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J. Biol. Chem., Vol. 279, Issue 5, 3160-3168, January 30, 2004

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On the Benzodiazepine Binding Pocket in GABA_A Receptors^*

Dmytro Berezhnoy, Yves Nyfeler, Anne Gonthier¶, Hervé Schwob¶, Maurice Goeldner¶, and Erwin Sigel||

From the Department of Pharmacology, University of Bern, CH-3010 Bern, Switzerland and the ^¶Laboratoire de Chimie Bioorganique, Unité Mixte de Recherche 7514 CNRS, Université Louis Pasteur, Strasbourg 67401, Illkirch Cedex, France

Benzodiazepines are used for their sedative/hypnotic, anxiolytic, muscle relaxant, and anticonvulsive effects. They exert their actions through a specific high affinity binding site on the major inhibitory neurotransmitter receptor, the -aminobutyric acid, type A (GABA_A) receptor channel, where they act as positive allosteric modulators. To start to elucidate the relative positioning of benzodiazepine binding site ligands in their binding pocket, GABA_A receptor residues thought to reside in the site were individually mutated to cysteine and combined with benzodiazepine analogs carrying substituents reactive to cysteine. Direct apposition of such reactive partners is expected to lead to an irreversible site-directed reaction. We describe here the covalent interaction of ₁H101C with a reactive group attached to the C-7 position of diazepam. This interaction was studied at the level of radioactive ligand binding and at the functional level using electrophysiological methods. Covalent reaction occurs concomitantly with occupancy of the binding pocket. It stabilizes the receptor in its allosterically stimulated conformation. Covalent modification is not observed in wild type receptors or when using mutated ₁H101C-containing receptors in combination with the reactive ligand pre-reacted with a sulfhydryl group, and the modification rate is reduced by the binding site ligand Ro15-1788. We present in addition evidence that ₂Ala-79 is probably located in the access pathway of the ligand to its binding pocket.

Received for publication, October 16, 2003 , and in revised form, November 11, 2003.

^* This work was supported by Swiss National Science Foundation Grant 3100-064789.01/1. 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.

Present address: Max Planck Institute of Immunobiology, D-79108 Freiburg, Germany.

^|| To whom correspondence should be addressed. Tel.: 41-31-632-3281; Fax: 41-31-632-4992; E-mail: erwin.sigel{at}pki.unibe.ch.

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