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Quantitation of cation transport by reconstituted membrane vesicles containing purified acetylcholine receptor

Wu, Wilson C.-S. and Moore, Hsiao-Ping H. and Raftery, Michael A. (1981) Quantitation of cation transport by reconstituted membrane vesicles containing purified acetylcholine receptor. Proceedings of the National Academy of Sciences of the United States of America, 78 (2). pp. 775-779. ISSN 0027-8424.

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A stopped-flow spectroscopic technique was used to study the kinetics of ion transport by reconstituted membrane preparations containing purified acetylcholine receptor. Influx of thallium (I) into membrane vesicles was monitored as a decrease, due to quenching by the thallous ion, in the fluorescence of an entrapped fluorophore. In a reproducible manner, the reconstituted receptor responded to cholinergic agonists by mediating rapid ion transport in the millisecond time range. The effect of agonists was blocked by receptor desensitization and by histrionicotoxin and was absent in membrane vesicles lacking receptor. Analysis of the fast kinetics of cation transport produced by saturating concentrations of agonists yielded an estimated rate of transport through a single reconstituted receptor channel. Comparison of this rate with those reported for in vivo preparations and for purified membranes shows that the reconstituted protein closely resembles the physiologically active receptor.

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Additional Information:Copyright © 1981 by the National Academy of Sciences. Communicated by Harry B. Gray, October 17, 1980. We appreciate the use of the computer programs written by Drs. Steven Blanchard and Susan Dunn. We thank Valerie Purvis for the typing and drafting of figures and John Racs for preparation of membranes. This research was supported by U.S. Public Health Service Grant NS-10298 and by National Research Service Predoctoral Award GM 07616 from the National Institute of General Medical Sciences (to W.C.-S.W.). The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Funding AgencyGrant Number
Public Health ServiceNS-10298
National Institute of General Medical SciencesGM 07616
Subject Keywords:reconstitution; cation flux; single-channel transport; stopped-flow spectroscopy
Issue or Number:2
Record Number:CaltechAUTHORS:WUWpnas81
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Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:12199
Deposited By: Archive Administrator
Deposited On:28 Oct 2008 23:42
Last Modified:03 Oct 2019 00:26

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