Distinct Modulation of Spontaneous and GABA-Evoked Gating by Flurazepam Shapes Cross-Talk Between Agonist-Free and Liganded GABAA Receptor Activity

GABA A receptors (GABA A Rs) play a crucial inhibitory role in the CNS. Benzodiazepines (BDZs) are positive modulators of specific subtypes of GABA A Rs, but the underlying mechanism remains obscure. Early studies demonstrated the major impact of BDZs on binding and more recent investigations indicated gating, but it is unclear which transitions are affected. Moreover, the upregulation of GABA A R spontaneous activity by BDZs indicates their impact on receptor gating but the underlying mechanisms remain unknown. Herein, we investigated the effect of a BDZ (flurazepam) on the spontaneous and GABA-induced activity for wild-type (WT, α 1 β 2 γ 2 ) and mutated (at the orthosteric binding site α 1 F64) GABA A Rs. Surprisingly, in spite of the localization at the binding site, these mutations increased the spontaneous activity. Flurazepam (FLU) upregulated this activity for mutants and WT receptors to a similar extent by affecting opening/closing transitions. Spontaneous activity affected GABA-evoked currents and is manifested as an overshoot after agonist removal that depended on the modulation by BDZs. We explain the mechanism of this phenomenon as a cross-desensitization of ligand-activated and spontaneously active receptors. Moreover, due to spontaneous activity, FLU-pretreatment and co-application (agonist + FLU) protocols yielded distinct results. We provide also the first evidence that GABA A R may enter the desensitized state in the absence of GABA in a FLU-dependent manner. Based on our data and model simulations, we propose that FLU affects agonist-induced gating by modifying primarily preactivation and desensitization. We conclude that the mechanisms of modulation of spontaneous and ligand-activated GABA A R activity concerns gating but distinct transitions are affected in spontaneous and agonist-evoked activity.