Received for publication January 3, 2008.
Revised April 17, 2008.
Accepted for publication April 18, 2008.

Individually monitoring ligand-induced changes in the structure of the GABA_A receptor at benzodiazepine binding site and non-binding site interfaces

Abstract

The mechanisms by which the GABA and benzodiazepine (BZD) binding sites of the GABA-A receptor are allosterically coupled remain elusive. Here, we separately monitored ligand-induced structural changes in the BZD binding site (/ interface) and at aligned positions in the / interface. ₁H101 and surrounding residues were individually mutated to cysteine and expressed with wild-type ₂ and ₂ subunits in Xenopus oocytes. The accessibilities of introduced cysteines to modification by 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, 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, SR-95531 (a GABA binding site antagonist) decreased the rate, whereas pentobarbital had no effect. Modification of ₁H101 at the / interface was significantly slower than modification of ₁H101 at the BZD-site and the presence of GABA or flurazepam had no effect on its accessibility indicating the physico-chemical environments of the / and / 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.

Key words: GABAA, GABAC, Ion channel regulation, Structure-activity relationships and modeling, Func. analysis receptor/ion channel mutants, Barbiturates, Benzodiazepines