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A fluorophore attached to nicotinic acetylcholine receptor beta M2 detects productive binding of agonist to the alpha delta site

Dahan, David S. and Dibas, Mohammed I. and Petersson, E. James and Auyeung, Vincent C. and Chanda, Baron and Bezanilla, Francisco and Dougherty, Dennis A. and Lester, Henry A. (2004) A fluorophore attached to nicotinic acetylcholine receptor beta M2 detects productive binding of agonist to the alpha delta site. Proceedings of the National Academy of Sciences of the United States of America, 101 (27). pp. 10195-10200. ISSN 0027-8424. http://resolver.caltech.edu/CaltechAUTHORS:DAHpnas04

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Abstract

To study conformational transitions at the muscle nicotinic acetylcholine (ACh) receptor (nAChR), a rhodamine fluorophore was tethered to a Cys side chain introduced at the beta-19' position in the M2 region of the nAChR expressed in Xenopus oocytes. This procedure led to only minor changes in receptor function. During agonist application, fluorescence increased by (Delta-F/F) approximate to 10%, and the emission peak shifted to lower wavelengths, indicating a more hydrophobic environment for the fluorophore. The dose-response relations for Delta-F agreed well with those for epibatidine-induced currents, but were shifted approximate to 100-fold to the left of those for ACh-induced currents. Because (i) epibatidine binds more tightly to the alpha-gamma-binding site than to the alpha-delta site and (ii) ACh binds with reverse-site selectivity, these data suggest that Delta-F monitors an event linked to binding specifically at the alpha-delta-subunit interface. In experiments with flash-applied agonists, the earliest detectable Delta-F occurs within milliseconds, i.e., during activation. At low [ACh] (less than or equal to 10 muM), a phase of Delta-F occurs with the same time constant as desensitization, presumably monitoring an increased population of agonist-bound receptors. However, recovery from Delta-F is complete before the slowest phase of recovery from desensitization (time constant approximate to 250 s), showing that one or more desensitized states have fluorescence like that of the resting channel. That conformational transitions at the alpha-delta-binding site are not tightly coupled to channel activation suggests that sequential rather than fully concerted transitions occur during receptor gating. Thus, time-resolved fluorescence changes provide a powerful probe of nAChR conformational changes.


Item Type:Article
Additional Information:© 2004 by The National Academy of Sciences of the USA. Published online before print June 24 2004. Edited by Arthur Karlin, Columbia University College of Physicians and Surgeons, New York, NY and approved April 19, 2004 (received for review April 1, 2003). We thank A. Karlin for providing the Beta-A19'C mutant and K. Kostenko and S. Kwoh for assistance with oocytes. This work was supported by National Institutes of Health Grants NS-11756, NS-34407, and GM-30376, and by a fellowship from the American Heart Association (to B.C.).
Subject Keywords:LIGAND-BINDING, CHANNEL, SUBUNIT, DESENSITIZATION, FLUORESCENCE, ACTIVATION, MECHANISM, AFFINITY, REVEALS
Record Number:CaltechAUTHORS:DAHpnas04
Persistent URL:http://resolver.caltech.edu/CaltechAUTHORS:DAHpnas04
Alternative URL:http://dx.doi.org/10.1073/pnas.0301885101
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:602
Collection:CaltechAUTHORS
Deposited By: Tony Diaz
Deposited On:01 Sep 2005
Last Modified:14 Nov 2014 19:18

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