Journal of Molecular Neuroscience

, Volume 7, Issue 3, pp 217–228 | Cite as

Comparative structure of human neuronal α2–α7 and β2–β4 nicotinic acetylcholine receptor subunits and functional expression of the α2, α3, α4, α7, β2, and β4 subunits

  • Kathryn J. Elliott
  • Steven B. Ellis
  • Kelly J. Berckhan
  • Arturo Urrutia
  • Laura E. Chavez-Noriega
  • Edwin C. Johnson
  • Gönül Veliçelebi
  • Michael M. Harpold
Original Articles

Abstract

cDNA clones encoding human neuronal nicotinic acetylcholine receptor α2, α3, α4, α5, α6, α7, β2, β3, and β4 subunits were isolated from brainstem, hippocampus, prefrontal cortex, substantia nigra, thalamus, and IMR32 libraries. Human α2 and α6 and full-length β3 and β4 clones have not been previously reported. Deduced amino acid sequences of the α2, α6, β3, and β4 predicted mature peptides are 503 residues (56.9 kDa), 464 residues (53.7 kDa), 440 residues (50.8 kDa), and 477 residues (54.1 kDa), respectively. These sequences show 84 (α2), 87 (α6), 89 (β3), and 84% (β4) identity to the corresponding rat sequences. The amino termini of the human α2 and β3 mature peptides contain 23 and six additional residues, respectively, compared to those of rat α2 and β3. Recombinant receptors were expressed inXenopus laevis oocytes injected with in vitro transcripts encoding either α7 alone or α2, α3, or α4 in pairwise combination with β2 or β4. Inward currents were elicited by the application of acetylcholine (1–100 µM) and other agonists; these responses were blocked 65–97% by application of 10 µM d-tubocurare, confirming functional expression of human nicotinic receptors.

Index Entries

Nicotinic acetylcholine receptor molecular cloning ligand-gated ion channel central nervous system human 

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. Anand R. and Lindstrom J. (1990) Nucleotide sequence of the human nicotinic acetylcholine receptor β2 subunit gene.Nucleic Acids Res. 18, 4272.PubMedCrossRefGoogle Scholar
  2. Anand R., Conroy W. G., Schoepfer R., Whiting P., and Lindstrom J. (1991) Neuronal nicotinic acetylcholine receptors expressed inXenopus oocytes have a pentameric quaternary structure.J. Biol. Chem. 266, 11,192–11,198.Google Scholar
  3. Ballivet M., Nef P., Couturier S., Rungger D., Bader C., Bertrand D., and Cooper E. (1988) Electrophysiology of a chick neuronal nicotinic acetylcholine receptor expressed inXenopus oocytes after cDNA injection.Neuron 1, 847–852.PubMedCrossRefGoogle Scholar
  4. Bertrand D., Bertrand S., and Ballivet M. (1992) Pharmacological properties of the homomeric α7 receptor.Neurosci. Lett. 146, 87–90.PubMedCrossRefGoogle Scholar
  5. Boulter J., Connolly J., Deneris E., Goldman D., Heinemann S., and Patrick J. (1987) Functional expression of two neuronal nicotinic acetylcholine receptors from cDNA clones identifies a gene family.Proc. Natl. Acad. Sci. USA 84, 7763–7767.PubMedCrossRefGoogle Scholar
  6. Boulter J., Evans K., Goldman D., Martin G., Treco D., Heinemann S., and Patrick J. (1986) Isolation of a cDNA clone coding for a possible neuronal nicotinic acetylcholine receptor α-subunit.Nature 319, 368–374.PubMedCrossRefGoogle Scholar
  7. Boulter J., O’Shea-Greenfield A., Duvoisin R. M., Connolly J. G., Wada E., Jensen A., Gardner P. D., Ballivet M., Deneris E. S., McKinnon D., Heinemann S., and Patrick J. (1990) α3, α5, and β4: three members of the rat neuronal nicotinic acetylcholine receptor-related gene family form a gene cluster.J. Biol. Chem. 265, 4472–4482.PubMedGoogle Scholar
  8. Chini B., Clementi F., Hukovic N., and Sher E. (1992) Neuronal-type α-bungarotoxin receptors and the α5-nicotinic receptor subunit gene are expressed in neuronal and nonneuronal human cell lines.Proc. Natl. Acad. Sci. USA 89, 1572–1576.PubMedCrossRefGoogle Scholar
  9. Chini B., Raimond E., Elgoyhen A. B., Moralli D., Balzaretti M., and Heinemann S. (1994) Molecular cloning and chromosomal localization of the human α7-nicotinic receptor subunit gene (CHRNA7).Genomics 19, 379–381.PubMedCrossRefGoogle Scholar
  10. Cooper E., Couturier S., and Ballivet M. (1991) Pentameric structure and subunit stoichiometry of a neuronal nicotinic acetylcholine receptor.Nature 350, 235–238.PubMedCrossRefGoogle Scholar
  11. Couturier S., Bertrand D., Matter J.-M., Hernandez M.-C., Bertrand S., Millar N., Valera S., Barkas T., and Ballivet M. (1990a) A neuronal nicotinic acetylcholine receptor subunit (α7) is developmentally regulated and forms a homo-oligomeric channel blocked by α-BTX.Neuron 5, 847–856.PubMedCrossRefGoogle Scholar
  12. Couturier S., Erkman L., Valera S., Rungger D., Bertrand S., Boulter J., Ballivet M., and Bertrand D. (1990b) α5, α3, and non-α3: three clustered avian genes encoding neuronal nicotinic acetylcholine receptor-related subunits.J. Biol. Chem. 265, 17,560–17,567.Google Scholar
  13. Deneris E. S., Connolly J., Boulter J., Wada E., Wada K., Swanson L. W., Patrick J., and Heinemann S. (1988) Primary structure and expression of β2: a novel subunit of neuronal nicotinic acetylcholine receptors.Neuron 1, 45–54.PubMedCrossRefGoogle Scholar
  14. Dennis M., Giraudat J., Kotzyba-Hilbert F., Goeldner M., Hirth C., Chang J.-Y., Lazure C., Chretien M., and Changeux J.-P. (1988) Amino acids of theTorpedo marmorata acetylcholine receptor α-subunit labeled by a photoaffinity ligand for the acetylcholine binding site.Biochemistry 27, 2346–2357.PubMedCrossRefGoogle Scholar
  15. Doucette-Stamm L., Monteggia L. M., Donnelly-Roberts D., Wang M. T., Lee J., Tian J., and Giordano T. (1993) Cloning and sequence of the human α7 nicotinic acetylcholine receptor.Drug Dev. Res. 30, 252–256.CrossRefGoogle Scholar
  16. Duvoisin R. M., Deneris E. S., Patrick J., and Heinemann S. (1989) The functional diversity of the neuronal nicotinic acetylcholine receptors is increased by a novel subunit: β4.Neuron 3, 487–496.PubMedCrossRefGoogle Scholar
  17. Elgoyhen A. B., Johnson D. S., Boulter J., Vetter D. E., and Heinemann S. (1994) α9: an acetylcholine receptor with novel pharmacological properties expressed in rat cochlear hair cells.Cell 79, 705–715.PubMedCrossRefGoogle Scholar
  18. Ellis S. B., Williams M. E., Ways N. R., Brenner R., Sharp A. H., Leung A. T., Campbell K. P., McKenna E., Koch W. J., Hui A., Schwartz A., and Harpold M. M. (1988) Sequence and expression of mRNAs encoding the α1 and α2 subunits of a DHP-sensitive calcium channel.Science 241, 1661–1664.PubMedCrossRefGoogle Scholar
  19. Fornasari D., Chini B., Tarroni P., and Clementi F. (1990) Molecular cloning of human neuronal nicotinic receptor α3-subunit.Neurosci. Lett. 111, 351–356.PubMedCrossRefGoogle Scholar
  20. Galzi J.-L., Revah F., Black D., Goeldner M., Hirth C., and Changeux J.-P. (1990) Identification of a novel amino acid α-tyrosine 93 within the cholinergic ligands-binding sites of the acetylcholine receptor by photoaffinity labeling. Additional evidence for a three-loop model of the cholinergic ligands-binding sites.J. Biol. Chem. 265, 10,430–10,437.Google Scholar
  21. Galzi J.-L., Bertrand D., Devillers-Thiery A., Revah F., Bertrand S., and Changeux J.-P. (1991) Functional significance of aromatic amino acids from three peptide loops of the α7 neuronal nicotinic receptor site investigated by site-directed mutagenesis.FEBS Lett. 294, 198–202.PubMedCrossRefGoogle Scholar
  22. Gerzanich V., Anand R., and Lindstrom J. (1994) Homomers of α8 and α7 subunits of nicotinic receptors exhibit similar channel but contrasting binding site properties.Mol. Pharmacol. 45, 212–220.PubMedGoogle Scholar
  23. Goldin A. L. and Sumikawa K. (1992) Preparation of RNA for injection intoXenopus oocytes, inMethods in Enzymology, vol. 207 (Rudy B. and Iverson L. E., eds.), Academic, New York, pp. 279–297.Google Scholar
  24. Goldman D., Deneris E., Luyten W., Kochhar A., Patrick J., and Heinemann S. (1987) Members of a nicotinic acetylcholine receptor gene family are expressed in different regions of the mammalian central nervous system.Cell 48, 965–973.PubMedCrossRefGoogle Scholar
  25. Hussy N., Ballivet M., and Bertrand D. (1994) Agonist and antagonist effects of nicotine on chick neuronal nicotinic receptors are defined by α and β subunits.J. Neurophysiol. 72, 1317–1326.PubMedGoogle Scholar
  26. Karlin A. (1993) Structure of nicotinic acetylcholine receptors.Curr. Opin. Neurobiol. 3, 299–309.PubMedCrossRefGoogle Scholar
  27. Kozak M. (1987) An analysis of 5′-noncoding sequences from 699 vertebrate messenger RNAs.Nucleic Acids Res. 15, 8125–8148.PubMedCrossRefGoogle Scholar
  28. Krieg P. A. and Melton D. A. (1984) Functional messenger RNAs are produced by SP6 in vitro transcription of cloned cDNAs.Nucleic Acids Res. 12, 7057–7070.PubMedCrossRefGoogle Scholar
  29. Luetje C. W. and Patrick J. (1991) Both α- and β-subunits contribute to the agonist sensitivity of neuronal nicotinic acetylcholine receptors.J. Neurosci. 11, 837–845.PubMedGoogle Scholar
  30. Maniatis T., Fritsch E. F., and Sambrook J. (1982)Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York.Google Scholar
  31. Monteggia L. M., Gopalakrishnan M., Touma E., Idler K. B., Nash N., Arneric S. P., Sullivan J. P., and Giordano T. (1995) Cloning and transient expression of genes encoding the human α4 and β2 neuronal nicotinic acetylcholine receptor (nAChR) subunits.Gene 155, 189–193.PubMedCrossRefGoogle Scholar
  32. Peng X., Katz M., Gerzanich V., Anand R., and Lindstrom J. (1994) Human α7 acetylcholine receptor: cloning of the α7 subunit from the SH-SY5Y cell line and determination of pharmacological properties of native receptors and functional α7 homomers expressed inXenopus oocytes.Mol. Pharmacol. 45, 546–554.PubMedGoogle Scholar
  33. Sargent P. B. (1993) The diversity of neuronal nicotinic acetylcholine receptors.Ann. Rev. Neurosci. 16, 403–443.PubMedCrossRefGoogle Scholar
  34. Schoepfer R., Conroy W. G., Whiting P., Gore M., and Lindstrom J. (1990) Brain α-bungarotoxin binding protein cDNAs and MAbs reveal subtypes of this branch of the ligand-gated ion channel gene superfamily.Neuron 5, 35–48.PubMedCrossRefGoogle Scholar
  35. Séguéla P., Wadiche J., Dineley-Miller K., Dani J. A., and Patrick J. W. (1993) Molecular cloning, functional properties, and distribution of rat brain α7: a nicotinic cation channel highly permeable to calcium.J. Neurosci. 13, 596–604.PubMedGoogle Scholar
  36. Tarroni P., Rubboli F., Chini B., Zwart R., Oortgiesen M., Sher E., and Clementi F. (1992) Neuronal-type nicotinic receptors in human neuroblastoma and small-cell lung carcinoma cell lines.FEBS Lett. 312, 66–70.PubMedCrossRefGoogle Scholar
  37. von Heijne G. (1986) A new method for predicting signal sequence cleavage sites.Nucleic Acids Res. 14, 4683–4691.CrossRefGoogle Scholar
  38. Wada K., Ballivet M., Boulter J., Connolly J., Wada E., Deneris E. S., Swanson L. W., Heinemann S., and Patrick J. (1988) Functional expression of a new pharmacological subtype of brain nicotinic acetylcholine receptor.Science 240, 330–334.PubMedCrossRefGoogle Scholar
  39. Wada E., Wada K., Boulter J., Deneris E., Heinemann S., Patrick J., and Swanson L. W. (1989) Distribution of α2, α3, α4, and β2 neuronal nicotinic receptor subunit mRNAs in the central nervous system: a hybridization histochemical study in the rat.J. Comp. Neurol. 284, 314–335.PubMedCrossRefGoogle Scholar
  40. Whiting P., Schoepfer R., Lindstrom J., and Priestley T. (1991) Structural and pharmacological characterization of the major brain nicotinic acetylcholine receptor subtype stably expressed in mouse fibroblasts.Mol. Pharmacol. 40, 463–472.PubMedGoogle Scholar
  41. Williams M. E., Feldman D. H., McCue A. F., Brenner R., Velicelebi G., Ellis S. B., and Harpold M. M. (1992) Structure and functional expression of α1, α2, and β subunits of a novel human neuronal calcium channel subtype.Neuron 8, 71–84.PubMedCrossRefGoogle Scholar
  42. Willoughby J. J., Ninkina N. N., Beech M. M., Latchman D. S., and Wood J. N. (1993) Molecular cloning of a human neuronal nicotinic acetylcholine receptor β3-like subunit.Neurosci. Lett. 155, 136–139PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press Inc 1996

Authors and Affiliations

  • Kathryn J. Elliott
    • 1
  • Steven B. Ellis
    • 1
  • Kelly J. Berckhan
    • 1
  • Arturo Urrutia
    • 1
  • Laura E. Chavez-Noriega
    • 1
  • Edwin C. Johnson
    • 1
  • Gönül Veliçelebi
    • 1
  • Michael M. Harpold
    • 1
  1. 1.SIBIA Neurosciences Inc.La Jolla

Personalised recommendations