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. 1983 Aug;80(16):5129–5133. doi: 10.1073/pnas.80.16.5129

Patch-recorded single-channel currents of the purified and reconstituted Torpedo acetylcholine receptor.

D W Tank, R L Huganir, P Greengard, W W Webb
PMCID: PMC384203  PMID: 6308673

Abstract

Small unilamellar vesicles containing purified and reconstituted nicotinic acetylcholine receptors from Torpedo electroplax have been fused by a simple freeze-thaw procedure to form large liposomes. Giga-seal patch-recording techniques were used to form isolated patches of liposome-membrane and to measure single-channel properties of the reconstituted receptor-ion channel complex. The observed properties are quantitatively similar to those reported for vertebrate muscle nicotinic acetylcholine receptor species recorded in situ. The results demonstrate that the pentameric complex consisting of the alpha 2 beta gamma delta subunits is fully functional. The methods used in these experiments should be useful in studying the effects of chemical alterations on the properties of acetylcholine receptor channels as well as other types of purified and reconstituted ion channels.

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Selected References

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

  1. Auerbach A., Sachs F. Flickering of a nicotinic ion channel to a subconductance state. Biophys J. 1983 Apr;42(1):1–10. doi: 10.1016/S0006-3495(83)84362-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Boheim G., Hanke W., Barrantes F. J., Eibl H., Sakmann B., Fels G., Maelicke A. Agonist-activated ionic channels in acetylcholine receptor reconstituted into planar lipid bilayers. Proc Natl Acad Sci U S A. 1981 Jun;78(6):3586–3590. doi: 10.1073/pnas.78.6.3586. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Changeux J. P., Heidmann T., Popot J. L., Sobel A. Reconstitution of a functional acetylcholine regulator under defined conditions. FEBS Lett. 1979 Sep 1;105(1):181–187. doi: 10.1016/0014-5793(79)80913-8. [DOI] [PubMed] [Google Scholar]
  4. Changeux J. P. The acetylcholine receptor: an "allosteric" membrane protein. Harvey Lect. 1979 1980;75:85–254. [PubMed] [Google Scholar]
  5. Colquhoun D., Sakmann B. Fluctuations in the microsecond time range of the current through single acetylcholine receptor ion channels. Nature. 1981 Dec 3;294(5840):464–466. doi: 10.1038/294464a0. [DOI] [PubMed] [Google Scholar]
  6. Conti-Tronconi B. M., Gotti C. M., Hunkapiller M. W., Raftery M. A. Mammalian muscle acetylcholine receptor: a supramolecular structure formed by four related proteins. Science. 1982 Dec 17;218(4578):1227–1229. doi: 10.1126/science.7146904. [DOI] [PubMed] [Google Scholar]
  7. Conti-Tronconi B. M., Raftery M. A. The nicotinic cholinergic receptor: correlation of molecular structure with functional properties. Annu Rev Biochem. 1982;51:491–530. doi: 10.1146/annurev.bi.51.070182.002423. [DOI] [PubMed] [Google Scholar]
  8. Fambrough D. M. Control of acetylcholine receptors in skeletal muscle. Physiol Rev. 1979 Jan;59(1):165–227. doi: 10.1152/physrev.1979.59.1.165. [DOI] [PubMed] [Google Scholar]
  9. 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]
  10. Hamill O. P., Sakmann B. Multiple conductance states of single acetylcholine receptor channels in embryonic muscle cells. Nature. 1981 Dec 3;294(5840):462–464. doi: 10.1038/294462a0. [DOI] [PubMed] [Google Scholar]
  11. Horn R., Patlak J. Single channel currents from excised patches of muscle membrane. Proc Natl Acad Sci U S A. 1980 Nov;77(11):6930–6934. doi: 10.1073/pnas.77.11.6930. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Huganir R. L., Greengard P. cAMP-dependent protein kinase phosphorylates the nicotinic acetylcholine receptor. Proc Natl Acad Sci U S A. 1983 Feb;80(4):1130–1134. doi: 10.1073/pnas.80.4.1130. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Huganir R. L., Racker E. Properties of proteoliposomes reconstituted with acetylcholine receptor from Torpedo californica. J Biol Chem. 1982 Aug 25;257(16):9372–9378. [PubMed] [Google Scholar]
  14. Huganir R. L., Schell M. A., Racker E. Reconstitution of the purified acetylcholine receptor from Torpedo californica. FEBS Lett. 1979 Dec 1;108(1):155–160. doi: 10.1016/0014-5793(79)81199-0. [DOI] [PubMed] [Google Scholar]
  15. Jackson M. B., Wong B. S., Morris C. E., Lecar H., Christian C. N. Successive openings of the same acetylcholine receptor channel are correlated in open time. Biophys J. 1983 Apr;42(1):109–114. doi: 10.1016/S0006-3495(83)84375-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Lindstrom J., Anholt R., Einarson B., Engel A., Osame M., Montal M. Purification of acetylcholine receptors, reconstitution into lipid vesicles, and study of agonist-induced cation channel regulation. J Biol Chem. 1980 Sep 10;255(17):8340–8350. [PubMed] [Google Scholar]
  17. Läuger P. Ion transport through pores: a rate-theory analysis. Biochim Biophys Acta. 1973 Jul 6;311(3):423–441. doi: 10.1016/0005-2736(73)90323-4. [DOI] [PubMed] [Google Scholar]
  18. Miledi R., Molinoff P., Potter L. T. Isolation of the cholinergic receptor protein of Torpedo electric tissue. Nature. 1971 Feb 19;229(5286):554–557. doi: 10.1038/229554a0. [DOI] [PubMed] [Google Scholar]
  19. Nelson N., Anholt R., Lindstrom J., Montal M. Reconstitution of purified acetylcholine receptors with functional ion channels in planar lipid bilayers. Proc Natl Acad Sci U S A. 1980 May;77(5):3057–3061. doi: 10.1073/pnas.77.5.3057. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Saitoh T., Changeux J. P. Change in state of phosphorylation of acetylcholine receptor during maturation of the electromotor synapse in Torpedo marmorata electric organ. Proc Natl Acad Sci U S A. 1981 Jul;78(7):4430–4434. doi: 10.1073/pnas.78.7.4430. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Sakmann B., Patlak J., Neher E. Single acetylcholine-activated channels show burst-kinetics in presence of desensitizing concentrations of agonist. Nature. 1980 Jul 3;286(5768):71–73. doi: 10.1038/286071a0. [DOI] [PubMed] [Google Scholar]
  22. Schindler H., Quast U. Functional acetylcholine receptor from Torpedo marmorata in planar membranes. Proc Natl Acad Sci U S A. 1980 May;77(5):3052–3056. doi: 10.1073/pnas.77.5.3052. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Tank D. W., Miller C., Webb W. W. Isolated-patch recording from liposomes containing functionally reconstituted chloride channels from Torpedo electroplax. Proc Natl Acad Sci U S A. 1982 Dec;79(24):7749–7753. doi: 10.1073/pnas.79.24.7749. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Trautmann A. Curare can open and block ionic channels associated with cholinergic receptors. Nature. 1982 Jul 15;298(5871):272–275. doi: 10.1038/298272a0. [DOI] [PubMed] [Google Scholar]
  25. Walker J. W., McNamee M. G., Pasquale E., Cash D. J., Hess G. P. Acetylcholine receptor inactivation in Torpedo californica electroplax membrane vesicles. Detection of two processes in the millisecond and second time regions. Biochem Biophys Res Commun. 1981 May 15;100(1):86–90. doi: 10.1016/s0006-291x(81)80066-6. [DOI] [PubMed] [Google Scholar]
  26. Walker J. W., Takeyasu K., McNamee M. G. Activation and inactivation kinetics of Torpedo californica acetylcholine receptor in reconstituted membranes. Biochemistry. 1982 Oct 26;21(22):5384–5389. doi: 10.1021/bi00265a001. [DOI] [PubMed] [Google Scholar]
  27. Wu W. C., Raftery M. A. Carbamylcholine-induced rapid cation efflux from reconstituted membrane vesicles containing purified acetylcholine receptor. Biochem Biophys Res Commun. 1979 Jul 12;89(1):26–35. doi: 10.1016/0006-291x(79)90938-0. [DOI] [PubMed] [Google Scholar]

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