Pregnenolone sulphate and Zn 2+ inhibit recombinant rat GABA A receptor through different channel property

Aims:  We compared the antagonistic effects of state‐dependent gamma‐aminobutyric acid A (GABA A ) receptor blockers picrotoxin, Zn 2+ and pregnenolone sulphate (PS) on GABA‐ and pentobarbital‐activated currents in recombinant rat GABA A receptors in Xenopus oocytes. Methods:  Experiments were performed with wild type rat α 1 β 2 γ 2L and α 1 β 2 receptors, mutants α 1V256S β 2 γ 2L and α 1 β 2A252S γ 2L receptors by the two‐electrode voltage‐clamp technique. Results:  In contrast to respective 3840‐ and 56‐fold increases in Zn 2+ potencies to inhibit GABA‐ and pentobarbital‐activated currents in the α 1 β 2 receptor, the corresponding potencies of PS remained unchanged in comparison with the α 1 β 2 γ 2L receptor. 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, abolished the inhibition of GABA A receptor by PS. In comparison with the wild type α 1 β 2 γ 2L receptor, mutants α 1V256S β 2 γ 2L and α 1 β 2A252S γ 2L receptors did not affect the Zn 2+ inhibition. Furthermore, a significant increase in GABA potency was observed in the mutant α 1V256S β 2 γ 2L receptor ( P  < 0.05), but not the mutant α 1 β 2A252S γ 2L receptor compared with the wild type receptor. Conclusions:  Pregnenolone sulphate was a γ 2‐subunit independent inhibitor in the GABA A receptor, whereas the Zn 2+ antagonism was profoundly influenced by the γ 2‐subunit. It is likely that the 2′ residue closest to the N‐terminus of the protein at M 2 helix on both α 1 and β 2 subunit are critical to the inhibitory actions of PS and the function of Cl − channels. These results are consistent with the hypothesis that PS behaves as a Cl − channel blocker that does not share with Zn 2+ , the coincident channel property in the GABA A receptors.