Different Residues in the GABAA Receptor Benzodiazepine Binding Pocket Mediate Benzodiazepine Efficacy and Binding□S

Benzodiazepines (BZDs) exert their therapeutic actions by binding to the GABAA receptor (GABAAR) and allosterically modulating GABA-induced chloride currents (IGABA). A variety of ligands with divergent structures bind to the BZD site, and the structural mechanisms that couple their binding to poten-tiation of IGABA 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 eszopi-clone, zolpidem, and flurazepam to potentiate IGABA. Wild-type and mutant 122 GABAARs were expressed in Xenopus lae-vis 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 bind-ing. We identified six residues whose mutation altered BZD maximal potentiation of IGABA (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 mecha-nisms underlying the ability of BZD ligands with divergent struc-tures to potentiate IGABA are distinct