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

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Abstract
Benzodiazepines allosterically modulate gamma-aminobutyric acid (GABA) evoked chloride currents of gamma-aminobutyric acid type A (GABAA) receptors. Coexpression of either rat gamma2 or gamma3, in combination with alpha1 and beta2 subunits, results both in receptors displaying high [3H]Ro 15-1788 affinity. However, receptors containing a gamma3 subunit display a 178-fold reduced affinity to zolpidem as compared with gamma2-containing receptors. Eight chimeras between gamma2 and gamma3 were constructed followed by nine different point mutations in gamma2, each to the homologous amino acid residue found in gamma3. Chimeric or mutant gamma subunits were coexpressed with alpha1 and beta2 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 gamma2 (gamma2M130L) 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 (gamma2M57I) was found to reduce zolpidem affinity by about 4-fold. Wild-type and gamma2M130L-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 gamma2M130L mutation. The identified amino acid residues may define part of the benzodiazepine binding pocket of GABAA receptors. As the modulatory site in the GABAA receptor is homologous to the GABA site, and to all agonist sites of related receptors, gamma2M130 may either point to a homologous region important for agonist binding in all receptors or define a new region not underlying this principle.