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

The extracellular a((+))/gamma((-))(2) interface in the alpha(1,2,3,4,5)beta gamma(2) 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 alpha(1)-preference exhibited by the hypnotic zolpidem (Ambien (R), Stilnox (R)) and the efficacy-based alpha(3)-over-alpha(1) selectivity displayed by the analgesic NS11394. Binding affinities and functional properties of the modulators were characterized at wild-type, concatenated, mutant and chimeric alpha(1,3)beta(2)gamma(2s) receptors expressed in tsA201 cells and Xenopus oocytes by [H-3]flumazenil binding and two-electrode voltage clamp electrophysiology. Substitution of G1y(201) in al with the corresponding Glu in alpha(3) completely eliminated the alpha(1)-over-alpha(3) preference exhibited by zolpidem. In contrast, the reverse alpha(3)-E225G mutation did not yield corresponding increases in the binding affinity or modulatory potency of zolpidem at alpha(3)beta(2)gamma(2s), and two additional molecular elements in the extracellular domain of the a-subunit were found also to contribute to its as-preference. Interestingly, the alpha(1)-G1y(201)/alpha(3)-Glu(225) residue was also a key determinant of the efficacy-based alpha(3)-over-alpha(1) selectivity exhibited by N511394, 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 alpha(1)-preferring modulator indiplon and the alpha(3)-over-alpha(1) selective modulator L-838,417, respectively. In conclusion, the molecular origins of subtype-selectivity exhibited by benzodiazepine-site modulators at the alpha(1,2,3,5)beta gamma(2) GABAA receptor seem more complex than previously appreciated, and the importance of the alpha(1)-Gly(201)/alpha(3)-Glu(225) residue for both potency- and efficacy-based subtype-selective modulation through this site is likely to be rooted in different molecular mechanisms.