Inhibition of the GABA_A Receptor by Sulfated Neurosteroids: A Mechanistic Comparison Study between Pregnenolone Sulfate and Dehydroepiandrosterone Sulfate

Abstract

The γ-aminobutyric acid type A receptor (GABA_AR) is negatively modulated by two structurally similar neurosteroids, pregnenolone sulfate (PS) and dehydroepiandrosterone sulfate (DHEAS). This study attempted to ascertain the molecular mechanisms of inhibition of the GABA-ergic current by neurosteroids. We demonstrated that the presence of the γ subunit in GABA_AR enhances the efficacy of DHEAS without altering its binding affinity. A saturating concentration of DHEAS blocked approximately 75 % of currents mediated by GABA_AR, which is composed of human α₁, β₁, and γ_2S subunits, whereas the inhibition was only 35 % in GABA_AR containing only α₁ and β₁ subunits. The IC₅₀ values of DHEAS with and without the γ subunit were almost identical. In contrast to DHEAS, neither the affinity nor the efficacy of PS was altered by the γ subunit. When Val256 of α₁ subunit was mutated to Ser, the mutant channel became resistant to inhibition by both DHEAS and PS. PS exerted its inhibitory effect by enhancing the desensitization kinetics of GABA_AR possibly through promoting the interaction between the M2-M3 linker and extracellular loop 7/loop 2. Mutant α₁, containing double Cys in loop 2/loop 7 and the M2-M3 linker, formed disulfide bonds three times as much fast, when treated with saturating GABA+PS, compared with GABA alone or with GABA+DHEAS. We demonstrated that PS, but not DHEAS, mediates GABA-ergic inhibition by promoting collisions between the structural elements involved in receptor desensitization, i.e., loop 2, loop 7, and the M2-M3 linker, thus following different inhibitory mechanisms.