A point mutation in the γ2 subunit of γ-aminobutyric acid type A receptors results in altered benzodiazepine binding site specificity

Benzodiazepines allosterically modulate γ-aminobutyric acid (GABA) evoked chloride currents of γ-aminobutyric acid type A (GABA_A) receptors. Coexpression of either rat γ₂ or γ₃, in combination with α₁ and β₂ subunits, results both in receptors displaying high [³H]Ro 15-1788 affinity. However, receptors containing a γ₃ subunit display a 178-fold reduced affinity to zolpidem as compared with γ₂-containing receptors. Eight chimeras between γ₂ and γ₃ were constructed followed by nine different point mutations in γ₂, each to the homologous amino acid residue found in γ₃. Chimeric or mutant γ subunits were coexpressed with α₁ and β₂ 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 γ₂ (γ₂M130L) resulted in a 51-fold reduction in zolpidem affinity whereas the affinity to [³H]Ro 15-1788 remained unchanged. The affinity for diazepam was only decreased by about 2-fold. The same mutation resulted in a 9-fold increase in Cl 218872 affinity. A second mutation (γ₂M57I) was found to reduce zolpidem affinity by about 4-fold. Wild-type and γ₂M130L-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 the zolpidem but not the diazepam apparent affinity is drastically reduced. The apparent GABA affinity is not significantly affected by the γ₂M130L 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, γ₂M130 may either point to a homologous region important for agonist binding in all receptors or define a new region not underlying this principle.