Skip to main content
PMC home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1991 Jul 1;114(1):125–141. doi: 10.1083/jcb.114.1.125

Neural factors regulate AChR subunit mRNAs at rat neuromuscular synapses

PMCID: PMC2289058  PMID: 1646821

Abstract

To elucidate the nature of signals that control the level and spatial distribution of mRNAs encoding acetylcholine receptor (AChR), alpha-, beta-, gamma-, delta- and epsilon-subunits in muscle fibers chronic paralysis was induced in rat leg muscles either by surgical denervation or by different neurotoxins that cause disuse of the muscle or selectively block neuromuscular transmission pre- or postsynaptically and cause an increase of AChRs in muscle membrane. After paralysis, the levels and the spatial distributions of the different subunit-specific mRNAs change discoordinately and seem to follow one of three different patterns depending on the subunit mRNA examined. The level of epsilon- subunit mRNA and its accumulation at the end-plate are largely independent on the presence of the nerve or electrical muscle activity. In contrast, the gamma-subunit mRNA level is tightly coupled to innervation. It is undetectable or low in innervated normally active muscle and in innervated but disused muscle, whereas it is abundant along the whole fiber length in denervated muscle or in muscle in which the neuromuscular contact is intact but the release of transmitter is blocked. The alpha-, beta-, and delta-subunit mRNA levels show a different pattern. Highest amounts are always found at end-plate nuclei irrespective of whether the muscle is innervated, denervated, active, or inactive, whereas in extrasynaptic regions they are tightly controlled by innervation partially through electrical muscle activity. The changes in the levels and distribution of gamma- and epsilon- subunit-specific mRNAs in toxin-paralyzed muscle correlate well with the spatial appearance of functional fetal and adult AChR channel subtypes along the muscle fiber. The results suggest that the focal accumulation at the synaptic region of mRNAs encoding the alpha-, beta- , delta-, and epsilon-subunits, which constitute the adult type end- plate channel, is largely determined by at least two different neural factors that act on AChR subunit gene expression of subsynaptic nuclei.

Full Text

The Full Text of this article is available as a PDF (2.4 MB).

Selected References

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

  1. Baldwin T. J., Yoshihara C. M., Blackmer K., Kintner C. R., Burden S. J. Regulation of acetylcholine receptor transcript expression during development in Xenopus laevis. J Cell Biol. 1988 Feb;106(2):469–478. doi: 10.1083/jcb.106.2.469. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bevan S., Steinbach J. H. Denervation increases the degradation rate of acetylcholine receptors at end-plates in vivo and in vitro. J Physiol. 1983 Mar;336:159–177. doi: 10.1113/jphysiol.1983.sp014574. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bray J. J., Hubbard J. I., Mills R. G. The trophic influence of tetrodotoxin-inactive nerves on normal and reinnervated rat skeletal muscles. J Physiol. 1979 Dec;297(0):479–491. doi: 10.1113/jphysiol.1979.sp013052. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Brehm P., Henderson L. Regulation of acetylcholine receptor channel function during development of skeletal muscle. Dev Biol. 1988 Sep;129(1):1–11. doi: 10.1016/0012-1606(88)90156-x. [DOI] [PubMed] [Google Scholar]
  5. Brenner H. R., Lømo T., Williamson R. Control of end-plate channel properties by neurotrophic effects and by muscle activity in rat. J Physiol. 1987 Jul;388:367–381. doi: 10.1113/jphysiol.1987.sp016619. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Brenner H. R., Rudin W. On the effect of muscle activity on the end-plate membrane in denervated mouse muscle. J Physiol. 1989 Mar;410:501–512. doi: 10.1113/jphysiol.1989.sp017546. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Brenner H. R., Witzemann V., Sakmann B. Imprinting of acetylcholine receptor messenger RNA accumulation in mammalian neuromuscular synapses. Nature. 1990 Apr 5;344(6266):544–547. doi: 10.1038/344544a0. [DOI] [PubMed] [Google Scholar]
  8. Cangiano A. Denervation supersensitivity as a model for the neural control of muscle. Neuroscience. 1985 Apr;14(4):963–971. doi: 10.1016/0306-4522(85)90268-4. [DOI] [PubMed] [Google Scholar]
  9. Chapron J., Koenig J. In vitro synaptic maturation. Neurosci Lett. 1989 Nov 20;106(1-2):19–22. doi: 10.1016/0304-3940(89)90195-x. [DOI] [PubMed] [Google Scholar]
  10. Cull-Candy S. G., Lundh H., Thesleff S. Effects of botulinum toxin on neuromuscular transmission in the rat. J Physiol. 1976 Aug;260(1):177–203. doi: 10.1113/jphysiol.1976.sp011510. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Drachman D. B., Stanley E. F., Pestronk A., Griffin J. W., Price D. L. Neurotrophic regulation of two properties of skeletal muscle by impulse-dependent and spontaneous acetylcholine transmission. J Neurosci. 1982 Feb;2(2):232–243. doi: 10.1523/JNEUROSCI.02-02-00232.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Feinberg A. P., Vogelstein B. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem. 1983 Jul 1;132(1):6–13. doi: 10.1016/0003-2697(83)90418-9. [DOI] [PubMed] [Google Scholar]
  13. Fontaine B., Changeux J. P. Localization of nicotinic acetylcholine receptor alpha-subunit transcripts during myogenesis and motor endplate development in the chick. J Cell Biol. 1989 Mar;108(3):1025–1037. doi: 10.1083/jcb.108.3.1025. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Fontaine B., Klarsfeld A., Changeux J. P. Calcitonin gene-related peptide and muscle activity regulate acetylcholine receptor alpha-subunit mRNA levels by distinct intracellular pathways. J Cell Biol. 1987 Sep;105(3):1337–1342. doi: 10.1083/jcb.105.3.1337. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Fontaine B., Sassoon D., Buckingham M., Changeux J. P. Detection of the nicotinic acetylcholine receptor alpha-subunit mRNA by in situ hybridization at neuromuscular junctions of 15-day-old chick striated muscles. EMBO J. 1988 Mar;7(3):603–609. doi: 10.1002/j.1460-2075.1988.tb02853.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Goldman D., Brenner H. R., Heinemann S. Acetylcholine receptor alpha-, beta-, gamma-, and delta-subunit mRNA levels are regulated by muscle activity. Neuron. 1988 Jun;1(4):329–333. doi: 10.1016/0896-6273(88)90081-5. [DOI] [PubMed] [Google Scholar]
  17. Goldman D., Staple J. Spatial and temporal expression of acetylcholine receptor RNAs in innervated and denervated rat soleus muscle. Neuron. 1989 Aug;3(2):219–228. doi: 10.1016/0896-6273(89)90035-4. [DOI] [PubMed] [Google Scholar]
  18. Gu Y., Hall Z. W. Immunological evidence for a change in subunits of the acetylcholine receptor in developing and denervated rat muscle. Neuron. 1988 Apr;1(2):117–125. doi: 10.1016/0896-6273(88)90195-x. [DOI] [PubMed] [Google Scholar]
  19. Habermann E., Dreyer F. Clostridial neurotoxins: handling and action at the cellular and molecular level. Curr Top Microbiol Immunol. 1986;129:93–179. doi: 10.1007/978-3-642-71399-6_2. [DOI] [PubMed] [Google Scholar]
  20. Hamill O. P., Marty A., Neher E., Sakmann B., Sigworth F. J. Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches. Pflugers Arch. 1981 Aug;391(2):85–100. doi: 10.1007/BF00656997. [DOI] [PubMed] [Google Scholar]
  21. Harris D. A., Falls D. L., Dill-Devor R. M., Fischbach G. D. Acetylcholine receptor-inducing factor from chicken brain increases the level of mRNA encoding the receptor alpha subunit. Proc Natl Acad Sci U S A. 1988 Mar;85(6):1983–1987. doi: 10.1073/pnas.85.6.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Harris D. A., Falls D. L., Fischbach G. D. Differential activation of myotube nuclei following exposure to an acetylcholine receptor-inducing factor. Nature. 1989 Jan 12;337(6203):173–176. doi: 10.1038/337173a0. [DOI] [PubMed] [Google Scholar]
  23. Horovitz O., Knaack D., Podleski T. R., Salpeter M. M. Acetylcholine receptor alpha-subunit mRNA is increased by ascorbic acid in cloned L5 muscle cells: Northern blot analysis and in situ hybridization. J Cell Biol. 1989 May;108(5):1823–1832. doi: 10.1083/jcb.108.5.1823. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Häggblad J., Heilbronn E. Externally applied adenosine-5'-triphosphate causes inositol triphosphate accumulation in cultured chick myotubes. Neurosci Lett. 1987 Feb 24;74(2):199–204. doi: 10.1016/0304-3940(87)90149-2. [DOI] [PubMed] [Google Scholar]
  25. Jackson M. B., Imoto K., Mishina M., Konno T., Numa S., Sakmann B. Spontaneous and agonist-induced openings of an acetylcholine receptor channel composed of bovine muscle alpha-, beta- and delta-subunits. Pflugers Arch. 1990 Oct;417(2):129–135. doi: 10.1007/BF00370689. [DOI] [PubMed] [Google Scholar]
  26. Kao I., Drachman D. B., Price D. L. Botulinum toxin: mechanism of presynaptic blockade. Science. 1976 Sep 24;193(4259):1256–1258. doi: 10.1126/science.785600. [DOI] [PubMed] [Google Scholar]
  27. Klarsfeld A., Laufer R., Fontaine B., Devillers-Thiéry A., Dubreuil C., Changeux J. P. Regulation of muscle AChR alpha subunit gene expression by electrical activity: involvement of protein kinase C and Ca2+. Neuron. 1989 Mar;2(3):1229–1236. doi: 10.1016/0896-6273(89)90307-3. [DOI] [PubMed] [Google Scholar]
  28. Kullberg R., Owens J. L., Vickers J. Development of synaptic currents in immobilized muscle of Xenopus laevis. J Physiol. 1985 Jul;364:57–68. doi: 10.1113/jphysiol.1985.sp015729. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Laufer R., Changeux J. P. Activity-dependent regulation of gene expression in muscle and neuronal cells. Mol Neurobiol. 1989 Spring-Summer;3(1-2):1–53. doi: 10.1007/BF02935587. [DOI] [PubMed] [Google Scholar]
  30. Lipsky N. G., Drachman D. B., Pestronk A., Shih P. J. Neural regulation of mRNA for the alpha-subunit of acetylcholine receptors: role of neuromuscular transmission. Exp Neurol. 1989 Aug;105(2):171–176. doi: 10.1016/0014-4886(89)90116-7. [DOI] [PubMed] [Google Scholar]
  31. Lomo T., Westgaard R. H. Further studies on the control of ACh sensitivity by muscle activity in the rat. J Physiol. 1975 Nov;252(3):603–626. doi: 10.1113/jphysiol.1975.sp011161. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Lømo T., Massoulié J., Vigny M. Stimulation of denervated rat soleus muscle with fast and slow activity patterns induces different expression of acetylcholinesterase molecular forms. J Neurosci. 1985 May;5(5):1180–1187. doi: 10.1523/JNEUROSCI.05-05-01180.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Matteoli M., Haimann C., Torri-Tarelli F., Polak J. M., Ceccarelli B., De Camilli P. Differential effect of alpha-latrotoxin on exocytosis from small synaptic vesicles and from large dense-core vesicles containing calcitonin gene-related peptide at the frog neuromuscular junction. Proc Natl Acad Sci U S A. 1988 Oct;85(19):7366–7370. doi: 10.1073/pnas.85.19.7366. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Melton D. A., Krieg P. A., Rebagliati M. R., Maniatis T., Zinn K., Green M. R. Efficient in vitro synthesis of biologically active RNA and RNA hybridization probes from plasmids containing a bacteriophage SP6 promoter. Nucleic Acids Res. 1984 Sep 25;12(18):7035–7056. doi: 10.1093/nar/12.18.7035. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Merlie J. P., Sanes J. R. Concentration of acetylcholine receptor mRNA in synaptic regions of adult muscle fibres. Nature. 1985 Sep 5;317(6032):66–68. doi: 10.1038/317066a0. [DOI] [PubMed] [Google Scholar]
  36. 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]
  37. Miledi R., Parker I., Schalow G. Transmitter induced calcium entry across the post-synaptic membrane at frog end-plates measured using arsenazo III. J Physiol. 1980 Mar;300:197–212. doi: 10.1113/jphysiol.1980.sp013158. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. 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]
  39. New H. V., Mudge A. W. Calcitonin gene-related peptide regulates muscle acetylcholine receptor synthesis. 1986 Oct 30-Nov 5Nature. 323(6091):809–811. doi: 10.1038/323809a0. [DOI] [PubMed] [Google Scholar]
  40. Nitkin R. M., Smith M. A., Magill C., Fallon J. R., Yao Y. M., Wallace B. G., McMahan U. J. Identification of agrin, a synaptic organizing protein from Torpedo electric organ. J Cell Biol. 1987 Dec;105(6 Pt 1):2471–2478. doi: 10.1083/jcb.105.6.2471. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Pennefather P., Quastel D. M. Relation between subsynaptic receptor blockade and response to quantal transmitter at the mouse neuromuscular junction. J Gen Physiol. 1981 Sep;78(3):313–344. doi: 10.1085/jgp.78.3.313. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Pestronk A., Drachman D. B., Griffin J. W. Effect of botulinum toxin on trophic regulation of acetylcholine receptors. Nature. 1976 Dec 23;264(5588):787–789. doi: 10.1038/264787a0. [DOI] [PubMed] [Google Scholar]
  43. Sassone-Corsi P., Lamph W. W., Verma I. M. Regulation of proto-oncogene fos: a paradigm for early response genes. Cold Spring Harb Symp Quant Biol. 1988;53(Pt 2):749–760. doi: 10.1101/sqb.1988.053.01.085. [DOI] [PubMed] [Google Scholar]
  44. Schmidt J., Raftery M. A. A simple assay for the study of solubilized acetylcholine receptors. Anal Biochem. 1973 Apr;52(2):349–354. doi: 10.1016/0003-2697(73)90036-5. [DOI] [PubMed] [Google Scholar]
  45. Schuetze S. M., Role L. W. Developmental regulation of nicotinic acetylcholine receptors. Annu Rev Neurosci. 1987;10:403–457. doi: 10.1146/annurev.ne.10.030187.002155. [DOI] [PubMed] [Google Scholar]
  46. Sellin L. C., Thesleff S. Pre- and post-synaptic actions of botulinum toxin at the rat neuromuscular junction. J Physiol. 1981 Aug;317:487–495. doi: 10.1113/jphysiol.1981.sp013838. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Shainberg A., Burstein M. Decrease of acetylcholine receptor synthesis in muscle cultures by electrical stimulation. Nature. 1976 Nov 25;264(5584):368–369. doi: 10.1038/264368a0. [DOI] [PubMed] [Google Scholar]
  48. Shieh B. H., Ballivet M., Schmidt J. Acetylcholine receptor synthesis rate and levels of receptor subunit messenger RNAs in chick muscle. Neuroscience. 1988 Jan;24(1):175–187. doi: 10.1016/0306-4522(88)90321-1. [DOI] [PubMed] [Google Scholar]
  49. Stadler H., Kiene M. L. Synaptic vesicles in electromotoneurones. II. Heterogeneity of populations is expressed in uptake properties; exocytosis and insertion of a core proteoglycan into the extracellular matrix. EMBO J. 1987 Aug;6(8):2217–2221. doi: 10.1002/j.1460-2075.1987.tb02493.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Stanley E. F., Drachman D. B. Botulinum toxin blocks quantal but not non-quantal release of ACh at the neuromuscular junction. Brain Res. 1983 Feb 14;261(1):172–175. doi: 10.1016/0006-8993(83)91300-8. [DOI] [PubMed] [Google Scholar]
  51. THESLEFF S. Supersensitivity of skeletal muscle produced by botulinum toxin. J Physiol. 1960 Jun;151:598–607. doi: 10.1113/jphysiol.1960.sp006463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Thesleff S. Botulinal neurotoxins as tools in studies of synaptic mechanisms. Q J Exp Physiol. 1989 Dec;74(7):1003–1017. doi: 10.1113/expphysiol.1989.sp003329. [DOI] [PubMed] [Google Scholar]
  53. Thomas P. S. Hybridization of denatured RNA and small DNA fragments transferred to nitrocellulose. Proc Natl Acad Sci U S A. 1980 Sep;77(9):5201–5205. doi: 10.1073/pnas.77.9.5201. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Tsujimoto T., Kuno M. Calcitonin gene-related peptide prevents disuse-induced sprouting of rat motor nerve terminals. J Neurosci. 1988 Oct;8(10):3951–3957. doi: 10.1523/JNEUROSCI.08-10-03951.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Volknandt W., Zimmermann H. Acetylcholine, ATP, and proteoglycan are common to synaptic vesicles isolated from the electric organs of electric eel and electric catfish as well as from rat diaphragm. J Neurochem. 1986 Nov;47(5):1449–1462. doi: 10.1111/j.1471-4159.1986.tb00778.x. [DOI] [PubMed] [Google Scholar]
  56. Witzemann V., Barg B., Criado M., Stein E., Sakmann B. Developmental regulation of five subunit specific mRNAs encoding acetylcholine receptor subtypes in rat muscle. FEBS Lett. 1989 Jan 2;242(2):419–424. doi: 10.1016/0014-5793(89)80514-9. [DOI] [PubMed] [Google Scholar]
  57. Witzemann V., Barg B., Nishikawa Y., Sakmann B., Numa S. Differential regulation of muscle acetylcholine receptor gamma- and epsilon-subunit mRNAs. FEBS Lett. 1987 Oct 19;223(1):104–112. doi: 10.1016/0014-5793(87)80518-5. [DOI] [PubMed] [Google Scholar]
  58. 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]

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

RESOURCES