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

Abstract

3β-hydroxysteroids are pregnenolone sulfate-like GABA A receptor antagonists. The aim of the current study was to compare the functional differences between 3β-hydroxysteroids and pregnenolone sulfate to inhibit GABA A receptors expressed in Xenopus oocytes. Recombinant rat GABA A receptors encoding wild type α 1β 2γ 2L receptor, mutant α 1V256Sβ 2γ 2L and α 1β 2A252Sγ 2L 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 M 2 helix to serine on both α 1 and β 2 subunit, α 1V256S and β 2A252S, 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 α 1V256Sβ 2γ 2L receptor ( P < 0.05), whereas it decreased moderately in the α 1β 2A252Sγ 2L 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 IC 50s 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 α 1V256Sβ 2γ 2L and α 1β 2A252Sγ 2L receptors ( P < 0.05), whereas the inhibitory effects of UC1019 and UC1020 were reduced only in the mutant α 1V256Sβ 2γ 2L 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 GABA A receptor inhibition by pregnenolone sulfate, but not 3β-hydroxysteroids, was dependent on desensitization kinetics of the Cl - channels. A point mutation at M 2 helix of the β 2-subunit (β 2A252S) can dramatically reduce the inhibitory effect of pregnenolone sulfate on the GABA A 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 GABA A receptor.