Point mutations of the alpha 1 beta 2 gamma 2 gamma-aminobutyric acid(A) receptor affecting modulation of the channel by ligands of the benzodiazepine binding site

Clinically relevant benzodiazepines allosterically stimulate neurotransmitter-evoked chloride currents at the gamma-aminobutyric acid type A (GABA(A)) receptor. Rat wild-type or mutated alpha 1, beta 2, and gamma 2S subunits were coexpressed in Xenopus oocytes and investigated with electrophysiological techniques. Point mutations in two subunits were identified that affect the response of gamma-aminobutyric acid (GABA)-induced currents by benzodiazepines. Mutation of one of three amino acid residues to alanine (alpha Tyr(161) and alpha Thr(206)) or leucine (gamma Phe(77)) resulted in a similar to 3-fold increase in potentiation by diazepam. The response to zolpidem was increased in two mutant channels containing the mutated alpha subunit but was nearly absent in channels containing the mutated gamma subunit. In the former cases, methyl-6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate (DMCM) acted as a negative allosteric modulator of the channel, much stronger than in the wild-type channel, whereas there was no significant difference to the wild-type channel in the latter case. Thus, the mutant gamma subunit has different functional consequences for the Various types of ligand of the benzodiazepine binding site. All three amino acid residues, alpha Tyr(161), alpha Thr(206), and gamma Phe(77), are let close or identical to homologous residues that are implicated in GABA binding. If the residues binding the channel agonist GABA are located at subunit interfaces, the residues influencing the benzodiazepine effects must also be located at subunit interfaces.