Skip to main content
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1979 Sep 1;82(3):626–643. doi: 10.1083/jcb.82.3.626

Dispersal and reformation of acetylcholine receptor clusters of cultured rat myotubes treated with inhibitors of energy metabolism

PMCID: PMC2110477  PMID: 511929

Abstract

The effects of energy metabolism inhibitors on the distribution of acetylcholine receptors (AChRs) in the surface membranes of non- innervated, cultured rat myotubes were studied by visualizing the AChRs with monotetramethylrhodamine-alpha-bungarotoxin. Incubation of myotubes with inhibitors of energy metabolism causes a large decrease in the fraction of myotubes displaying clusters of AChR. This decrease is reversible, and is dependent on temperature, the concentration of inhibitor, and the duration of treatment. Cluster dispersal is probably not the result of secondary effects on Ca++ or cyclic nucleotide metabolism, membrane potential, cytoskeletal elements, or protein synthesis. Sequential observations of identified cells treated with sodium azide showed that clusters appear to disperse by movements of receptors within the sarcolemma without accompanying changes in cell shape. AChR clusters dispersed by pretreating cells with sodium azide rapidly reform upon removal of the inhibitor. Reclustering involves the formation of small aggregates of AChR, which act as foci for further aggregation and which appear to be precursors of large AChR clusters. Small AChR aggregates also appear to be precursors of clusters which form on myotubes never exposed to azide. Reclustering after azide treatment does not necessarily occur at the same sites occupied by clusters before dispersal, nor does it employ only receptors which had previously been in clusters. Cluster reformation can be blocked by cycloheximide, colchicine, and drugs which alter the intracellular cation composition.

Full Text

The Full Text of this article is available as a PDF (1.5 MB).

Selected References

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

  1. Anderson M. J., Cohen M. W. Fluorescent staining of acetylcholine receptors in vertebrate skeletal muscle. J Physiol. 1974 Mar;237(2):385–400. doi: 10.1113/jphysiol.1974.sp010487. [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. Axelrod D., Ravdin P. M., Podleski T. R. Control of acetylcholine receptor mobility and distribution in cultured muscle membranes. A fluorescence study. Biochim Biophys Acta. 1978 Jul 20;511(1):23–38. doi: 10.1016/0005-2736(78)90062-7. [DOI] [PubMed] [Google Scholar]
  5. Axelrod D., Ravdin P., Koppel D. E., Schlessinger J., Webb W. W., Elson E. L., Podleski T. R. Lateral motion of fluorescently labeled acetylcholine receptors in membranes of developing muscle fibers. Proc Natl Acad Sci U S A. 1976 Dec;73(12):4594–4598. doi: 10.1073/pnas.73.12.4594. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. BRIERLEY G. P., MURER E., BACHMANN E. STUDIES ON ION TRANSPORT. III. THE ACCUMULATION OF CALCIUM AND INORGANIC PHOSPHATE BY HEART MITOCHONDRIA. Arch Biochem Biophys. 1964 Apr;105:89–102. doi: 10.1016/0003-9861(64)90239-5. [DOI] [PubMed] [Google Scholar]
  7. Barnard E. A., Wieckowski J., Chiu T. H. Cholinergic receptor molecules and cholinesterase molecules at mouse skeletal muscle junctions. Nature. 1971 Nov 26;234(5326):207–209. doi: 10.1038/234207a0. [DOI] [PubMed] [Google Scholar]
  8. Berg D. K., Hall Z. W. Fate of alpha-bungarotoxin bound to acetylcholine receptors of normal and denervated muscle. Science. 1974 Apr 26;184(4135):473–475. doi: 10.1126/science.184.4135.473. [DOI] [PubMed] [Google Scholar]
  9. Bevan S., Steinbach J. H. The distribution of alpha-bungarotoxin binding sites of mammalian skeletal muscle developing in vivo. J Physiol. 1977 May;267(1):195–213. doi: 10.1113/jphysiol.1977.sp011808. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. CHANCE B. THE ENERGY-LINKED REACTION OF CALCIUM WITH MITOCHONDRIA. J Biol Chem. 1965 Jun;240:2729–2748. [PubMed] [Google Scholar]
  11. DIAMOND J., MILEDI R. A study of foetal and new-born rat muscle fibres. J Physiol. 1962 Aug;162:393–408. doi: 10.1113/jphysiol.1962.sp006941. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. De Petris S. Inhibition and reversal of capping by cytochalasin B, vinblastine and colchicine. Nature. 1974 Jul 5;250(461):54–56. doi: 10.1038/250054a0. [DOI] [PubMed] [Google Scholar]
  13. Devreotes P. N., Fambrough D. M. Acetylcholine receptor turnover in membranes of developing muscle fibers. J Cell Biol. 1975 May;65(2):335–358. doi: 10.1083/jcb.65.2.335. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Earp H. S., Utsinger P. D., Yount W. J., Logue M., Steiner A. L. Lymphocyte surface modulation and cyclic nucleotides I. Topographic correlation of cyclic adenosine 3':5'-monophosphate and immunoglobulin immunofluorescence during lymphocyte capping. J Exp Med. 1977 Apr 1;145(4):1087–1092. doi: 10.1084/jem.145.4.1087. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Fambrough D. M., Hartzell H. C. Acetylcholine receptors: number and distribution at neuromuscular junctions in rat diaphragm. Science. 1972 Apr 14;176(4031):189–191. doi: 10.1126/science.176.4031.189. [DOI] [PubMed] [Google Scholar]
  16. Fertuck H. C., Salpeter M. M. Quantitation of junctional and extrajunctional acetylcholine receptors by electron microscope autoradiography after 125I-alpha-bungarotoxin binding at mouse neuromuscular junctions. J Cell Biol. 1976 Apr;69(1):144–158. doi: 10.1083/jcb.69.1.144. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Fischbach G. D., Cohen S. A. The distribution of acetylcholine sensitivity over uninnervated and innervated muscle fibers grown in cell culture. Dev Biol. 1973 Mar;31(1):147–162. doi: 10.1016/0012-1606(73)90326-6. [DOI] [PubMed] [Google Scholar]
  18. Fischbach G. D. Synapse formation between dissociated nerve and muscle cells in low density cell cultures. Dev Biol. 1972 Jun;28(2):407–429. doi: 10.1016/0012-1606(72)90023-1. [DOI] [PubMed] [Google Scholar]
  19. HEYTLER P. G. uncoupling of oxidative phosphorylation by carbonyl cyanide phenylhydrazones. I. Some characteristics of m-Cl-CCP action on mitochondria and chloroplasts. Biochemistry. 1963 Mar-Apr;2:357–361. doi: 10.1021/bi00902a031. [DOI] [PubMed] [Google Scholar]
  20. Hartzell H. C., Fambrough D. M. Acetycholine receptor production and incorporation into membranes of developing muscle fibers. Dev Biol. 1973 Jan;30(1):153–165. doi: 10.1016/0012-1606(73)90054-7. [DOI] [PubMed] [Google Scholar]
  21. Heinemann S., Bevan S., Kullberg R., Lindstrom J., Rice J. Modulation of acetylcholine receptor by antibody against the receptor. Proc Natl Acad Sci U S A. 1977 Jul;74(7):3090–3094. doi: 10.1073/pnas.74.7.3090. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Ko P. K., Anderson M. J., Cohen M. W. Denervated skeletal muscle fibers develop discrete patches of high acetylcholine receptor density. Science. 1977 Apr 29;196(4289):540–542. doi: 10.1126/science.850796. [DOI] [PubMed] [Google Scholar]
  23. LARDY H. A., JOHNSON D., McMURRAY W. C. Antibiotics as tools for metabolic studies. I. A survey of toxic antibiotics in respiratory, phosphorylative and glycolytic systems. Arch Biochem Biophys. 1958 Dec;78(2):587–597. doi: 10.1016/0003-9861(58)90383-7. [DOI] [PubMed] [Google Scholar]
  24. LEHNINGER A. L., ROSSI C. S., GREENAWALT J. W. Respiration-dependent accumulation of inorganic phosphate and Ca ions by rat liver mitochondria. Biochem Biophys Res Commun. 1963 Mar 25;10:444–448. doi: 10.1016/0006-291x(63)90377-2. [DOI] [PubMed] [Google Scholar]
  25. LOOMIS W. F., LIPMANN F. Inhibition of phosphorylation by azide in kidney homogenate. J Biol Chem. 1949 May;179(1):503–503. [PubMed] [Google Scholar]
  26. Land B. R., Podleski T. R., Salpeter E. E., Salpeter M. M. Acetylcholine receptor distribution on myotubes in culture correlated to acetylcholine sensitivity. J Physiol. 1977 Jul;269(1):155–176. doi: 10.1113/jphysiol.1977.sp011897. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Loor F., Forni L., Pernis B. The dynamic state of the lymphocyte membrane. Factors affecting the distribution and turnover of surface immunoglobulins. Eur J Immunol. 1972 Jun;2(3):203–212. doi: 10.1002/eji.1830020304. [DOI] [PubMed] [Google Scholar]
  28. Patrick J., McMillan J., Wolfson H., O'Brien J. C. Acetylcholine receptor metabolism in a nonfusing muscle cell line. J Biol Chem. 1977 Mar 25;252(6):2143–2153. [PubMed] [Google Scholar]
  29. Porter C. W., Barnard E. A. The density of cholinergic receptors at the endplate postsynaptic membrane: ultrastructural studies in two mammalian species. J Membr Biol. 1975;20(1-2):31–49. doi: 10.1007/BF01870626. [DOI] [PubMed] [Google Scholar]
  30. Schreiner G. F., Fujiwara K., Pollard T. D., Unanue E. R. Redistribution of myosin accompanying capping of surface Ig. J Exp Med. 1977 May 1;145(5):1393–1398. doi: 10.1084/jem.145.5.1393. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Schreiner G. F., Unanue E. R. Calcium-sensitive modulation of Ig capping: evidence supporting a cytoplasmic control of ligand-receptor complexes. J Exp Med. 1976 Jan 1;143(1):15–31. doi: 10.1084/jem.143.1.15. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Schreiner G. F., Unanue E. R. Membrane and cytoplasmic changes in B lymphocytes induced by ligand-surface immunoglobulin interaction. Adv Immunol. 1976;24:37–165. doi: 10.1016/s0065-2776(08)60329-6. [DOI] [PubMed] [Google Scholar]
  33. Stallcup W. B., Cohn M. Electrical properties of a clonal cell line as determined by measurement of ion fluxes. Exp Cell Res. 1976 Mar 15;98(2):277–284. doi: 10.1016/0014-4827(76)90439-0. [DOI] [PubMed] [Google Scholar]
  34. Sytkowski A. J., Vogel Z., Nirenberg M. W. Development of acetylcholine receptor clusters on cultured muscle cells. Proc Natl Acad Sci U S A. 1973 Jan;70(1):270–274. doi: 10.1073/pnas.70.1.270. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Sällström J. F., Alm G. V. Binding of concanavalin A to thymic and bursal chicken lymphoid cells. Exp Cell Res. 1972 Nov;75(1):63–72. doi: 10.1016/0014-4827(72)90520-4. [DOI] [PubMed] [Google Scholar]
  36. Vlodavsky I., Sachs L. Difference in the calcium regulation of concanavalin A agglutinability and surface microvilli in normal and transformed cells. Relationship to membrane--cytoskeleton interaction. Exp Cell Res. 1977 Mar 1;105(1):179–189. doi: 10.1016/0014-4827(77)90164-1. [DOI] [PubMed] [Google Scholar]
  37. Vogel Z., Sytkowski A. J., Nirenberg M. W. Acetylcholine receptors of muscle grown in vitro. Proc Natl Acad Sci U S A. 1972 Nov;69(11):3180–3184. doi: 10.1073/pnas.69.11.3180. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Yahara I., Edelman G. M. Modulation of lymphocyte receptor redistribution by concanavalin A, anti-mitotic agents and alterations of pH. Nature. 1973 Nov 16;246(5429):152–155. doi: 10.1038/246152a0. [DOI] [PubMed] [Google Scholar]
  39. de Petris S. Concanavalin A receptors, immunoglobulins, and theta antigen of the lymphocyte surface. Interactions with concanavalin A and with Cytoplasmic structures. J Cell Biol. 1975 Apr;65(1):123–146. doi: 10.1083/jcb.65.1.123. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from The Journal of Cell Biology are provided here courtesy of The Rockefeller University Press

RESOURCES