The extracellular α/γ interface in the αβγ GABA 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 α-preference exhibited by the hypnotic zolpidem (Ambien®， Stilnox®) and the efficacy-based α-over-α selectivity displayed by the analgesic NS11394. Binding affinities and functional properties of the modulators were characterized at wild-type， concatenated， mutant and chimeric αβγ receptors expressed in tsA201 cells and Xenopus oocytes by [H]flumazenil binding and two-electrode voltage clamp electrophysiology. Substitution of Gly in α with the co... More
The extracellular α/γ interface in the αβγ GABA 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 α-preference exhibited by the hypnotic zolpidem (Ambien®， Stilnox®) and the efficacy-based α-over-α selectivity displayed by the analgesic NS11394. Binding affinities and functional properties of the modulators were characterized at wild-type， concatenated， mutant and chimeric αβγ receptors expressed in tsA201 cells and Xenopus oocytes by [H]flumazenil binding and two-electrode voltage clamp electrophysiology. Substitution of Gly in α with the corresponding Glu in α completely eliminated the α-over-α preference exhibited by zolpidem. In contrast， the reverse α-E225G mutation did not yield corresponding increases in the binding affinity or modulatory potency of zolpidem at αβγ， and two additional molecular elements in the extracellular domain of the α-subunit were found also to contribute to its α-preference. Interestingly， the α-Gly/α-Glu residue was also a key determinant of the efficacy-based α-over-α 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 α-preferring modulator indiplon and the α-over-α selective modulator L-838，417， respectively. In conclusion， the molecular origins of subtype-selectivity exhibited by benzodiazepine-site modulators at the αβγ GABA receptor seem more complex than previously appreciated， and the importance of the α-Gly/α-Glu residue for both potency- and efficacy-based subtype-selective modulation through this site is likely to be rooted in different molecular mechanisms.
