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The Journal of Neuroscience, November 8, 2006, 26(45):11599-11605; doi:10.1523/JNEUROSCI.3467-06.2006

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Cellular/Molecular
Identification of a GABA_A Receptor Anesthetic Binding Site at Subunit Interfaces by Photolabeling with an Etomidate Analog

Guo-Dong Li,^1 * David C. Chiara,^2 * Gregory W. Sawyer,¹ S. Shaukat Husain,³ Richard W. Olsen,¹ and Jonathan B. Cohen²

¹Department of Molecular and Medical Pharmacology, Geffen School of Medicine, University of California at Los Angeles, Los Angeles, California 90095, ²Department of Neurobiology, Harvard Medical School, Boston, Massachusetts 02115, and ³Department of Anesthesia and Critical Care, Massachusetts General Hospital, Boston, Massachusetts 02114

Correspondence should be addressed to either of the following: Richard W. Olsen, Department of Molecular and Medical Pharmacology, Geffen School of Medicine, University of California at Los Angeles, Los Angeles, CA 90095, Email: rolsen{at}mednet.ucla.edu; or Jonathan B. Cohen, Department of Neurobiology, Harvard Medical School, Boston, MA 02115, Email: jonathan_cohen{at}hms.harvard.edu

General anesthetics, including etomidate, act by binding to and enhancing the function of GABA type A receptors (GABA_ARs), which mediate inhibitory neurotransmission in the brain. Here, we used a radiolabeled, photoreactive etomidate analog ([³H]azietomidate), which retains anesthetic potency in vivo and enhances GABA_AR function in vitro, to identify directly, for the first time, amino acids that contribute to a GABA_AR anesthetic binding site. For GABA_ARs purified by affinity chromatography from detergent extracts of bovine cortex, [³H]azietomidate photoincorporation was increased by GABA and inhibited by etomidate in a concentration-dependent manner (IC₅₀ = 30 µM). Protein microsequencing of fragments isolated from proteolytic digests established photolabeling of two residues: one within the M1 transmembrane helix at 1Met-236 (and/or the homologous methionines in 2,3,5), not previously implicated in etomidate function, and one within the M3 transmembrane helix at 3Met-286 (and/or the homologous methionines in 1,2), an etomidate sensitivity determinant. The pharmacological specificity of labeling indicates that these methionines contribute to a single binding pocket for etomidate located in the transmembrane domain at the interface between and subunits, in what is predicted by structural models based on homology with the nicotinic acetylcholine receptor to be a water-filled pocket 50 Å below the GABA binding site. The localization of the etomidate binding site to an intersubunit, not an intrasubunit, binding pocket is a novel conclusion that suggests more generally that the localization of drug binding sites to subunit interfaces may be a feature not only for GABA and benzodiazepines but also for etomidate and other intravenous and volatile anesthetics.

Key words: GABA_A receptor; anesthesia; structure; photolabeling; nicotinic receptor; muscimol; binding; etomidate

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Received Aug. 10, 2006; revised Sept. 25, 2006; accepted Sept. 28, 2006.

Correspondence should be addressed to either of the following: Richard W. Olsen, Department of Molecular and Medical Pharmacology, Geffen School of Medicine, University of California at Los Angeles, Los Angeles, CA 90095, Email: rolsen{at}mednet.ucla.edu; or Jonathan B. Cohen, Department of Neurobiology, Harvard Medical School, Boston, MA 02115, Email: jonathan_cohen{at}hms.harvard.edu

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