Skip to main content
NCBI home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1974 Dec;71(12):4768–4772. doi: 10.1073/pnas.71.12.4768

Purified Acetylcholine Receptor: Its Reconstitution to a Chemically Excitable Membrane*

Daniel M Michaelson 1, Michael A Raftery 1
PMCID: PMC433978  PMID: 4531016

Abstract

Association of purified acetylcholine receptor from Torpedo californica electroplax with lipids from the same organism results in a vesicular membrane system in which the receptor protein is oriented so that all neurotoxin binding sites appear to be on the outer surface. The reconstituted system is chemically excitable by acetylcholine and carbamylcholine, as measured by 22Na+ efflux. This excitability is specifically blocked by the antagonist α-bungarotoxin. These results demonstrate that the purified receptor macromolecule contains not only the specific neurotransmitter binding site but also the molecular elements necessary for ion translocation in order to effect postsynaptic depolarization.

Keywords: acetylcholine binding, ion translocation

Full text

PDF
4768

Selected References

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

  1. BLIGH E. G., DYER W. J. A rapid method of total lipid extraction and purification. Can J Biochem Physiol. 1959 Aug;37(8):911–917. doi: 10.1139/o59-099. [DOI] [PubMed] [Google Scholar]
  2. Biesecker G. Molecular properties of the cholinergic receptor purified from Electrophorus electricus. Biochemistry. 1973 Oct 23;12(22):4403–4409. doi: 10.1021/bi00746a017. [DOI] [PubMed] [Google Scholar]
  3. Chang H. W. Purification and characterization of acetylcholine receptor-I from Electrophorus electricus. Proc Natl Acad Sci U S A. 1974 May;71(5):2113–2117. doi: 10.1073/pnas.71.5.2113. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. DEL CASTILLO J., KATZ B. On the localization of acetylcholine receptors. J Physiol. 1955 Apr 28;128(1):157–181. doi: 10.1113/jphysiol.1955.sp005297. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. 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]
  6. Eldefrawi M. E., Eldefrawi A. T. Purification and molecular properties of the acetylcholine receptor from Torpedo electroplax. Arch Biochem Biophys. 1973 Nov;159(1):362–373. doi: 10.1016/0003-9861(73)90462-1. [DOI] [PubMed] [Google Scholar]
  7. Goodall M. C., Bradley R. J., Saccomani G., Romine W. O. Quantum conductance changes in lipid bilayer membranes associated with incorporation of acetylcholine receptors. Nature. 1974 Jul 5;250(461):68–70. doi: 10.1038/250068a0. [DOI] [PubMed] [Google Scholar]
  8. Hazelbauer G. L., Changeux J. P. Reconstitution of a chemically excitable membrane. Proc Natl Acad Sci U S A. 1974 Apr;71(4):1479–1483. doi: 10.1073/pnas.71.4.1479. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Hong K., Hubbell W. L. Lipid requirements for Rhodopsin regenerability. Biochemistry. 1973 Oct 23;12(22):4517–4523. doi: 10.1021/bi00746a033. [DOI] [PubMed] [Google Scholar]
  10. Jain M. K., Mehl L. E., Cordes E. H. Incorporation of eel electroplax acetylcholinesterase into black lipid membranes. A possible model for the cholinergic receptor. Biochem Biophys Res Commun. 1973 Mar 5;51(1):192–197. doi: 10.1016/0006-291x(73)90527-5. [DOI] [PubMed] [Google Scholar]
  11. Kagawa Y. Reconstitution of oxidative phosphorylation. Biochim Biophys Acta. 1972 Aug 4;265(3):297–338. [PubMed] [Google Scholar]
  12. Karlsson E., Heilbronn E., Widlund L. Isolation of the nicotinic acetylcholine receptor by biospecific chromatography on insolubilized Naja naja neurotoxin. FEBS Lett. 1972 Nov 15;28(1):107–111. doi: 10.1016/0014-5793(72)80688-4. [DOI] [PubMed] [Google Scholar]
  13. Kemp G., Dolly J. O., Barnard E. A., Wenner C. E. Reconstitution of a partially purified endplate acetylcholine receptor preparation in lipid bilayer membranes. Biochem Biophys Res Commun. 1973 Sep 18;54(2):607–613. doi: 10.1016/0006-291x(73)91466-6. [DOI] [PubMed] [Google Scholar]
  14. Klett R. P., Fulpius B. W., Cooper D., Smith M., Reich E., Possani L. D. The acetylcholine receptor. I. Purification and characterization of a macromolecule isolated from Electrophorus electricus. J Biol Chem. 1973 Oct 10;248(19):6841–6853. [PubMed] [Google Scholar]
  15. Martinez-Carrion M., Raftery M. A. Use of a fluorescent probe for the study of ligand binding by the isolated cholinergic receptor of Torpedo californica. Biochem Biophys Res Commun. 1973 Dec 19;55(4):1156–1164. doi: 10.1016/s0006-291x(73)80016-6. [DOI] [PubMed] [Google Scholar]
  16. Meissner G., Fleischer S. Ca 2+ uptake in reconstituted sarcoplasmic reticulum vesicles. Biochem Biophys Res Commun. 1973 Jun 8;52(3):913–920. doi: 10.1016/0006-291x(73)91024-3. [DOI] [PubMed] [Google Scholar]
  17. Meunier J. C., Sealock R., Olsen R., Changeux J. P. Purification and properties of the cholinergic receptor protein from Electrophorus electricus electric tissue. Eur J Biochem. 1974 Jun 15;45(2):371–394. doi: 10.1111/j.1432-1033.1974.tb03563.x. [DOI] [PubMed] [Google Scholar]
  18. Michaelson D. M., Horwitz A. F., Klein M. P. Transbilayer asymmetry and surface homogeneity of mixed phospholipids in cosonicated vesicles. Biochemistry. 1973 Jul 3;12(14):2637–2645. doi: 10.1021/bi00738a014. [DOI] [PubMed] [Google Scholar]
  19. Moody T., Schmidt J., Raftery M. A. Binding of acetylcholine and related compounds to purified acetylcholine receptor from Torpedo Californica electroplax. Biochem Biophys Res Commun. 1973 Aug 6;53(3):761–772. doi: 10.1016/0006-291x(73)90158-7. [DOI] [PubMed] [Google Scholar]
  20. Racker E. Reconstitution of a calcium pump with phospholipids and a purified Ca ++ - adenosine triphosphatase from sacroplasmic reticulum. J Biol Chem. 1972 Dec 25;247(24):8198–8200. [PubMed] [Google Scholar]
  21. Racker E., Stoeckenius W. Reconstitution of purple membrane vesicles catalyzing light-driven proton uptake and adenosine triphosphate formation. J Biol Chem. 1974 Jan 25;249(2):662–663. [PubMed] [Google Scholar]
  22. Raftery M. A. Isolation of acetylcholine receptor-- -bungarotoxin complexes from Torpedo californica electroplax. Arch Biochem Biophys. 1973 Jan;154(1):270–276. doi: 10.1016/0003-9861(73)90057-x. [DOI] [PubMed] [Google Scholar]
  23. Razin S. Reconstruction of biological membranes. Biochim Biophys Acta. 1972 Apr 18;265(2):241–296. [PubMed] [Google Scholar]
  24. 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]
  25. Schmidt J., Raftery M. A. Use of affinity chromatography for acetylcholine receptor purification. Biochem Biophys Res Commun. 1972 Oct 17;49(2):572–578. doi: 10.1016/0006-291x(72)90449-4. [DOI] [PubMed] [Google Scholar]
  26. Warren G. B., Toon P. A., Birdsall N. J., Lee A. G., Metcalfe J. C. Reconstitution of a calcium pump using defined membrane components. Proc Natl Acad Sci U S A. 1974 Mar;71(3):622–626. doi: 10.1073/pnas.71.3.622. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES