THE UNIQUE EXTRACELLULAR DISULFIDE LOOP OF THE GLYCINE RECEPTOR IS A PRINCIPAL LIGAND-BINDING ELEMENT

A loop structure, formed by the putative disulfide bridging of Cys198 and Cys209, is a principal element of the ligand binding site in the g lycine receptor (GlyR). Disruption of the loop's tertiary structure by Ser mutations of these Cys residues either prevented receptor assembl y on the cell surface, or created receptors unable to be activated by agonists or to bind the competitive antagonist, strychnine. Mutation o f residues Lys200, Tyr202 and Thr204 within this loop reduced agonist binding and channel activation sensitivities by up to 55-, 520- and 19 0-fold, respectively, without altering maximal current sizes, and muta tions of Lys200 and Tyr202 abolished strychnine binding to the recepto r. Removal of the hydroxyl moiety from Try202 by mutation to Phe profo undly reduced agonist sensitivity, whilst removal of the benzene ring abolished strychnine binding, thus demonstrating that Tyr202 is crucia l for both agonist and antagonist binding to the GlyR. Tyr202 also inf luences receptor assembly on the cell surface, with only large chain s ubstitutions (Phe, Leu and Arg, but not Thr, Ser and Ala) forming func tional receptors. Our data demonstrate the presence of a second ligand binding site in the GlyR, consistent with the three-loop model of lig and binding to the ligand-gated ion channel superfamily.