Abstract

Ligands of the benzodiazepine binding site allosterically modulate γ-aminobutyric acid_A receptors. Their binding pocket is made up of amino acid residues located on both α and γ subunits. We transiently expressed wild-type α1β2γ2 and mutant GABA_A receptors in human embryonic kidney 293 cells and determined their binding properties. Receptors containing the mutant αY209A showed ∼40-fold decrease in affinity for [³H]Ro 15–1788 and diazepam, whereas zolpidem displayed no measurable affinity. Receptors containing the mutant αY209F showed a small-to-moderate decrease in affinity for [³H]Ro 15–1788, diazepam, zolpidem, methyl-6,7-dimethoxy-4-ethyl-β-carboline-3-carboxylate, and Cl 218872, amounting to 2–8-fold. Receptors containing the mutant αY209Q appeared in the surface membrane of transfected cells, bound [³H]muscimol with wild-type affinity, but failed to bind [³H]Ro 15–1788 or [³H]flunitrazepam with detectable affinity. If these mutant receptors were expressed inXenopus laevis oocytes, the apparent affinity for GABA was only slightly decreased, whereas the ability of the currents to be stimulated by low concentrations of flunitrazepam was abolished. Receptors containing a point mutant of another amino acid residue, αT206A, surprisingly showed an increase in affinity of 5- and 16-fold, for the negative allosteric modulator methyl-6,7-dimethoxy-4-ethyl-β-carboline-3-carboxylate and the partial positive allosteric modulator Cl 218872, respectively, whereas there was only a small decrease in affinity for Ro 15–1788, diazepam, and zolpidem, amounting to 2-, 4-, and 5-fold. Both α206 and α209 are thus both important in determining the binding affinities for ligands of the benzodiazepine binding site. The residues are spaced at an interval of three amino acids and may be part of an α helix.