Aims: We compared the antagonistic effects of state‐dependent gamma‐aminobutyric acid A (GABA_A) receptor blockers picrotoxin, Zn²⁺ 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 ratα1β2γ2L and α1β2 receptors, mutants α1V256Sβ2γ2L and α1β2A252Sγ2L receptors by the two‐electrode voltage‐clamp technique.Results: In contrast to respective 3840‐ and 56‐fold increases in Zn²⁺ potencies to inhibit GABA‐ and pentobarbital‐activated currents in the α1β2 receptor, the corresponding potencies of PS remained unchanged in comparison with the α1β2γ2L receptor. 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, abolished the inhibition of GABA_A receptor by PS. In comparison with the wild type α1β2γ2L receptor, mutants α1V256Sβ2γ2L and α1β2A252Sγ2L receptors did not affect the Zn²⁺ inhibition. Furthermore, a significant increase in GABA potency was observed in the mutant α1V256Sβ2γ2L receptor (P < 0.05), but not the mutant α1β2A252Sγ2L receptor compared with the wild type receptor.Conclusions: Pregnenolone sulphate was a γ2‐subunit independent inhibitor in the GABA_A receptor, whereas the Zn²⁺ antagonism was profoundly influenced by the γ2‐subunit. It is likely that the 2′ residue closest to the N‐terminus of the protein at M₂ helix on both α1 and β2 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 Zn²⁺, the coincident channel property in the GABA_A receptors.

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