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J. Biol. Chem., Vol. 283, Issue 32, 22051-22062, August 8, 2008

This Article Full Text Full Text (PDF) Supplemental Data All Versions of this Article: 283/32/22051    most recent M801332200v1 Submit a Letter to Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Nirthanan, S. Articles by Cohen, J. B. Search for Related Content PubMed PubMed Citation Articles by Nirthanan, S. Articles by Cohen, J. B. Related Collections Related Webpages Social Bookmarking                      What's this?

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Identification of Binding Sites in the Nicotinic Acetylcholine Receptor for TDBzl-etomidate, a Photoreactive Positive Allosteric Effector^*

Selvanayagam Nirthanan¹, Galo Garcia, III, David C. Chiara, S. Shaukat Husain, and Jonathan B. Cohen²

From the Department of Neurobiology, Harvard Medical School, Boston, Massachusetts 02115 and Department of Anesthesia and Critical Care, Massachusetts General Hospital, Boston, Massachusetts 02114

Etomidate, one of the most potent general anesthetics used clinically, acts at micromolar concentrations as an anesthetic and positive allosteric modulator of -aminobutyric acid responses, whereas it inhibits muscle-type nicotinic acetylcholine receptors (nAChRs) at concentrations above 10 µM. 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 µM) increased the submaximal response to acetylcholine (10 µM) with a 2.5-fold increase at 60 µM. At higher concentrations, it inhibited the binding of the noncompetitive antagonists [³H]tetracaine and [³H]phencyclidine to Torpedo nAChR-rich membranes (IC₅₀ values of 0. 8 mM). nAChR-rich membranes were photolabeled with [³H]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 (Leu-265) and M2-13 (Val-255 and Val-269), whereas labeling of M2-10 (Ser-252) was not inhibited by tetracaine but was enhanced 10-fold by proadifen or phencyclidine. In addition, there was labeling in M3 (Met-299), a residue that contributes to the same pocket in the nAChR structure as M2-10. 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 and subunits (labeling of M2-10 and Met-299) likely to be a site for positive allosteric modulation.

Received for publication, February 20, 2008 , and in revised form, May 30, 2008.

^* This work was supported, in whole or in part, by National Institutes of Health Grant GM-58448 from the NIGMS. This work was also supported by an award to Harvard Medical School from the Howard Hughes Biomedical Research Support Program for Medical Schools. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1–S6.

¹ Supported by a Brooks Foundation Fellowship in Neurobiology.

² To whom correspondence should be addressed: Dept. of Neurobiology, Harvard Medical School, 220 Longwood Ave., Boston, MA 02115. Tel.: 617-432-1728; Fax: 617-734-7557; E-mail: jonathan_cohen{at}hms.harvard.edu.

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