3Beta-hydroxysteroids are pregnenolone sulfate-like GABA(A) receptor antagonists. The aim of the current study was to compare the functional differences between 3beta-hydroxysteroids and pregnenolone sulfate to inhibit GABA(A) receptors expressed in Xenopus oocytes. Recombinant rat GABA(A) receptors encoding wild type alpha1 beta2 gamma2L receptor, mutant alpha1V256S beta2 gamma2L and alpha1 beta2A252S gamma2L 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 alpha1 and beta2 subunit, alpha1V256S and beta2A252S, 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 alpha1V256S beta2 gamma2L receptor (P<0.05), whereas it decreased moderately in the alpha1 beta2A252S gamma2L receptor. We found that 5alpha-pregnan-3beta, 20(S)-diol (UC1019) and 5beta-pregnan-3beta, 20(R)-diol (UC1020) were the most effective blockers of maximal GABA responses among a panel of 3beta-hydroxysteroids. Pregnenolone sulfate, UC1019 and UC1020 were potent antagonists in the wild type receptor with calculated IC50s of 0.20+/-0.07 microM; 1.88+/-0.32 microM and 2.58+/-0.58 microM, respectively. The inhibitory effect of pregnenolone sulfate was significantly reduced in both mutants alpha1V256S beta2 gamma2L and alpha1 beta2A252S gamma2L receptors (P<0.05), whereas the inhibitory effects of UC1019 and UC1020 were reduced only in the mutant alpha1V256S beta2 gamma2L 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 (< or = 20 microM) did not promote desensitization in both wild type and mutant receptors. In conclusion, the GABA(A) receptor inhibition by pregnenolone sulfate, but not 3beta-hydroxysteroids, was dependent on desensitization kinetics of the Cl- channels. A point mutation at M2 helix of the beta2-subunit (beta2A252S) can dramatically reduce the inhibitory effect of pregnenolone sulfate on the GABA(A) receptors without affecting the inhibitory properties of 3beta-hydroxysteroids. These results are consistent with the hypothesis that pregnenolone sulfate-inhibition does not share with 3beta-hydroxysteroids the coincident channel property at the GABA(A) receptor.