Zolpidem is a potent stoichiometry-selective modulator of α1β3 GABAA receptors: evidence of a novel benzodiazepine site in the α1-α1 interface

Abstract

Zolpidem is not a typical GABAA receptor hypnotic. Unlike benzodiazepines, zolpidem modulates tonic GABA currents in the rat dorsal motor nucleus of the vagus, exhibits residual effects in mice lacking the benzodiazepine binding site, and improves speech, cognitive and motor function in human patients with severe brain injury. The receptor by which zolpidem mediates these effects is not known. In this study we evaluated binary α1β3 GABAA receptors in either the 3α1:2β3 or 2α1:3β3 subunit stoichiometry, which differ by the existence of either an α1-α1 interface, or a β3-β3 interface, respectively. Both receptor stoichiometries are readily expressed in Xenopus oocytes, distinguished from each other by using GABA, zolpidem, diazepam and Zn2+. At the 3α1:2β3 receptor, clinically relevant concentrations of zolpidem enhanced GABA in a flumazenil-sensitive manner. The efficacy of diazepam was significantly lower compared to zolpidem. No modulation by either zolpidem or diazepam was detected at the 2α1:3β3 receptor, indicating that the binding site for zolpidem is at the α1-α1 interface, a site mimicking the classical α1-γ2 benzodiazepine site. Activating α1β3 (3α1:2β3) receptors may, in part, mediate the physiological effects of zolpidem observed under distinct physiological and clinical conditions, constituting a potentially attractive drug target.

Flumazenil-Insensitive Benzodiazepine Effects in Recombinant αβ and Neuronal GABAA Receptors

“…similar to the results from α1β2 receptor expressed in xenopus oocytes [17]. The α2β2, α3β2, α4β2, α5β2 and α1β3 receptors were also activated by GABA in a concentration-dependent manner, with EC50 values of 7.05, 7.94, 0.37, 7.12 and 1.13 μmol/L (Figure 2c-g, Table 1), which were similar to those estimated in previous studies [19,[23][24][25][26]. Hill coefficient values were different among distinct receptors ( Table 1).…”

“…Binary αβ GABA A receptors are gaining increasing attention as the receptors have been reported to be expressed in the mammalian central nervous system [8,27] and certain benzodiazepines have been demonstrated to modulate some of these receptors [17][18][19][20][21]. Although functional properties of some αβ receptors have been reported [23][24][25][26][27], the variations in expression systems (Xenopus oocytes, HEK293 cells or Sf9 cells) and the methods of transfection and drug application make it difficult to compare the results from different studies.…”

“…Additionally, our previous study showed evidence of non-classical binding sites in vivo [16]. Moreover, benzodiazepines modulate α1β2 [17,18] and α1β3 [19][20][21] receptors and these two receptor subtypes were used as experimental models to study certain non-classical benzodiazepine binding sites [17,20]. However, the non-classical benzodiazepine effects are not systemically studied in αβ receptor subtypes, and the contribution of the subunits in forming the non-classical binding sites are unclear.…”

Molecular tools for GABAA receptors: High affinity ligands for β1-containing subtypes

“…At the experimental conditions used in this study, the α1βl (l = 1, 2, 3) receptors formed in the oocyte are thought to be of α1(2)βl(3) stoichiometry 27 .…”

Reducing GABAA-mediated inhibition improves forelimb motor function after focal cortical stroke in mice

“…The complex post-stroke changes in composition and localization of the GABA receptors make it diffcult to clarify the precise site of action of the GABA-acting drugs. In particular zolpidem, even if believed to work at the synapse, can act also at the extrasynaptic level81, adding complexity to the interpretation of its mechanism of action. In our experiments, reducing GABA A signaling via DMCM results in a significant gain of forelimb function after stroke, possibly by adjusting the excitation/inhibition balance in specific cortical microcircuits20.…”