Fluorescent Staining of Acetylcholine Receptors in Vertebrate Skeletal Muscle

Overview

Journal J Physiol

Specialty Physiology

Date 1974 Mar 1

PMID 4133039

Citations 45

Authors

M J Anderson

M W Cohen

Affiliations

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Abstract

1. alpha-Bungarotoxin was labelled with fluorescent dyes and used as a stain for visualizing the distribution of acetylcholine receptors in vertebrate skeletal muscle fibres.2. Dye-toxin conjugates had the same pharmacological properties as native toxin, but their potencies were lower.3. Fluorescent staining was examined in teased muscle fibres. The stain was found to be confined to the neuromuscular junction and associated with the subsynaptic membrane.4. Staining intensity was reduced by curare and even more so by carbachol, but not by atropine or neostigmine. Pre-treatment of muscles with unlabelled alpha-bungarotoxin entirely prevented staining.5. The staining at amphibian neuromuscular junctions was characterized by a pattern of intense transverse bands occurring at intervals of approximately 0.5-1 mum, with fluorescence of lower intensity between them. Fluorescent staining was not detected on adjacent, extrasynaptic, muscle membrane. In side views the staining appeared as a fine line with small protuberances occurring at the same intervals as the intense bands seen face-on. These results indicate that acetylcholine receptors are associated with the entire subsynaptic membrane, including the membrane of the junctional folds and that their density changes abruptly at the border between synaptic and extrasynaptic muscle membrane.

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References

Feltz A, Mallart A . An analysis of acetylcholine responses of junctional and extrajunctional receptors of frog muscle fibres. J Physiol. 1971; 218(1):85-100. PMC: 1331585. DOI: 10.1113/jphysiol.1971.sp009605. View

Chang C, Chen T, Lee C . Studies of the presynaptic effect of -bungarotoxin on neuromuscular transmission. J Pharmacol Exp Ther. 1973; 184(2):339-45. View

Edidin M, Fambrough D . Fluidity of the surface of cultured muscle fibers. Rapid lateral diffusion of marked surface antigens. J Cell Biol. 1973; 57(1):27-37. PMC: 2108958. DOI: 10.1083/jcb.57.1.27. View

Bosmann H . Acetylcholine receptor. I. Identification and biochemical characteristics of a cholinergic receptor of guinea pig cerebral cortex. J Biol Chem. 1972; 247(1):130-45. View

Hartzell H, Fambrough D . Acetylcholine receptors. Distribution and extrajunctional density in rat diaphragm after denervation correlated with acetylcholine sensitivity. J Gen Physiol. 1972; 60(3):248-62. PMC: 2226074. DOI: 10.1085/jgp.60.3.248. View

Lester H . Postsynaptic action of cobra toxin at the myoneural junction. Nature. 1970; 227(5259):727-8. DOI: 10.1038/227727a0. View

Marshall J, EVELAND W, Smith C . Superiority of fluorescein isothiocyanate (Riggs) for fluorescent-antibody technic with a modification of its application. Proc Soc Exp Biol Med. 1958; 98(4):898-900. DOI: 10.3181/00379727-98-24222. View

Greene L, Sytkowski A, Vogel Z, Nirenberg M . -Bungarotoxin used as a probe for acetylcholine receptors of cultured neurones. Nature. 1973; 243(5403):163-6. DOI: 10.1038/243163a0. View

Miledi R, Zelena J . Sensitivity to acetylcholine in rat slow muscle. Nature. 1966; 210(5038):855-6. DOI: 10.1038/210855a0. View

10.

Mebs D, Narita K, Iwanaga S, Samejima Y, Lee C . Amino acid sequence of -bungarotoxin from the venom of Bungarus multicinctus. Biochem Biophys Res Commun. 1971; 44(3):711-6. DOI: 10.1016/s0006-291x(71)80141-9. View

11.

HUBBARD J, Schmidt R, Yokota T . The effect of acetylcholine upon mammalian motor nerve terminals. J Physiol. 1965; 181(4):810-29. PMC: 1357685. DOI: 10.1113/jphysiol.1965.sp007799. View

12.

Lee C, Chang S, Kau S, Luh S . Chromatographic separation of the venom of Bungarus multicinctus and characterization of its components. J Chromatogr. 1972; 72(1):71-82. DOI: 10.1016/0021-9673(72)80009-8. View

13.

ECCLES J, JAEGER J . The relationship between the mode of operation and the dimensions of the junctional regions at synapses and motor end-organs. Proc R Soc Lond B Biol Sci. 1958; 148(930):38-56. DOI: 10.1098/rspb.1958.0003. View

14.

Miledi R, Potter L . Acetylcholine receptors in muscle fibres. Nature. 1971; 233(5322):599-603. DOI: 10.1038/233599a0. View

15.

Miledi R, Molinoff P, Potter L . Isolation of the cholinergic receptor protein of Torpedo electric tissue. Nature. 1971; 229(5286):554-7. DOI: 10.1038/229554a0. View

16.

Birks R, Huxley H, Katz B . The fine structure of the neuromuscular junction of the frog. J Physiol. 1960; 150:134-44. PMC: 1363152. DOI: 10.1113/jphysiol.1960.sp006378. View

17.

Berg D, Kelly R, Sargent P, Williamson P, Hall Z . Binding of -bungarotoxin to acetylcholine receptors in mammalian muscle (snake venom-denervated muscle-neonatal muscle-rat diaphragm-SDS-polyacrylamide gel electrophoresis). Proc Natl Acad Sci U S A. 1972; 69(1):147-51. PMC: 427564. DOI: 10.1073/pnas.69.1.147. View

18.

Tamiya N, Arai H . Studies on sea-snake venoms. Crystallization of erabutoxins a and b from Laticauda semifasciata venom. Biochem J. 1966; 99(3):624-30. PMC: 1265051. DOI: 10.1042/bj0990624. View

19.

Porter C, Chiu T, Wieckowski J, Barnard E . Types and locations of cholinergic receptor-like molecules in muscle fibres. Nat New Biol. 1973; 241(105):3-7. DOI: 10.1038/newbio241003a0. View

20.

Miledi R . Junctional and extra-junctional acetylcholine receptors in skeletal muscle fibres. J Physiol. 1960; 151:24-30. PMC: 1363215. View