Probing the molecular basis for affinity/potency- and efficacy-based subtype-selectivity exhibited by benzodiazepine-site modulators at GABAA receptors

The extracellular α⁽⁺⁾/γ₂^(-) interface in the α_1,2,3,5βγ₂ GABA_A receptor harbours the allosteric binding site targeted by benzodiazepines and newer generations of subtype-selective modulators. We have probed the molecular determinants for the affinity/potency-based α₁-preference exhibited by the hypnotic zolpidem (Ambien®, Stilnox®) and the efficacy-based α₃-over-α₁ selectivity displayed by the analgesic NS11394. Binding affinities and functional properties of the modulators were characterized at wild-type, concatenated, mutant and chimeric α_1,3β₂γ_2S receptors expressed in tsA201 cells and Xenopus oocytes by [³H]flumazenil binding and two-electrode voltage clamp electrophysiology. Substitution of Gly²⁰¹ in α₁ with the corresponding Glu in α₃ completely eliminated the α₁-over-α₃ preference exhibited by zolpidem. In contrast, the reverse α₃-E225G mutation did not yield corresponding increases in the binding affinity or modulatory potency of zolpidem at α₃β₂γ_2S, and two additional molecular elements in the extracellular domain of the α-subunit were found also to contribute to its α₁-preference. Interestingly, the α₁-Gly²⁰¹/α₃-Glu²²⁵ residue was also a key determinant of the efficacy-based α₃-over-α₁ selectivity exhibited by NS11394, and a pronounced correlation existed between the side-chain bulkiness of this residue and the modulatory efficacy of NS11394 at the receptor. The subtype-selectivity determinants identified for zolpidem and NS11394 were found also to apply in different degrees to the α₁-preferring modulator indiplon and the α₃-over-α₁ selective modulator L-838,417, respectively. In conclusion, the molecular origins of subtype-selectivity exhibited by benzodiazepine-site modulators at the α_1,2,3,5βγ₂ GABA_A receptor seem more complex than previously appreciated, and the importance of the α₁-Gly²⁰¹/α₃-Glu²²⁵ residue for both potency- and efficacy-based subtype-selective modulation through this site is likely to be rooted in different molecular mechanisms.