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Direct spectroscopic studies of cation translocation by Torpedo acetylcholine receptor on a time scale of physiological relevance

Moore, Hsiao-Ping H. and Raftery, Michael A. (1980) Direct spectroscopic studies of cation translocation by Torpedo acetylcholine receptor on a time scale of physiological relevance. Proceedings of the National Academy of Sciences of the United States of America, 77 (8). pp. 4509-4513. ISSN 0027-8424.

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The kinetics of carbamoylcholine-mediated cation transport across the membrane of vesicles containing acetylcholine receptor have been measured on the physiologically relevant time scale of a few milliseconds. The stopped-flow spectroscopic approach utilizes thallium(I) as the cation transported into sealed vesicles containing a water-soluble fluorophore. Upon entry of thallium(I), fluorescence quenching occurs by a heavy atom effect. Rapid thallium translocation into the vesicles is mediated by cholinergic agonists and is blocked by antagonists and neurotoxins and by desensitization. The kinetics of thallium transport are used to demonstrate that the four polypeptides known to comprise the receptor are the only protein components necessary for cation translocation. The kinetics of thallium(I) transport at saturating agonist concentrations are also used to calculate the apparent ion transport rate for a single receptor. The minimal value obtained is close to that for a single activated channel determined in vivo. This demonstrates that the physiological receptor has been isolated in intact form.

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Additional Information:© 1980 by the National Academy of Sciences. Communicated by Harden M. McConnell, April 28, 1980. We thank Dr. Paul Hartig for initial suggestions and discussions regarding the use of heavy atom effects. We thank Steven Blanchard for suggesting the freeze-thaw method for loading, Steven Blanchard and Dr. Susan Dunn for helpful discussions and much assistance in operating the stopped-flow instrument and the computer, Valerie Purvis for the typing and artwork, and John Racs for making the membrane preparations. This research was supported by U.S. Public Health Service Grant NS-10294, by a grant from the Muscular Dystrophy Association, and by a grant from the Pew Charitable Trust. 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.
Subject Keywords:cation flux; functional polypeptides; single-channel transport; spectroscopic transport assay
Issue or Number:8
Record Number:CaltechAUTHORS:MOOpnas80
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Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:8614
Deposited By: Tony Diaz
Deposited On:23 Aug 2007
Last Modified:02 Oct 2019 23:52

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