A point mutation in the gamma(2) subunit of gamma-aminobutyric acid type A receptors results in altered benzodiazepine binding site specificity

Benzodiazepines allosterically modulate gamma-aminobutyric acid (GABA) evoked chloride currents of gamma-aminobutyric acid type A (GABA(A)) receptors, Coexpression of either rat gamma(2) or gamma(3), in combination with alpha(1) and beta(2) subunits, results both in receptors displaying high [H-3]Ro 15-1788 affinity, However, receptors containing a gamma 3 subunit display a 178-fold reduced affinity to zolpidem as compared with gamma(2)-containing receptors. Eight chimeras between gamma(2) and gamma(3) were constructed followed by nine different point mutations in gamma(2), each to the homologous amino acid residue found in gamma(3). Chimeric or mutant gamma subunits were coexpressed with alpha(1) and beta(2) in human embryonic kidney 293 cells to localize amino acid residues responsible for the reduced zolpidem affinity, Substitution of a methionine-to-leucine at position 130 of gamma(2) (gamma(2)M130L) resulted in a 51-fold reduction in zolpidem affinity whereas the affinity to [H-3]Ro 15-1788 remained unchanged, The affinity for diazepam was only decreased by about 2-fold, The saute mutation resulted in a 9-fold increase in Cl 213572 affinity, A second mutation (gamma(2)M57I) was found to reduce zolpidem affinity by about 4-fold. Wild-type and gamma(2)M130L-containing receptors were functionally expressed in Xenopus oocytes. Upon mutation allosteric coupling between agonist and modulatory sites is preserved, Dose-response curves for zolpidem and for diazepam showed that I-he zolpidem but not the diazepam apparent affinity is drastically reduced, The apparent GABA affinity is not significantly affected by the gamma(2)M130L mutation, The identified amino acid residues may define part of the benzodiazepine binding pocket of GABA(A) receptors. As the modulatory site in the GABA(A) receptor is homologous to the GABA site, and to all agonist sites of related receptors, gamma(2)M130 may either point to a homologous region important for agonist binding in all receptors or define a new region not underlying this principle.