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J. Biol. Chem., Vol. 280, Issue 14, 13631-13640, April 8, 2005

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Gating-enhanced Accessibility of Hydrophobic Sites within the Transmembrane Region of the Nicotinic Acetylcholine Receptor's -Subunit

A TIME-RESOLVED PHOTOLABELING STUDY^*

Enrique Arevalo¶, David C. Chiara||¶, Stuart A. Forman, Jonathan B. Cohen||**, and Keith W. Miller**

From the Department of Anesthesia and Critical Care, Massachusetts General Hospital, Boston, Massachusetts 02114 and the Departments of Biological Chemistry and Molecular Pharmacology and ^||Neurobiology, Harvard Medical School, Boston, Massachusetts 02115

General anesthetics often interact more strongly with sites on open than on closed states of ligand-gated ion channels. To seek such sites, Torpedo membranes enriched in nicotinic acetylcholine receptors (nAChRs) were preincubated with the hydrophobic probe 3-(trifluoromethyl)-3-(m-iodophenyl) diazirine ([¹²⁵I]TID) and exposed to agonist for either 0 ms (closed state), 1.5 and 10 ms (activated states), 1 s (fast desensitized state), or 1 h (equilibrium or slow desensitized state) and then rapidly frozen (<1 ms) and photolabeled. Within 1.5 ms, the fractional change in photoincorporation relative to the closed state decreased to 0.7 in the - and -subunits, whereas in the -subunit, it changed little. The most dramatic change occurred in the -subunit, where it increased to 1.6 within 10 ms but fell to 0.7 during fast desensitization. Four residues in the -subunit's transmembrane domain accounted for the enhanced photoincorporation induced by a 10-ms agonist exposure both when TID was added simultaneously with agonist and when it was preincubated with membranes. In the published closed state structure, two residues (Thr²⁷⁴ and Leu²⁷⁸) are situated toward the extracellular end of helix M2, both contralateral to the ion channel and adjacent to the third residue (Phe²³²) on M1. The fourth labeled residue (Ile²⁸⁸) is toward the end of the M2-M3 loop. Contact with these residues occurs on the time scale of a rapid phase of TID inhibition in Torpedo nAChRs, suggesting the formation of a transient hydrophobic pocket between M1, M2, and M3 in the -subunit during gating.

Received for publication, December 10, 2004 , and in revised form, January 20, 2005.

^* This research was supported by National Institutes of Health Grant GM-58448, by the Department of Anesthesia and Critical Care, Massachusetts General Hospital, and by an award to the Harvard Medical School from the Howard Hughes Biomedical Research Support Program. 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.

^¶ These two authors contributed equally to this work.

^** To whom correspondence may be addressed: Jonathan B. Cohen, Dept. of Neurobiology, Harvard Medical School, Boston, MA 02115. Tel.: 617-432-1728; Fax: 617-734-7557; E-mail: jonathan_cohen{at}hms.harvard.edu. To whom correspondence may be addressed: Dept. of Anesthesia and Critical Care, Massachusetts General Hospital, 32 Fruit St., Boston, MA 02114. Tel.: 617-726-8985; Fax: 617-724-8644; E-mail: k_miller{at}helix.mgh.harvard.edu.

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