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J. Biol. Chem., Vol. 283, Issue 22, 15250-15257, May 30, 2008

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-Aminobutyric Acid (GABA) and Pentobarbital Induce Different Conformational Rearrangements in the GABA_A Receptor ₁ and β₂ Pre-M1 Regions^*

Jose Mercado and Cynthia Czajkowski¹

From the Department of Physiology and the Neuroscience Training Program, University of Wisconsin-Madison, Madison, Wisconsin 53706

-Aminobutyric acid (GABA) binding to GABA_A receptors (GABA_ARs) triggers conformational movements in the ₁ and β₂ pre-M1 regions that are associated with channel gating. At high concentrations, the barbiturate pentobarbital opens GABA_AR channels with similar conductances as GABA, suggesting that their open state structures are alike. Little, however, is known about the structural rearrangements induced by barbiturates. Here, we examined whether pentobarbital activation triggers movements in the GABA_AR pre-M1 regions. ₁β₂ GABA_ARs containing cysteine substitutions in the pre-M1 ₁ (K219C, K221C) and β₂ (K213C, K215C) subunits were expressed in Xenopus oocytes and analyzed using two-electrode voltage clamp. The cysteine substitutions had little to no effect on GABA and pentobarbital EC₅₀ values. Tethering chemically diverse thiol-reactive methanethiosulfonate reagents onto ₁K219C and ₁K221C affected GABA- and pentobarbital-activated currents differently, suggesting that the pre-M1 structural elements important for GABA and pentobarbital current activation are distinct. Moreover, pentobarbital altered the rates of cysteine modification by methanethiosulfonate reagents differently than GABA. For ₁K221Cβ₂ receptors, pentobarbital decreased the rate of cysteine modification whereas GABA had no effect. For ₁β₂K215C receptors, pentobarbital had no effect whereas GABA increased the modification rate. The competitive GABA antagonist SR-95531 and a low, non-activating concentration of pentobarbital did not alter their modification rates, suggesting that the GABA- and pentobarbital-mediated changes in rates reflect gating movements. Overall, the data indicate that the pre-M1 region is involved in both GABA- and pentobarbital-mediated gating transitions. Pentobarbital, however, triggers different movements in this region than GABA, suggesting their activation mechanisms differ.

Received for publication, October 18, 2007 , and in revised form, March 12, 2008.

^* This work was supported, in whole or in part, by National Institutes of Health Grant NS34727 from NINDS (to C. C.). This work was also supported by the Diversity Program in Neuroscience of the American Psychological Association (to J. M.). 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 To whom correspondence should be addressed: University of Wisconsin-Madison, 601 Science Dr., Madison, WI 53711. Tel.: 608-265-5863; E-mail: czajkowski{at}physiology.wisc.edu.

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