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J. Biol. Chem., Vol. 281, Issue 25, 17034-17043, June 23, 2006

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An Asymmetric Contribution to -Aminobutyric Type A Receptor Function of a Conserved Lysine within TM2–3 of 1, 2, and 2 Subunits^*

Tim G. Hales¹, Tarek Z. Deeb², Haiyan Tang^¶2, Karen A. Bollan^¶, Dale P. King^¶, Sara J. Johnson^¶, and Christopher N. Connolly^¶

From the Department of Pharmacology & Physiology and the Department of Anesthesiology & Critical Care Medicine, The George Washington University, Washington, D. C. 20037 and the ^¶Department of Pharmacology & Neuroscience, Ninewells Medical School, University of Dundee, Dundee DD1 9SY, Scotland, United Kingdom

Mutations that impair the expression and/or function of -aminobutyric acid type A (GABA_A) receptors can lead to epilepsy. The familial epilepsy 2(K289M) mutation affects a basic residue conserved in the TM2–3 linker of most GABA_A subunits. We investigated the effect on expression and function of the Lys Met mutation in mouse 1(K278M), 2(K274M), and 2(K289M) subunits. Compared with cells expressing wild-type and 122(K289M) receptors, cells expressing 1(K278M)22 and 12(K274M)2 receptors exhibited reduced agonist-evoked current density and reduced GABA potency, with no change in single channel conductance. The low current density of 12(K274M)2 receptors coincided with reduced surface expression. By contrast the surface expression of 1(K278M)22 receptors was similar to wild-type and 122(K289M) receptors suggesting that the 1(K278M) impairs function. In keeping with this interpretation GABA-activated channels mediated by 1(K278M)22 receptors had brief open times. To a lesser extent 2(K289M) also reduced mean open time, whereas 2(K274M) had no effect. We used propofol as an alternative GABA_A receptor agonist to test whether the functional deficits of mutant subunits were specific to GABA activation. Propofol was less potent as an activator of 1(K278M)22 receptors. By contrast, neither 2(K274M) nor 2(K289M) affected the potency of propofol. The 2(K274M) construct was unique in that it reduced the efficacy of propofol activation relative to GABA. These data suggest that the 1 subunit Lys-278 residue plays a pivotal role in channel gating that is not dependent on occupancy of the GABA binding site. Moreover, the conserved TM2–3 loop lysine has an asymmetric function in different GABA_A subunits.

Received for publication, April 13, 2006

^* This work has been supported by the Biotechnology and Biological Sciences and Research Council (Grant 94/C17336 awarded to C. N. C.), Tenovus Scotland (to C. N. C.), Anonymous Trust (to C. N. C.), and the National Institutes of Health (Grant GM058037 awarded to T. G. H.). 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.

² Both authors contributed equally to this work.

¹ To whom correspondence should be addressed: Dept. of Pharmacology, Medical Center, The George Washington University, 2300 Eye St. NW, Washington, D. C. 20037. Tel.: 202-994-3546; Fax: 202-994-2870; E-mail: phmtgh{at}gwumc.edu.

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