Abstract
Alcohols and inhaled anesthetics modulate GABA(A) receptor (GABA(A)R) function via putative binding sites within the transmembrane regions. The relative position of the amino acids lining these sites could be either inter- or intra-subunit. We introduced cysteines in relevant TM locations and tested the proximity of cysteine pairs using oxidizing and reducing agents to induce or break disulfide bridges between cysteines, and thus change GABA-mediated currents in wild-type and mutant α1β2γ2 GABA(A)Rs expressed in Xenopus laevis oocytes. We tested for: (i) inter-subunit cross-linking: a cysteine located in α1TM1 [either α1(Q229C) or α1(L232C)] was paired with a cysteine in different positions of β2TM2 and TM3; (ii) intra-subunit cross-linking: a cysteine located either in β2TM1 [β2(T225C)] or in TM2 [β2(N265C)] was paired with a cysteine in different locations along β2TM3. Three inter-subunit cysteine pairs and four intra-subunits cross-linked. In three intra-subunit cysteine combinations, the alcohol effect was reduced by oxidizing agents, suggesting intra-subunit alcohol binding. We conclude that the structure of the alcohol binding site changes during activation and that potentiation or inhibition by binding at inter- or intra-subunit sites is determined by the specific receptor and ligand.