Abstract

Benzodiazepines (BZDs) exert their therapeutic actions by binding to the GABA_A receptor (GABA_AR) and allosterically modulating GABA-induced chloride currents (I_GABA). A variety of ligands with divergent structures bind to the BZD site, and the structural mechanisms that couple their binding to potentiation of I_GABA are not well understood. In this study, we measured the effects of individually mutating 22 residues throughout the BZD binding pocket on the abilities of eszopiclone, zolpidem, and flurazepam to potentiate I_GABA. Wild-type and mutant α₁β₂γ₂ GABA_ARs were expressed in Xenopus laevis oocytes and analyzed using a two-electrode voltage clamp. GABA EC₅₀, BZD EC₅₀, and BZD maximal potentiation were measured. These data, combined with previous radioligand binding data describing the mutations' effects on BZD apparent binding affinities (J Neurosci 28:3490–3499, 2008; J Med Chem 51:7243–7252, 2008), were used to distinguish residues within the BZD pocket that contribute to BZD efficacy and BZD binding. We identified six residues whose mutation altered BZD maximal potentiation of I_GABA (BZD efficacy) without altering BZD binding apparent affinity, three residues whose mutation altered binding but had no effect on BZD efficacy, and four residues whose mutation affected both binding and efficacy. Moreover, depending on the BZD ligand, the effects of some mutations were different, indicating that the structural mechanisms underlying the ability of BZD ligands with divergent structures to potentiate I_GABA are distinct.