Classic Benzodiazepines Modulate the Open–Close Equilibrium in α1β2γ2Lγ-Aminobutyric Acid Type A Receptors

Background Classic benzodiazepine agonists induce their clinical effects by binding to a site on gamma-aminobutyric acid type A (GABAA) receptors and enhancing receptor activity. There are conflicting data regarding whether the benzodiazepine site is allosterically coupled to gamma-aminobutyric acid binding versus the channel open-close (gating) equilibrium. The authors tested the hypothesis that benzodiazepine site ligands modulate alpha1beta2gamma2L GABAA receptor gating both in the absence of orthosteric agonists and when the orthosteric sites are occupied. Methods GABAA receptors were recombinantly expressed in Xenopus oocytes and studied using two-microelectrode voltage clamp electrophysiology. To test gating effects in the absence of orthosteric agonist, the authors used spontaneously active GABAA receptors containing a leucine-to-threonine mutation at residue 264 on the alpha1 subunit. To examine effects on gating when orthosteric sites were fully occupied, they activated wild-type receptors with high concentrations of a partial agonist, piperidine-4-sulfonic acid. Results In the absence of orthosteric agonists, the channel activity of alpha1L264Tbeta2gamma2L receptors was increased by diazepam and midazolam and reduced by the inverse benzodiazepine agonist FG7142. Flumazenil displayed very weak agonism and blocked midazolam from further activating mutant channels. In wild-type receptors activated with saturating concentrations of piperidine-4-sulfonic acid, midazolam increased maximal efficacy. Conclusions Independent of orthosteric site occupancy, classic benzodiazepines modulate the gating equilibrium in alpha1beta2gamma2L GABAA receptors and are therefore allosteric coagonists. A Monod-Wyman-Changeux coagonist gating model quantitatively predicts these effects, suggesting that benzodiazepines minimally alter orthosteric ligand binding.