3β-hydroxysteroids and pregnenolone sulfate inhibit recombinant rat GABAA receptor through different channel property

3β-hydroxysteroids are pregnenolone sulfate-like GABAA receptor antagonists. The aim of the current study was to compare the functional differences between 3β-hydroxysteroids and pregnenolone sulfate to inhibit GABAA receptors expressed in Xenopus oocytes. Recombinant rat GABAA receptors encoding wild type α1β2γ2L receptor, mutant α1V256Sβ2γ2L and α1β2A252Sγ2L receptors were examined using a two-electrode voltage-clamp technique. A homologous mutation of the residue at 2′position closest to the cytoplasmic end of the M2 helix to serine on both α1 and β2 subunit, α1V256S and β2A252S, reduced the slow desensitization components of GABA-activated currents at saturating doses. Compared to the wild type receptor, the potency of GABA increased significantly in the α1V256Sβ2γ2L receptor (P < 0.05), whereas it decreased moderately in the α1β2A252Sγ2L receptor. We found that 5α-pregnan-3β, 20(S)-diol (UC1019) and 5β-pregnan-3β, 20(R)-diol (UC1020) were the most effective blockers of maximal GABA responses among a panel of 3β-hydroxysteroids. Pregnenolone sulfate, UC1019 and UC1020 were potent antagonists in the wild type receptor with calculated IC50s of 0.20 ± 0.07 μM; 1.88 ± 0.32 μM and 2.58 ± 0.58 μM, respectively. The inhibitory effect of pregnenolone sulfate was significantly reduced in both mutants α1V256Sβ2γ2L and α1β2A252Sγ2L receptors (P < 0.05), whereas the inhibitory effects of UC1019 and UC1020 were reduced only in the mutant α1V256Sβ2γ2L receptor. Pregnenolone sulfate promoted slow desensitization with prolonged GABA application in a dose-dependent manner in the wild type receptor, but not mutant receptors. On the contrary, UC1019 and UC1020 (≤ 20 μM) did not promote desensitization in both wild type and mutant receptors. In conclusion, the GABAA receptor inhibition by pregnenolone sulfate, but not 3β-hydroxysteroids, was dependent on desensitization kinetics of the Cl- channels. A point mutation at M2 helix of the β2-subunit (β2A252S) can dramatically reduce the inhibitory effect of pregnenolone sulfate on the GABAA receptors without affecting the inhibitory properties of 3β-hydroxysteroids. These results are consistent with the hypothesis that pregnenolone sulfate-inhibition does not share with 3β-hydroxysteroids the coincident channel property at the GABAA receptor.