Skip to main content
NCBI home page
The Journal of Physiology logoLink to The Journal of Physiology
. 1995 Dec 15;489(Pt 3):767–778. doi: 10.1113/jphysiol.1995.sp021090

Functional and non-functional isoforms of the human muscle acetylcholine receptor.

C F Newland 1, D Beeson 1, A Vincent 1, J Newsom-Davis 1
PMCID: PMC1156846  PMID: 8788941

Abstract

1. The properties of a recently identified isoform of the human muscle nicotinic acetylcholine receptor (AChR) alpha subunit (alpha +), which in muscle is expressed at similar levels to the alpha subunit, were investigated by both electrophysiological and biochemical approaches following expression in Xenopus laevis oocytes. The single-channel properties of adult (alpha 2 beta delta epsilon) and fetal (alpha 2 beta delta gamma) forms of the human AChR were also investigated. 2. The mean burst duration of adult channels (4.1 +/- 0.3 ms, mean +/- S.E.M., n = 5) is half that of fetal channels (7.9 +/- 0.6 ms, n = 4), while the single-channel conductance is larger (62.2 +/- 0.8 and 37.9 +/- 1.6 pS for adult and fetal channels, respectively), comparable to the developmental changes in single-channel properties observed for other mammalian species. 3. In contrast to the alpha isoform, the alpha + subunit does not bind 125I-labelled alpha-bungarotoxin or monoclonal antibodies directed against the AChR 'main immunogenic region' (MIR), illustrating why the alpha + subunit was first detected through screening of cDNA libraries. 4. By using site-directed mutagenesis to produce subunits that conferred different single-channel conductances on the AChR, we demonstrate that the alpha + isoform is not integrated into functional AChRs. 5. The mutagenesis experiments also revealed that the two alpha subunits within an AChR pentamer are not equivalent within the pore lining region.

Full text

PDF
767

Images in this article

769Figure 1
on p.769
770Figure 2
on p.770

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Beeson D., Brydson M., Betty M., Jeremiah S., Povey S., Vincent A., Newsom-Davis J. Primary structure of the human muscle acetylcholine receptor. cDNA cloning of the gamma and epsilon subunits. Eur J Biochem. 1993 Jul 15;215(2):229–238. doi: 10.1111/j.1432-1033.1993.tb18027.x. [DOI] [PubMed] [Google Scholar]
  2. Beeson D., Morris A., Vincent A., Newsom-Davis J. The human muscle nicotinic acetylcholine receptor alpha-subunit exist as two isoforms: a novel exon. EMBO J. 1990 Jul;9(7):2101–2106. doi: 10.1002/j.1460-2075.1990.tb07378.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Chavez R. A., Maloof J., Beeson D., Newsom-Davis J., Hall Z. W. Subunit folding and alpha delta heterodimer formation in the assembly of the nicotinic acetylcholine receptor. Comparison of the mouse and human alpha subunits. J Biol Chem. 1992 Nov 15;267(32):23028–23034. [PubMed] [Google Scholar]
  4. Colquhoun D., Sakmann B. Fast events in single-channel currents activated by acetylcholine and its analogues at the frog muscle end-plate. J Physiol. 1985 Dec;369:501–557. doi: 10.1113/jphysiol.1985.sp015912. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Cooper E., Couturier S., Ballivet M. Pentameric structure and subunit stoichiometry of a neuronal nicotinic acetylcholine receptor. Nature. 1991 Mar 21;350(6315):235–238. doi: 10.1038/350235a0. [DOI] [PubMed] [Google Scholar]
  6. Cull-Candy S. G., Miledi R., Uchitel O. D. Properties of junctional and extrajunctional acetylcholine-receptor channels in organ cultured human muscle fibres. J Physiol. 1982 Dec;333:251–267. doi: 10.1113/jphysiol.1982.sp014452. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Deng W. P., Nickoloff J. A. Site-directed mutagenesis of virtually any plasmid by eliminating a unique site. Anal Biochem. 1992 Jan;200(1):81–88. doi: 10.1016/0003-2697(92)90280-k. [DOI] [PubMed] [Google Scholar]
  8. Engel A. G., Hutchinson D. O., Nakano S., Murphy L., Griggs R. C., Gu Y., Hall Z. W., Lindstrom J. Myasthenic syndromes attributed to mutations affecting the epsilon subunit of the acetylcholine receptor. Ann N Y Acad Sci. 1993 Jun 21;681:496–508. doi: 10.1111/j.1749-6632.1993.tb22933.x. [DOI] [PubMed] [Google Scholar]
  9. Green W. N., Claudio T. Acetylcholine receptor assembly: subunit folding and oligomerization occur sequentially. Cell. 1993 Jul 16;74(1):57–69. doi: 10.1016/0092-8674(93)90294-z. [DOI] [PubMed] [Google Scholar]
  10. Gu Y., Forsayeth J. R., Verrall S., Yu X. M., Hall Z. W. Assembly of the mammalian muscle acetylcholine receptor in transfected COS cells. J Cell Biol. 1991 Aug;114(4):799–807. doi: 10.1083/jcb.114.4.799. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Gu Y., Franco A., Jr, Gardner P. D., Lansman J. B., Forsayeth J. R., Hall Z. W. Properties of embryonic and adult muscle acetylcholine receptors transiently expressed in COS cells. Neuron. 1990 Aug;5(2):147–157. doi: 10.1016/0896-6273(90)90305-y. [DOI] [PubMed] [Google Scholar]
  12. Harcourt G., Batocchi A. P., Hawke S., Beeson D., Pantic N., Jacobson L., Willcox N., Vincent A., Newsom-Davis J. Detection of alpha-subunit isoforms in human muscle acetylcholine receptor by specific T cells from a myasthenia gravis patient. Proc Biol Sci. 1993 Oct 22;254(1339):1–6. doi: 10.1098/rspb.1993.0118. [DOI] [PubMed] [Google Scholar]
  13. Imoto K., Busch C., Sakmann B., Mishina M., Konno T., Nakai J., Bujo H., Mori Y., Fukuda K., Numa S. Rings of negatively charged amino acids determine the acetylcholine receptor channel conductance. Nature. 1988 Oct 13;335(6191):645–648. doi: 10.1038/335645a0. [DOI] [PubMed] [Google Scholar]
  14. Imoto K., Konno T., Nakai J., Wang F., Mishina M., Numa S. A ring of uncharged polar amino acids as a component of channel constriction in the nicotinic acetylcholine receptor. FEBS Lett. 1991 Sep 9;289(2):193–200. doi: 10.1016/0014-5793(91)81068-j. [DOI] [PubMed] [Google Scholar]
  15. Kurosaki T., Fukuda K., Konno T., Mori Y., Tanaka K., Mishina M., Numa S. Functional properties of nicotinic acetylcholine receptor subunits expressed in various combinations. FEBS Lett. 1987 Apr 20;214(2):253–258. doi: 10.1016/0014-5793(87)80065-0. [DOI] [PubMed] [Google Scholar]
  16. Lindstrom J., Gullick W., Conti-Tronconi B., Ellisman M. Proteolytic nicking of the acetylcholine receptor. Biochemistry. 1980 Oct 14;19(21):4791–4795. doi: 10.1021/bi00562a012. [DOI] [PubMed] [Google Scholar]
  17. Luther M. A., Schoepfer R., Whiting P., Casey B., Blatt Y., Montal M. S., Montal M., Linstrom J. A muscle acetylcholine receptor is expressed in the human cerebellar medulloblastoma cell line TE671. J Neurosci. 1989 Mar;9(3):1082–1096. doi: 10.1523/JNEUROSCI.09-03-01082.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. MacLennan C., Beeson D., Vincent A., Newsom-Davis J. Human nicotinic acetylcholine receptor alpha-subunit isoforms: origins and expression. Nucleic Acids Res. 1993 Nov 25;21(23):5463–5467. doi: 10.1093/nar/21.23.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Methfessel C., Witzemann V., Takahashi T., Mishina M., Numa S., Sakmann B. Patch clamp measurements on Xenopus laevis oocytes: currents through endogenous channels and implanted acetylcholine receptor and sodium channels. Pflugers Arch. 1986 Dec;407(6):577–588. doi: 10.1007/BF00582635. [DOI] [PubMed] [Google Scholar]
  20. Mishina M., Takai T., Imoto K., Noda M., Takahashi T., Numa S., Methfessel C., Sakmann B. Molecular distinction between fetal and adult forms of muscle acetylcholine receptor. Nature. 1986 May 22;321(6068):406–411. doi: 10.1038/321406a0. [DOI] [PubMed] [Google Scholar]
  21. Morris A., Beeson D., Jacobson L., Baggi F., Vincent A., Newsom-Davis J. Two isoforms of the muscle acetylcholine receptor alpha-subunit are translated in the human cell line TE671. FEBS Lett. 1991 Dec 16;295(1-3):116–118. doi: 10.1016/0014-5793(91)81399-s. [DOI] [PubMed] [Google Scholar]
  22. Sine S. M. Functional properties of human skeletal muscle acetylcholine receptors expressed by the TE671 cell line. J Biol Chem. 1988 Dec 5;263(34):18052–18062. [PubMed] [Google Scholar]
  23. Sumikawa K., Miledi R. Assembly and N-glycosylation of all ACh receptor subunits are required for their efficient insertion into plasma membranes. Brain Res Mol Brain Res. 1989 May;5(3):183–192. doi: 10.1016/0169-328x(89)90034-x. [DOI] [PubMed] [Google Scholar]
  24. Tzartos S. J., Barkas T., Cung M. T., Kordossi A., Loutrari H., Marraud M., Papadouli I., Sakarellos C., Sophianos D., Tsikaris V. The main immunogenic region of the acetylcholine receptor. Structure and role in myasthenia gravis. Autoimmunity. 1991;8(4):259–270. doi: 10.3109/08916939109007633. [DOI] [PubMed] [Google Scholar]
  25. Tzartos S., Langeberg L., Hochschwender S., Lindstrom J. Demonstration of a main immunogenic region on acetylcholine receptors from human muscle using monoclonal antibodies to human receptor. FEBS Lett. 1983 Jul 11;158(1):116–118. doi: 10.1016/0014-5793(83)80688-7. [DOI] [PubMed] [Google Scholar]
  26. Verrall S., Hall Z. W. The N-terminal domains of acetylcholine receptor subunits contain recognition signals for the initial steps of receptor assembly. Cell. 1992 Jan 10;68(1):23–31. doi: 10.1016/0092-8674(92)90203-o. [DOI] [PubMed] [Google Scholar]
  27. Villarroel A., Herlitze S., Witzemann V., Koenen M., Sakmann B. Asymmetry of the rat acetylcholine receptor subunits in the narrow region of the pore. Proc Biol Sci. 1992 Sep 22;249(1326):317–324. doi: 10.1098/rspb.1992.0121. [DOI] [PubMed] [Google Scholar]
  28. Whiting P. J., Vincent A., Schluep M., Newsom-Davis J. Monoclonal antibodies that distinguish between normal and denervated human acetylcholine receptor. J Neuroimmunol. 1986 May;11(3):223–235. doi: 10.1016/0165-5728(86)90006-8. [DOI] [PubMed] [Google Scholar]
  29. Witzemann V., Stein E., Barg B., Konno T., Koenen M., Kues W., Criado M., Hofmann M., Sakmann B. Primary structure and functional expression of the alpha-, beta-, gamma-, delta- and epsilon-subunits of the acetylcholine receptor from rat muscle. Eur J Biochem. 1990 Dec 12;194(2):437–448. doi: 10.1111/j.1432-1033.1990.tb15637.x. [DOI] [PubMed] [Google Scholar]
  30. Yu X. M., Hall Z. W. Extracellular domains mediating epsilon subunit interactions of muscle acetylcholine receptor. Nature. 1991 Jul 4;352(6330):64–67. doi: 10.1038/352064a0. [DOI] [PubMed] [Google Scholar]

Articles from The Journal of Physiology are provided here courtesy of The Physiological Society

RESOURCES