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J. Biol. Chem., Vol. 280, Issue 43, 35836-35843, October 28, 2005

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A Picrotoxin-specific Conformational Change in the Glycine Receptor M2–M3 Loop^*

From the School of Biomedical Sciences, University of Queensland, Brisbane, Queensland 4072, Australia

The external loop linking the M2 and M3 transmembrane domains is crucial for coupling agonist binding to channel gating in the glycine receptor chloride channel (GlyR). A substituted cysteine accessibility scan previously showed that glycine activation increased the surface accessibility of 6 contiguous residues (Arg²⁷¹– Lys²⁷⁶) toward the N-terminal end of the homomeric 1 GlyR M2–M3 loop. In the present study we used a similar approach to determine whether the allosteric antagonist, picrotoxin, could impose conformational changes to this domain that cannot be induced by varying agonist concentrations alone. Picrotoxin slowed the reaction rate of a sulfhydryl-containing compound (MTSET) with A272C, S273C, and L274C. Before interpreting this as a picrotoxin-specific conformational change, it was necessary to eliminate the possibility of steric competition between picrotoxin and MTSET. Accordingly, we showed that picrotoxin and the structurally unrelated blocker, bilobalide, were both trapped in the R271C GlyR in the closed state and that a point mutation to the pore-lining Thr⁶' residue abolished inhibition by both compounds. We also demonstrated that the picrotoxin dissociation rate was linearly related to the channel open probability. These observations constitute a strong case for picrotoxin binding in the pore. We thus conclude that the picrotoxin-specific effects on the M2–M3 loop are mediated allosterically. This suggests that the M2–M3 loop responds differently to the occupation of different binding sites.

Received for publication, June 20, 2005 , and in revised form, August 16, 2005.

^* This work was supported by the Australian Research Council. 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. Tel.: 617-3365-3157; Fax: 617-3365-1766; E-mail: j.lynch{at}uq.edu.au.

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