Identification of binding sites in the nicotinic acetylcholine receptor for TDBzl-etomidate, a photoreactive positive allosteric effector.

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Title Identification of binding sites in the nicotinic acetylcholine receptor for TDBzl-etomidate, a photoreactive positive allosteric effector.
Author Nirthanan, S. Niru; Garcia III, Galo; Chiara, David C.; Husain, S. Shaukat; Cohen, Jonathan B.
Journal Name Journal of Biological Chemistry
Year Published 2008
Place of publication Bethesda
Publisher American Society for Biochemistry and Molecular Biology
Abstract Etomidate, one of the most potent general anesthetics used clinically, acts at micromolar concentrations as an anesthetic and positive allosteric modulator of gamma-aminobutyric acid responses, whereas it inhibits muscle-type nicotinic acetylcholine receptors (nAChRs) at concentrations above 10 microm. We report here that TDBzl-etomidate, a photoreactive etomidate analog, acts as a positive allosteric nAChR modulator rather than an inhibitor, and we identify its binding sites by photoaffinity labeling. TDBzl-etomidate (>10 microm) increased the submaximal response to acetylcholine (10 microm) with a 2.5-fold increase at 60 microm. At higher concentrations, it inhibited the binding of the noncompetitive antagonists [(3)H]tetracaine and [(3)H]phencyclidine to Torpedo nAChR-rich membranes (IC(50) values of 0. 8 mm). nAChR-rich membranes were photolabeled with [(3)H]TDBzl-etomidate, and labeled amino acids were identified by Edman degradation. For nAChRs photolabeled in the absence of agonist (resting state), there was tetracaine-inhibitable photolabeling of amino acids in the ion channel at positions M2-9 (deltaLeu-265) and M2-13 (alphaVal-255 and deltaVal-269), whereas labeling of alphaM2-10 (alphaSer-252) was not inhibited by tetracaine but was enhanced 10-fold by proadifen or phencyclidine. In addition, there was labeling in gammaM3 (gammaMet-299), a residue that contributes to the same pocket in the nAChR structure as alphaM2-10. The pharmacological specificity of labeling of residues, together with their locations in the nAChR structure, indicate that TDBzl-etomidate binds at two distinct sites: one within the lumen of the ion channel (labeling of M2-9 and -13), an inhibitory site, and another at the interface between the alpha and gamma subunits (labeling of alphaM2-10 and gammaMet-299) likely to be a site for positive allosteric modulation.
Peer Reviewed Yes
Published Yes
Alternative URI http://dx.doi.org/10.1074/jbc.M801332200
Copyright Statement This research was originally published in Journal of Biological Chemistry (JBC). Selvanayagam Nirthanan, Galo Garcia III, David C. Chiara, S. Shaukat Husain§, Jonathan B. Cohen, Identification of binding sites in the nicotinic acetylcholine receptor for TDBzl-etomidate, a photoreactive positive allosteric effector Journal of Biological Chemistry (JBC), 2008; 283: 22051-22062. Copyright the American Society for Biochemistry and Molecular Biology. This is the author-manuscript version of this paper. Reproduced in accordance with the copyright policy of the publisher. Please refer to the journal's website for access to the definitve version.
Volume 283
Issue Number 32
Page from 22051
Page to 22062
ISSN 1083-351X
Date Accessioned 2009-07-09
Language en_US
Research Centre Griffith Health Institute
Faculty Griffith Health Faculty
Subject Anaesthesiology; Cellular Nervous System
URI http://hdl.handle.net/10072/26747
Publication Type Journal Articles (Refereed Article)
Publication Type Code c1x

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