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. 1980 Oct;77(10):5807–5811. doi: 10.1073/pnas.77.10.5807

Topographic studies of Torpedo acetylcholine receptor subunits as a transmembrane complex.

C D Strader, M A Raftery
PMCID: PMC350160  PMID: 6934512

Abstract

The exposure of the four subunits of the acetylcholine receptor from Torpedo californica on both the extracellular and cytoplasmic faces of the postsynaptic membranes of the electroplaque cells has been investigated. Sealed membrane vesicles containing no protein components other than the receptor were isolated and were shown to have 95% of their synaptic surfaces facing the medium. The susceptibility of the four receptor subunits in these preparations to hydrolysis by trypsin both from the external and from the internal medium was used to investigate the exposure of the subunits on the synaptic and cytoplasmic surfaces of the membrane. It was shown by sodium dodecyl sulfate gel electrophoresis of the tryptic products that all four subunits are exposed on the extracellular surface to a similar degree. All four subunits are also exposed on the internal surface of the membrane, but the apparent degree of exposure varies with the subunit size, the larger subunits being more exposed. The results are discussed in terms of a possible topographic model of the receptor as a transmembrane protein complex.

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Selected References

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

  1. Blanchard S. G., Elliott J., Raftery M. A. Interaction of local anesthetics with Torpedo californica membrane-bound acetylcholine receptor. Biochemistry. 1979 Dec 25;18(26):5880–5885. doi: 10.1021/bi00593a019. [DOI] [PubMed] [Google Scholar]
  2. Blanchard S. G., Quast U., Reed K., Lee T., Schimerlik M. I., Vandlen R., Claudio T., Strader C. D., Moore H. P., Raftery M. A. Interaction of [125I]-alpha-bungarotoxin with acetylcholine receptor from Torpedo californica. Biochemistry. 1979 May 15;18(10):1875–1883. doi: 10.1021/bi00577a005. [DOI] [PubMed] [Google Scholar]
  3. Cartaud J., Benedetti E. L., Cohen J. B., Meunier J. C., Changeux J. P. Presence of a lattice structure in membrane fragments rich in nicotinic receptor protein from the electric organ of Torpedo marmorata. FEBS Lett. 1973 Jun 15;33(1):109–113. doi: 10.1016/0014-5793(73)80171-1. [DOI] [PubMed] [Google Scholar]
  4. Chang H. W., Bock E. Molecular forms of acetylcholine receptor. Effects of calcium ions and a sulfhydryl reagent on the occurrence of oligomers. Biochemistry. 1977 Oct 4;16(20):4513–4520. doi: 10.1021/bi00639a028. [DOI] [PubMed] [Google Scholar]
  5. Chang R. S., Potter L. T., Smith D. S. Postsynaptic membranes in the electric tissue of Narcine: IV. Isolation and characterization of the nicotinic receptor protein. Tissue Cell. 1977;9(4):623–644. doi: 10.1016/0040-8166(77)90031-3. [DOI] [PubMed] [Google Scholar]
  6. Clark D. G., Macmurchie D. D., Elliott E., Wolcott R. G., Landel A. M., Raftery M. A. Elapid neurotoxins. Purification, characterization, and immunochemical studies of -bungarotoxin. Biochemistry. 1972 Apr 25;11(9):1663–1668. doi: 10.1021/bi00759a020. [DOI] [PubMed] [Google Scholar]
  7. Cohen J. B., Weber M., Huchet M., Changeux J. P. Purification from Torpedo marmorata electric tissue of membrane fragments particularly rich in cholinergic receptor protein. FEBS Lett. 1972 Oct 1;26(1):43–47. doi: 10.1016/0014-5793(72)80538-6. [DOI] [PubMed] [Google Scholar]
  8. Damle V. N., McLaughlin M., Karlin A. Bromoacetylcholine as an affinity label of the acetylcholine receptor from Torpedo californica. Biochem Biophys Res Commun. 1978 Oct 30;84(4):845–851. doi: 10.1016/0006-291x(78)91661-3. [DOI] [PubMed] [Google Scholar]
  9. Duguid J. R., Raftery M. A. Fractionation and partial characterization of membrane particles from Torpedo californica electroplax. Biochemistry. 1973 Sep 11;12(19):3593–3597. doi: 10.1021/bi00743a003. [DOI] [PubMed] [Google Scholar]
  10. Eldefrawi A. T., Eldefrawi M. E., Albuquerque E. X., Oliveira A. C., Mansour N., Adler M., Daly J. W., Brown G. B., Burgermeister W., Witkop B. Perhydrohistrionicotoxin: a potential ligand for the ion conductance modulator of the acetylcholine receptor. Proc Natl Acad Sci U S A. 1977 May;74(5):2172–2176. doi: 10.1073/pnas.74.5.2172. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Elliot J., Raftery M. A. Interactions of perhydrohistrionicotoxin with postsynaptic membranes. Biochem Biophys Res Commun. 1977 Aug 22;77(4):1347–1353. doi: 10.1016/s0006-291x(77)80127-7. [DOI] [PubMed] [Google Scholar]
  12. Elliott J., Blanchard S. G., Wu W., Miller J., Strader C. D., Hartig P., Moore H. P., Racs J., Raftery M. A. Purification of Torpedo californica post-synaptic membranes and fractionation of their constituent proteins. Biochem J. 1980 Mar 1;185(3):667–677. doi: 10.1042/bj1850667. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Froehner S. C., Rafto S. Comparison of the subunits of Torpedo californica acetylcholine receptor by peptide mapping. Biochemistry. 1979 Jan 23;18(2):301–307. doi: 10.1021/bi00569a011. [DOI] [PubMed] [Google Scholar]
  14. Hartig P. R., Raftery M. A. Lactoperoxidase catalyzed membrane surface labeling of the acetylcholine receptor from Torpedo californica. Biochem Biophys Res Commun. 1977 Sep 9;78(1):16–22. doi: 10.1016/0006-291x(77)91215-3. [DOI] [PubMed] [Google Scholar]
  15. Hartig P. R., Raftery M. A. Preparation of right-side-out, acetylcholine receptor enriched intact vesicles from Torpedo californica electroplaque membranes. Biochemistry. 1979 Apr 3;18(7):1146–1150. doi: 10.1021/bi00574a004. [DOI] [PubMed] [Google Scholar]
  16. Hess G. P., Andrews J. P. Functional acetylcholine receptor--electroplax membrane microsacs (vesicles): purification and characterization. Proc Natl Acad Sci U S A. 1977 Feb;74(2):482–486. doi: 10.1073/pnas.74.2.482. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Hunkapiller M. W., Strader C. D., Hood L., Raftery M. A. Amino terminal amino acid sequence of the major polypeptide subunit of Torpedo californica acetylcholine receptor. Biochem Biophys Res Commun. 1979 Nov 14;91(1):164–169. doi: 10.1016/0006-291x(79)90598-9. [DOI] [PubMed] [Google Scholar]
  18. Klymkowsky M. W., Stroud R. M. Immunospecific identification and three-dimensional structure of a membrane-bound acetylcholine receptor from Torpedo californica. J Mol Biol. 1979 Mar 5;128(3):319–334. doi: 10.1016/0022-2836(79)90091-3. [DOI] [PubMed] [Google Scholar]
  19. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  20. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  21. Lindstrom J., Einarson B., Merlie J. Immunization of rats with polypeptide chains from torpedo acetylcholine receptor causes an autoimmune response to receptors in rat muscle. Proc Natl Acad Sci U S A. 1978 Feb;75(2):769–773. doi: 10.1073/pnas.75.2.769. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Lindstrom J., Merlie J., Yogeeswaran G. Biochemical properties of acteylcholine receptor subunits from Torpedo californica. Biochemistry. 1979 Oct 16;18(21):4465–4470. doi: 10.1021/bi00588a003. [DOI] [PubMed] [Google Scholar]
  23. Martinez-Carrion M., Sator V., Raftery M. A. The molecular weight of an acetylcholine receptor isolated from Torpedo californica. Biochem Biophys Res Commun. 1975 Jul 8;65(1):129–137. doi: 10.1016/s0006-291x(75)80070-2. [DOI] [PubMed] [Google Scholar]
  24. Miller D. L., Moore H. P., Hartig P. R., Raftery M. A. Fast cation flux from Torpedo californica membrane preparations: implications for a functional role for acetylcholine receptor dimers. Biochem Biophys Res Commun. 1978 Nov 29;85(2):632–640. doi: 10.1016/0006-291x(78)91209-3. [DOI] [PubMed] [Google Scholar]
  25. Moore H. P., Hartig P. R., Raftery M. A. Correlation of polypeptide composition with functional events in acetylcholine receptor-enriched membranes from Torpedo californica. Proc Natl Acad Sci U S A. 1979 Dec;76(12):6265–6269. doi: 10.1073/pnas.76.12.6265. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Moore H. P., Raftery M. A. Direct spectroscopic studies of cation translocation by Torpedo acetylcholine receptor on a time scale of physiological relevance. Proc Natl Acad Sci U S A. 1980 Aug;77(8):4509–4513. doi: 10.1073/pnas.77.8.4509. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Moore H. P., Raftery M. A. Studies of reversible and irreversible interactions of an alkylating agonist with Torpedo californica acetylcholine receptor in membrane-bound and purified states. Biochemistry. 1979 May 15;18(10):1862–1867. doi: 10.1021/bi00577a003. [DOI] [PubMed] [Google Scholar]
  28. Neubig R. R., Krodel E. K., Boyd N. D., Cohen J. B. Acetylcholine and local anesthetic binding to Torpedo nicotinic postsynaptic membranes after removal of nonreceptor peptides. Proc Natl Acad Sci U S A. 1979 Feb;76(2):690–694. doi: 10.1073/pnas.76.2.690. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Nickel E., Potter L. T. Ultrastructure of isolated membranes of Torpedo electric tissue. Brain Res. 1973 Jul 27;57(2):508–517. doi: 10.1016/0006-8993(73)90158-3. [DOI] [PubMed] [Google Scholar]
  30. Raftery M. A., Schmidt J., Clark D. G. Specificity of -bungarotoxin binding to Torpedo californica electroplax. Arch Biochem Biophys. 1972 Oct;152(2):882–886. doi: 10.1016/0003-9861(72)90285-8. [DOI] [PubMed] [Google Scholar]
  31. Raftery M. A., Vandlen R. L., Reed K. L., Lee T. Characterization of Torpedo californica acetylcholine receptor: its subunit composition and ligand-binding properties. Cold Spring Harb Symp Quant Biol. 1976;40:193–202. doi: 10.1101/sqb.1976.040.01.021. [DOI] [PubMed] [Google Scholar]
  32. Raftery M. A., Vandlen R., Michaelson D., Bode J., Moody T., Chao Y., Reed K., Deutsch J., Duguid J. The biochemistry of an acetylcholine receptor. J Supramol Struct. 1974;2(5-6):582–592. doi: 10.1002/jss.400020506. [DOI] [PubMed] [Google Scholar]
  33. Reynolds J. A., Karlin A. Molecular weight in detergent solution of acetylcholine receptor from Torpedo californica. Biochemistry. 1978 May 30;17(11):2035–2038. doi: 10.1021/bi00604a001. [DOI] [PubMed] [Google Scholar]
  34. Sator V., Gonzalez-Ros J. M., Calvo-Fernandez P., Martinez-Carrion M. Pyrenesulfonyl azide: a marker of acetylcholine receptor subunits in contact with membrane hydrophobic environment. Biochemistry. 1979 Apr 3;18(7):1200–1206. doi: 10.1021/bi00574a013. [DOI] [PubMed] [Google Scholar]
  35. Schimerlik M., Raftery M. A. A fluorescence probe of acetylcholine receptor conformation and local anesthetic binding. Biochem Biophys Res Commun. 1976 Dec 6;73(3):607–613. doi: 10.1016/0006-291x(76)90853-6. [DOI] [PubMed] [Google Scholar]
  36. 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]
  37. Sobel A., Weber M., Changeux J. P. Large-scale purification of the acetylcholine-receptor protein in its membrane-bound and detergent-extracted forms from Torpedo marmorata electric organ. Eur J Biochem. 1977 Oct 17;80(1):215–224. doi: 10.1111/j.1432-1033.1977.tb11874.x. [DOI] [PubMed] [Google Scholar]
  38. Strader C. B., Revel J. P., Raftery M. A. Demonstration of the transmembrane nature of the acetylcholine receptor by labeling with anti-receptor antibodies. J Cell Biol. 1979 Nov;83(2 Pt 1):499–510. doi: 10.1083/jcb.83.2.499. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Tarrab-Hazdai R., Bercovici T., Goldfarb V., Gitler C. Identification of the acetylcholine receptor subunit in the lipid bilayer of Torpedo electric organ excitable membranes. J Biol Chem. 1980 Feb 10;255(3):1204–1209. [PubMed] [Google Scholar]
  40. Tarrab-Hazdai R., Geiger B., Fuchs S., Amsterdam A. Localization of acetylcholine receptor in excitable membrane from the electric organ of Torpedo: Evidence for exposure of receptor antigenic sites on both sides of the membrane. Proc Natl Acad Sci U S A. 1978 May;75(5):2497–2501. doi: 10.1073/pnas.75.5.2497. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Vandlen R. L., Wu W. C., Eisenach J. C., Raftery M. A. Studies of the composition of purified Torpedo californica acetylcholine receptor and of its subunits. Biochemistry. 1979 May 15;18(10):1845–1854. doi: 10.1021/bi00577a001. [DOI] [PubMed] [Google Scholar]
  42. Weber M., Changeux J. P. Binding of Naja nigricollis (3H)alpha-toxin to membrane fragments from Electrophorus and Torpedo electric organs. II. Effect of cholinergic agonists and antagonists on the binding of the tritiated alpha-neurotoxin. Mol Pharmacol. 1974 Jan;10(1):15–34. [PubMed] [Google Scholar]
  43. Weill C. L., McNamee M. G., Karlin A. Affinity-labeling of purified acetylcholine receptor from Torpedo californica. Biochem Biophys Res Commun. 1974 Dec 11;61(3):997–1003. doi: 10.1016/0006-291x(74)90254-x. [DOI] [PubMed] [Google Scholar]
  44. Witzemann V., Muchmore D., Raftery M. A. Affinity-directed cross-linking of membrane-bound acetylcholine receptor polypeptides with photolabile alpha-bungarotoxin derivatives. Biochemistry. 1979 Nov 27;18(24):5511–5518. doi: 10.1021/bi00591a039. [DOI] [PubMed] [Google Scholar]
  45. Witzemann V., Raftery M. A. Affinity directed crosslinking of acetylcholine receptor polypeptide components in post-synaptic membranes. Biochem Biophys Res Commun. 1978 Nov 29;85(2):623–631. doi: 10.1016/0006-291x(78)91208-1. [DOI] [PubMed] [Google Scholar]
  46. Witzemann V., Raftery M. A. Selective photoaffinity labeling of acetylcholine receptor using a cholinergic analogue. Biochemistry. 1977 Dec 27;16(26):5862–5868. doi: 10.1021/bi00645a034. [DOI] [PubMed] [Google Scholar]
  47. Yu J., Fischman D. A., Steck T. L. Selective solubilization of proteins and phospholipids from red blood cell membranes by nonionic detergents. J Supramol Struct. 1973;1(3):233–248. doi: 10.1002/jss.400010308. [DOI] [PubMed] [Google Scholar]

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