Diazepam enhancement of GABA-gated currents in binary and ternary GABAA receptors

Abstract

Although the predominant GABA_A receptor isoform in the adult rodent central nervous system is a ternary complex composed of α₁β_2/3γ₂-subunits, small populations of binary receptors lacking β-subunits (i.e., complexes containing αγ-subunits) have also been identified. When expressed in HEK 293 cells, recombinant GABA_A receptors composed of either α₁β_2/3γ₂- or α₁γ₂-subunits form benzodiazepine-responsive, GABA-gated chloride channels. The objective of this study was to compare the ability of a prototypic benzodiazepine (diazepam) to augment GABA-gated chloride currents in these binary and ternary receptor isoforms. The potency of GABA was characteristically increased by diazepam (1μM) in both receptor isoforms, but this increase was significantly greater (p1β₂γ₂-subunits (approximately five-to sixfold) compared to α₁γ₂-subunits (∼2.2-fold). At GABA concentrations approximating its EC₅₀ value (5 μM), the greater augmentation observed in ternary receptors was attributable to a higher efficacy of diazepam. Radioligand binding studies revealed that theB _max of [³H]flunitrazepam was increased ∼1.8- and 3.5-fold in cells expressing α₁β₂γ₂- and α₁β₃γ₂-subunits, respectively, compared to cells expressing α₁γ₂-subunits. A similar increase (∼3.8-fold) in theB _max of [³H]Ro 15-4513 was observed in HEK 293 cells transiently transfected with cDNAs encodign α₆β₃γ₂- compared to α₆γ₂-subunits. TheK _d values of these radioligands were not different in binary and ternary receptor isoforms. It is hypothesized that the greater efficacy of diazepam in α₁β₂γ₂ compared to α₁γ₂ GABA_A receptors results from the higher benzodiazepine binding site density produced by the formation of a ternary complex.