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J. Biol. Chem., Vol. 281, Issue 50, 38871-38878, December 15, 2006

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Spontaneous Mobility of GABA_A Receptor M2 Extracellular Half Relative to Noncompetitive Antagonist Action^*

Ligong Chen, Kathleen A. Durkin, and John E. Casida¹

From the Environmental Chemistry and Toxicology Laboratory, Department of Environmental Science, Policy, and Management and the Molecular Graphics Facility, College of Chemistry, University of California, Berkeley, California 94720

The -aminobutyric acid type A receptor ₃ homopentamer is spontaneously open and highly sensitive to many noncompetitive antagonists(NCAs) and Zn²⁺.OurearlierstudyoftheM2cytoplasmic half (-1' to 10') established a model in which NCAs bind at porelining residues Ala²', Thr⁶', and Leu⁹'. To further define transmembrane 2 (M2) structure relative to NCA action, we extended the Cys scanning to the extra cellular half of the ₃ homopentamer (11' to 20'). Spontaneous disulfides formed with T13'C, L18'C, and E20'C from M2/M2 cross-linking and with I14'C (weak), H17'C, and R19'Con bridging M2/M3 intersubunits, based on single (M2 Cys only) and dual (M2 Cys plus M3 C289S) mutations. Induced disulfides also formed with T16'C, but there were few or none with M11'C, T12'C, and N15'C. These findings show conformational flexibility/mobility in the M2 extracellular half 17' to 20' region interpreted as a deformed -like conformation in the open channel. The NCA radioligands used were [³H]1-(4-ethynylphenyl)-4-n-propyl-2,6,7-trioxabicyclo[2.2.2]octane ([³H]EBOB) and [³H]3,3-bis-trifluoromethylbicyclo[2.2.1]heptane-2,2-dicarbonitrile with essentially the same results. NCA binding was disrupted by individual Cys substitutions at 13',14',16',17', and 19'. The inactivity of T13'C/T13'S may have been due to disturbance of the channel gate; I14'S and T16'S showed much better binding activity than their Cys counterparts, and the low activities of H17'C and R19'C were reversed by dithiothreitol. Zn²⁺ potency for inhibition of [³H]EBOB binding was lowered 346-fold by the mutation H17'A. We propose that NCAs enter their binding site both directly, through the channel pore, and indirectly, through the water cavity of adjacent subunits.

Received for publication, August 30, 2006 , and in revised form, October 6, 2006.

^* This work was supported by the William Mureice Hoskins Chair in Chemical and Molecular Entomology (to J. E. C.), NIEHS, National Institutes of Health, Grant ES08419 (to J. E. C.), and National Science Foundation Grant CHE-0233882 (to K. A. D.). 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: Environmental Chemistry and Toxicology Laboratory, 114 Wellman Hall, University of California, Berkeley, CA 94720-3112. Tel.: 510-642-5424; Fax: 510-642-6497; E-mail: ectl{at}nature.berkeley.edu.

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