Relative location of transmembrane regions of GABAA receptors probed by cysteine substitution and crosslinking

Abstract

The GABAA receptor is a likely target for alcohols and volatile anesthetics. The transmembrane regions (TMs) 1, 2 and 3 provide one amino acid each that line a putative alcohol and volatile anesthetic binding site. However, the relative position of these amino acids is still uncertain, as the pocket they line could be located between or within receptor subunits. Introducing cysteines in key TM locations, we tested the proximity of cysteine pairs by applying reducing and oxidizing agents that may break or form disulfide bridges between cysteines that are close enough, usually altering GABA‐induced currents through the receptor. Wild‐type and cysteine mutant α1 and β2 GABAA receptor subunits were expressed along with wild‐type γ2 in Xenopus laevis oocytes. A cysteine located in either α1 TM1 or β2 TM2 was paired with a cysteine in different positions along β2 TM3. EC50 GABA‐induced currents were recorded before and after application of dithiothreitol or copper:phenanthroline. Three pairs of cysteines appeared to crosslink. We will test alcohol and volatile anesthetics in GABAA receptors containing these cysteine combinations, before and after application of reducing or oxidizing agents. We expect that, if the cysteines that crosslink are located in the drug binding site, the drug's effect will be decreased. The results will be used to inform structural models based on recent high‐resolution structures of related channels.