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. 1984 Feb 1;98(2):550–557. doi: 10.1083/jcb.98.2.550

Participation of calcium and calmodulin in the formation of acetylcholine receptor clusters

PMCID: PMC2113098  PMID: 6363424

Abstract

The formation of acetylcholine receptor (AChR) clusters can be experimentally induced in cultured Xenopus myotomal muscle cells by positive polypeptide-coated latex beads (Peng, H.B., P.-C. Cheng, and P.W. Luther, 1981, Nature [Lond.], 292:831-834). This provides a simple procedure for studying the cellular process of AChR clustering. In this study, the involvement of calcium and calmodulin in this process was examined. A deprivation in extracellular calcium by calcium-free medium or by the addition of calcium antagonists such as divalent cations Co2+ and Ni2+ (1-5 mM) or organic compounds verapamil and D-600 (0.1-0.5 mM) suppressed the formation of AChR clusters induced by the latex beads in a largely reversible manner. Antagonists against calmodulin, including trifluoperazine (1-5 microM) and the naphthalene sulfonamide W-7 (20 microM), also suppressed AChR clustering. However, the effect of W-7 was much weaker than that of trifluoperazine (TFP). Although the formation of AChR clusters is inhibited by these drugs, the stability of the existent clusters is relatively insensitive to them. These data suggest that the clustering of AChR involves a Ca2+ and calmodulin- activated process. Immunofluorescence studies using an antibody against calmodulin indicate that calmodulin is diffusely distributed in the cytoplasm in addition to its localization at the I-bands. Thus I propose that a local rise in intracellular calcium caused by a locally applied stimulus, exemplified here by the polypeptide-coated latex beads, may trigger the formation of AChR clusters. Furthermore, the cellular machinery for this process may involve calmodulin and is diffusely distributed in the muscle cell.

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Selected References

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  1. Almers W., Palade P. T. Slow calcium and potassium currents across frog muscle membrane: measurements with a vaseline-gap technique. J Physiol. 1981 Mar;312:159–176. doi: 10.1113/jphysiol.1981.sp013622. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Anderson M. J., Cohen M. W. Nerve-induced and spontaneous redistribution of acetylcholine receptors on cultured muscle cells. J Physiol. 1977 Jul;268(3):757–773. doi: 10.1113/jphysiol.1977.sp011880. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Anderson M. J., Cohen M. W., Zorychta E. Effects of innervation on the distribution of acetylcholine receptors on cultured muscle cells. J Physiol. 1977 Jul;268(3):731–756. doi: 10.1113/jphysiol.1977.sp011879. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bloch R. J. Dispersal and reformation of acetylcholine receptor clusters of cultured rat myotubes treated with inhibitors of energy metabolism. J Cell Biol. 1979 Sep;82(3):626–643. doi: 10.1083/jcb.82.3.626. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Brehm P., Yeh E., Patrick J., Kidokoro Y. Metabolism of acetylcholine receptors on embryonic amphibian muscle. J Neurosci. 1983 Jan;3(1):101–107. doi: 10.1523/JNEUROSCI.03-01-00101.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Cheung W. Y. Calmodulin plays a pivotal role in cellular regulation. Science. 1980 Jan 4;207(4426):19–27. doi: 10.1126/science.6243188. [DOI] [PubMed] [Google Scholar]
  7. Cohen M. W., Weldon P. R. Localization of acetylcholine receptors and synaptic ultrastructure at nerve-muscle contacts in culture: dependence on nerve type. J Cell Biol. 1980 Aug;86(2):388–401. doi: 10.1083/jcb.86.2.388. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Douglas W. W., Nemeth E. F. On the calcium receptor activating exocytosis: inhibitory effects of calmodulin-interacting drugs on rat mast cells. J Physiol. 1982 Feb;323:229–244. doi: 10.1113/jphysiol.1982.sp014070. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Eisenberg R. S., McCarthy R. T., Milton R. L. Paralysis of frog skeletal muscle fibres by the calcium antagonist D-600. J Physiol. 1983 Aug;341:495–505. doi: 10.1113/jphysiol.1983.sp014819. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Fambrough D., Hartzell H. C., Rash J. E., Ritchie A. K. Trophic functions of the neuron. I. Development of neural connections. Receptor properties of developing muscle. Ann N Y Acad Sci. 1974 Mar 22;228(0):47–62. doi: 10.1111/j.1749-6632.1974.tb20501.x. [DOI] [PubMed] [Google Scholar]
  11. Fleckenstein A. Specific pharmacology of calcium in myocardium, cardiac pacemakers, and vascular smooth muscle. Annu Rev Pharmacol Toxicol. 1977;17:149–166. doi: 10.1146/annurev.pa.17.040177.001053. [DOI] [PubMed] [Google Scholar]
  12. Frank E., Fischbach G. D. Early events in neuromuscular junction formation in vitro: induction of acetylcholine receptor clusters in the postsynaptic membrane and morphology of newly formed synapses. J Cell Biol. 1979 Oct;83(1):143–158. doi: 10.1083/jcb.83.1.143. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Gingell D. Contractile responses at the surface of an amphibian egg. J Embryol Exp Morphol. 1970 Jun;23(3):583–609. [PubMed] [Google Scholar]
  14. Gingell D., Palmer J. F. Changes in membrane impedance associated with a cortical contraction in the egg of Xenopus laevis. Nature. 1968 Jan 6;217(5123):98–102. doi: 10.1038/217098a0. [DOI] [PubMed] [Google Scholar]
  15. Guerriero V., Jr, Rowley D. R., Means A. R. Production and characterization of an antibody to myosin light chain kinase and intracellular localization of the enzyme. Cell. 1981 Dec;27(3 Pt 2):449–458. doi: 10.1016/0092-8674(81)90386-x. [DOI] [PubMed] [Google Scholar]
  16. Hagiwara S., Byerly L. Calcium channel. Annu Rev Neurosci. 1981;4:69–125. doi: 10.1146/annurev.ne.04.030181.000441. [DOI] [PubMed] [Google Scholar]
  17. Hall Z. W., Lubit B. W., Schwartz J. H. Cytoplasmic actin in postsynaptic structures at the neuromuscular junction. J Cell Biol. 1981 Sep;90(3):789–792. doi: 10.1083/jcb.90.3.789. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Heuser J. E., Salpeter S. R. Organization of acetylcholine receptors in quick-frozen, deep-etched, and rotary-replicated Torpedo postsynaptic membrane. J Cell Biol. 1979 Jul;82(1):150–173. doi: 10.1083/jcb.82.1.150. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Hidaka H., Sasaki Y., Tanaka T., Endo T., Ohno S., Fujii Y., Nagata T. N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide, a calmodulin antagonist, inhibits cell proliferation. Proc Natl Acad Sci U S A. 1981 Jul;78(7):4354–4357. doi: 10.1073/pnas.78.7.4354. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Horwitz S. B., Chia G. H., Harracksingh C., Orlow S., Pifko-Hirst S., Schneck J., Sorbara L., Speaker M., Wilk E. W., Rosen O. M. Trifluoperazine inhibits phagocytosis in a macrophagelike cultured cell line. J Cell Biol. 1981 Dec;91(3 Pt 1):798–802. doi: 10.1083/jcb.91.3.798. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Kidokoro Y., Anderson M. J., Gruener R. Changes in synaptic potential properties during acetylcholine receptor accumulation and neurospecific interactions in Xenopus nerve-muscle cell culture. Dev Biol. 1980 Aug;78(2):464–483. doi: 10.1016/0012-1606(80)90347-4. [DOI] [PubMed] [Google Scholar]
  22. Kidokoro Y., Heinemann S., Schubert D., Brandt B. L., Klier F. G. Synapse formation and neurotrophic effects on muscle cell lines. Cold Spring Harb Symp Quant Biol. 1976;40:373–388. doi: 10.1101/sqb.1976.040.01.036. [DOI] [PubMed] [Google Scholar]
  23. McManaman J. L., Blosser J. C., Appel S. H. The effect of calcium on acetylcholine receptor synthesis. J Neurosci. 1981 Jul;1(7):771–776. doi: 10.1523/JNEUROSCI.01-07-00771.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Means A. R., Dedman J. R. Calmodulin--an intracellular calcium receptor. Nature. 1980 May 8;285(5760):73–77. doi: 10.1038/285073a0. [DOI] [PubMed] [Google Scholar]
  25. Meijer L., Guerrier P. Calmodulin in starfish oocytes. I. Calmodulin antagonists inhibit meiosis reinitiation. Dev Biol. 1981 Dec;88(2):318–324. doi: 10.1016/0012-1606(81)90175-5. [DOI] [PubMed] [Google Scholar]
  26. Nelson G. A., Andrews M. L., Karnovsky M. J. Participation of calmodulin in immunoglobulin capping. J Cell Biol. 1982 Dec;95(3):771–780. doi: 10.1083/jcb.95.3.771. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Obata K. Development of neuromuscular transmission in culture with a variety of neurons and in the presence of cholinergic substances and tetrodotoxin. Brain Res. 1977 Jan 1;119(1):141–153. doi: 10.1016/0006-8993(77)90096-8. [DOI] [PubMed] [Google Scholar]
  28. Peng H. B., Cheng P. C. Formation of postsynaptic specializations induced by latex beads in cultured muscle cells. J Neurosci. 1982 Dec;2(12):1760–1774. doi: 10.1523/JNEUROSCI.02-12-01760.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Peng H. B., Cheng P. C., Luther P. W. Formation of ACh receptor clusters induced by positively charged latex beads. Nature. 1981 Aug 27;292(5826):831–834. doi: 10.1038/292831a0. [DOI] [PubMed] [Google Scholar]
  30. Peng H. B. Cytoskeletal organization of the presynaptic nerve terminal and the acetylcholine receptor cluster in cell cultures. J Cell Biol. 1983 Aug;97(2):489–498. doi: 10.1083/jcb.97.2.489. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Peng H. B., Nakajima Y., Bridgman P. C. Development of the postsynaptic membrane in Xenopus neuromuscular cultures observed by freeze-fracture and thin-section electron microscopy. Brain Res. 1980 Aug 25;196(1):11–31. doi: 10.1016/0006-8993(80)90713-1. [DOI] [PubMed] [Google Scholar]
  32. Peng H. B., Nakajima Y. Membrane particle aggregates in innervated and noninnervated cultures of Xenopus embryonic muscle cells. Proc Natl Acad Sci U S A. 1978 Jan;75(1):500–504. doi: 10.1073/pnas.75.1.500. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Poo M. Rapid lateral diffusion of functional A Ch receptors in embryonic muscle cell membrane. Nature. 1982 Jan 28;295(5847):332–334. doi: 10.1038/295332a0. [DOI] [PubMed] [Google Scholar]
  34. Prives J., Fulton A. B., Penman S., Daniels M. P., Christian C. N. Interaction of the cytoskeletal framework with acetylcholine receptor on th surface of embryonic muscle cells in culture. J Cell Biol. 1982 Jan;92(1):231–236. doi: 10.1083/jcb.92.1.231. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Puro D. G., De Mello F. G., Nirenberg M. Synapse turnover: the formation and termination of transient synapses. Proc Natl Acad Sci U S A. 1977 Nov;74(11):4977–4981. doi: 10.1073/pnas.74.11.4977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Ravdin P., Axelrod D. Fluorescent tetramethyl rhodamine derivatives of alpha-bungarotoxin: preparation, separation, and characterization. Anal Biochem. 1977 Jun;80(2):585–592. doi: 10.1016/0003-2697(77)90682-0. [DOI] [PubMed] [Google Scholar]
  37. Salisbury J. L., Condeelis J. S., Satir P. Role of coated vesicles, microfilaments, and calmodulin in receptor-mediated endocytosis by cultured B lymphoblastoid cells. J Cell Biol. 1980 Oct;87(1):132–141. doi: 10.1083/jcb.87.1.132. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Weeds A. Actin-binding proteins--regulators of cell architecture and motility. Nature. 1982 Apr 29;296(5860):811–816. doi: 10.1038/296811a0. [DOI] [PubMed] [Google Scholar]
  39. Weiss B., Prozialeck W., Cimino M., Barnette M. S., Wallace T. L. Pharmacological regulation of calmodulin. Ann N Y Acad Sci. 1980;356:319–345. doi: 10.1111/j.1749-6632.1980.tb29621.x. [DOI] [PubMed] [Google Scholar]

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