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Individually Monitoring Ligand-Induced Changes in the Structure of the GABA_A Receptor at Benzodiazepine Binding Site and Non-Binding-Site Interfaces

Neuroscience Training Program (L.M.S., C.C.), and Department of Physiology (C.C.), University of Wisconsin-Madison, Madison, Wisconsin

The mechanisms by which the GABA and benzodiazepine (BZD) binding sites of the GABA-A receptor are allosterically coupled remain elusive. In this study, we separately monitored ligand-induced structural changes in the BZD binding site (/ interface) and at aligned positions in the /β interface. ₁His101 and surrounding residues were individually mutated to cysteine and expressed with wild-type β2 and 2 subunits in Xenopus laevis oocytes. The accessibilities of introduced cysteines to modification by methanethiosulfonate ethylammonium (MTSEA)-Biotin were measured in the presence and absence of GABA-site agonists, antagonists, BZDs, and pentobarbital. The presence of flurazepam or the BZD-site antagonist flumazenil (Ro15-1788) decreased the rate of modification of ₁H101C at the BZD binding site. GABA and muscimol each increased MTSEA-Biotin modification of ₁H101C located at the BZD-site, gabazine (SR-95531, a GABA binding site antagonist) decreased the rate, whereas pentobarbital had no effect. Modification of ₁H101C at the /β interface was significantly slower than modification of ₁H101C at the BZD site, and the presence of GABA or flurazepam had no effect on its accessibility, indicating the physicochemical environments of the / and /β interfaces are different. The data are consistent with the idea that GABA-binding site occupation by agonists causes a GABA binding cavity closure that is directly coupled to BZD binding cavity opening, and GABA-site antagonist binding causes a movement linked to BZD binding cavity closure. Pentobarbital binding/gating resulted in no observable movements in the BZD binding site near ₁H101C, indicating that structural mechanisms underlying allosteric coupling between the GABA and BZD binding sites are distinct.

Address correspondence to: Dr. Cynthia Czajkowski, Dept. of Physiology, University of Wisconsin, 601 Science Drive, Madison, WI 53711. E-mail: czajkowski{at}physiology.wisc.edu