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

The gamma-aminobutyric acid type A receptor (GABA(A)R) 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 gamma subunit in GABA(A)R enhances the efficacy of DHEAS without altering its binding affinity. A saturating concentration of DHEAS blocked approximately 75% of currents mediated by GABA(A)R, which is composed of human alpha(1), beta(1), and gamma(2S) subunits, whereas the inhibition was only 35 % in GABA(A)R containing only alpha(1) and beta(1) subunits. The IC50 values of DHEAS with and without the gamma subunit were almost identical. In contrast to DHEAS, neither the affinity nor the efficacy of PS was altered by the gamma subunit. When Val256 of alpha(1) 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(A)R possibly through promoting the interaction between the M2-M3 linker and extracellular loop 7/loop 2. Mutant alpha(1), 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.