargent P. B., Sarin V., et al. (1986a) Location of antigenic determinants on primary sequences of subunits of nicotinic acetylcholine receptor by peptide mapping. Biochemistry 25, 2621–2632.PubMedCrossRefGoogle Scholar
  28. Ratnam M., Nguyen D. L., Rivier J., Sargent P. B., and Lindstrom J. (1986b) Transmembrane topography of nicotinic acetylcholine receptor: immunochemical tests contradict theoretical predictions based on hydrophobicity plots. Biochemistry 25, 2633–2643.PubMedCrossRefGoogle Scholar
  29. Ruiz-Gomez A., Vaello M.-L., Valdivieso F., and Mayor F. Jr. (1991) Phosphorylation of the 48 kDa subunit of the glycine receptor by protein kinase C. J. Biol. Chem. 266, 559–566.PubMedGoogle Scholar
  30. Spier A. D., Wotherspoon G., Nayak S. V., Nichols R. A., Priestley J. V., and Lummis S. C. R. (1999) Antibodies against the extracellular domain of the 5-HT3 receptor label both native and recombinant receptors. Mol. Brain Res. 67, 221–230.PubMedCrossRefGoogle Scholar
  31. Spier A. D. and Lummis S. C. R. (2000) The role of tryptophan residues in the 5-Hydroxytryptamine(3) receptor ligand-binding domain. J. Biol. Chem. 275, 5620–5625.PubMedCrossRefGoogle Scholar
  32. Steward L. J., Boess F. G., Steele J. A., Liu D., Wong N., and Martin I. L. (2000) Importance of phenylalanine 107 in agonist recognition by the 5-hydroxytryptamine(3A) receptor. Mol. Pharmacol. 57, 1249–1255.PubMedGoogle Scholar
  33. Tobimatsu T., Fujita Y., Fukuda K., et al. (1987) Effects of substitution of putative transmembrane segments on nicotinic acetylcholine receptor function. FEBS Lett. 222, 56–62.PubMedCrossRefGoogle Scholar
  34. Turton S., Gillard N. P., Stephenson F. A., and McKernon R. M. (1993) Antibodies against the 5-HT3-A receptor identify a 54 kDa protein affinity-purified from NCB20 cells. Mol. Neuropharmacol. 3, 167–171.Google Scholar
  35. Unwin N. (1993) Nicotinic acetylcholine receptor at 9Å resolution. J. Mol. Biol. 229, 1101–1124.PubMedCrossRefGoogle Scholar
  36. Unwin N. (2000) The Croonian Lecture 2000. Nicotinic acetylcholine receptor and the structural basis of fast synaptic transmission. Phil. Trans. R. Soc. Lond. B 355, 1813–1829.CrossRefGoogle Scholar
  37. Yan D., Schulte M. K., Bloom K. E., and White M. M. (1999) Structural features of the ligand-binding domain of the serotonin 5HT3 receptor. J. Biol. Chem. 274, 5537–5541.PubMedCrossRefGoogle Scholar
  38. Yee G. H. and Huganir R. L. (1987) Determination of the sites of cAMP-dependent phosphorylation on the nicotinic acetylcholine receptor. J. Biol. Chem. 262, 16748–16753.PubMedGoogle Scholar
  39. Young E. F., Ralston E., Blake J., Ramachandran J., Hall Z. W., and Stroud R. M. (1985) Topological mapping of acetylcholine receptor: evidence for a model with five transmembrane segments and a cytoplasmic COOH-terminal peptide. Proc. Natl. Acad. Sci. USA 82, 626–630.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press Inc 2002

Authors and Affiliations

  1. 1.Division of Neurobiology, Laboratory of Molecular BiologyMRC CentreCambridgeUK
  2. 2.Department of BiochemistryUniversity of CambridgeCambridgeUK

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