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J. Biol. Chem., Vol. 283, Issue 5, 2702-2708, February 1, 2008

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The F-loop of the GABA_A Receptor ₂ Subunit Contributes to Benzodiazepine Modulation^*

From the Department of Biochemistry, University of Cambridge, Cambridge CB2 1QW, United Kingdom

GABA_A receptors can be modulated by benzodiazepines, although these compounds do not directly activate or inhibit the receptors. The prototypic benzodiazepine, diazepam, potentiates responses to GABA in GABA_A receptors that contain a subunit. Here we have used mutagenesis, radioligand binding, voltage clamp electrophysiology, and homology modeling to probe the role of the F-loop residues Asp¹⁹²-Arg¹⁹⁷ in the GABA_A receptor ₂ subunit in diazepam potentiation of the GABA response. Substitution of all of these residues with Ala and/or a residue with similar chemical properties to the wild type residue decreased the level of diazepam potentiation, and one mutation (D192A) resulted in its complete ablation. None of the mutations changed the GABA EC₅₀ or the [³H]flumazenil binding affinity, suggesting they do not affect GABA or benzodiazepine binding characteristics; we therefore propose that they are involved in the diazepam-mediated conformational change that results in an increased response to GABA. Homology models of the receptor binding pocket in agonist-bound and unbound states suggest that the F-loop is flexible and has different orientations in the two states. Considering our data in relation to these models, we find that the F-loop residues could contribute to hydrogen bond networks and hydrophobic interactions with neighboring residues that change during receptor activation.

Received for publication, July 11, 2007 , and in revised form, October 30, 2007.

^* This work was supported in part by the Wellcome Trust (to S. C. R. L.). 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.

¹ Recipient of a CASE studentship with Merck Sharp and Dohme from the Biotechnology and Biological Sciences Research Council. Present address: The Salk Institute, La Jolla, CA 92037.

² To whom correspondence should be addressed: Dept. of Biochemistry, University of Cambridge, Tennis Court Rd., Cambridge, UK. Tel.: 44-1223-765950; Fax: 44-1223-333345; E-mail: sl120{at}cam.ac.uk.

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