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J. Biol. Chem., Vol. 283, Issue 41, 27698-27706, October 10, 2008
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Roles for Loop 2 Residues of 
1 Glycine Receptors in Agonist Activation*
1
From the
Alcohol and Brain Research Laboratory, Department of Pharmacology and Pharmaceutical Sciences, School of Pharmacy and Neuroscience Graduate Program, University of Southern California, Los Angeles, California 90089, the ¶Department of Anesthesia and Beckman Program for Molecular and Genetic Medicine, Stanford School of Medicine, Stanford, California 94305, and the ||Department of Anesthesia, Palo Alto Veterans Affairs Health Care System, Palo Alto, California 94304
The present study tested the hypothesis that several residues in Loop 2 of 
1 glycine receptors (GlyRs) play important roles in mediating the transduction of agonist activation to channel gating. This was accomplished by investigating the effect of cysteine point mutations at positions 50–60 on glycine responses in 1GlyRs using two-electrode voltage clamp of Xenopus oocytes. Cysteine substitutions produced position-specific changes in glycine sensitivity that were consistent with a β-turn structure of Loop 2, with odd-numbered residues in the β-turn interacting with other agonist-activation elements at the interface between extracellular and transmembrane domains. We also tested the hypothesis that the charge at position 53 is important for agonist activation by measuring the glycine response of wild type (WT) and E53C GlyRs exposed to methanethiosulfonate reagents. As earlier, E53C GlyRs have a significantly higher EC50 than WT GlyRs. Exposing E53C GlyRs to the negatively charged 2-sulfonatoethyl methanethiosulfonate, but not neutral 2-hydroxyethyl methanethiosulfonate, positively charged 2-aminoethyl methanethiosulfonate, or 2-trimethylammonioethyl methanethiosulfonate, decreased the glycine EC50 to resemble WT GlyR responses. Exposure to these reagents did not significantly alter the glycine EC50 for WT GlyRs. The latter findings suggest that the negative charge at position 53 is important for activation of GlyRs through its interaction with positive charge(s) in other neighboring agonist activation elements. Collectively, the findings provide the basis for a refined molecular model of 1GlyRs based on the recent x-ray structure of a prokaryotic pentameric ligand-gated ion channel and offer insight into the structure-function relationships in GlyRs and possibly other ligand-gated ion channels.
Received for publication, March 27, 2008 , and in revised form, June 20, 2008.
* This work was supported, in whole or in part, by National Institutes of Health, NIAAA, Grants AA03972 (to R. L. A.), AA013890 (to D. L. D.), AA013922 (to D. L. D.), and AA013378 (to J. R. T.) and the University of Southern California School of Pharmacy. This work was conducted as partial fulfillment of the requirements for the Ph.D. degree in Neuroscience, University of Southern California (D. K. C.). Portions of these findings were presented at the Annual Meeting of the Society for Neuroscience in Atlanta, GA (Program 233.7/D32, 2006 Neuroscience Meeting Planner, Atlanta, GA, Society for Neuroscience). 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 Southern California, School of Pharmacy, 1985 Zonal Ave. PSC 500, Los Angeles, CA 90089. Tel.: 323-442-1427; Fax: 323-442-1704; E-mail: ddavies{at}usc.edu.
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