Pregnenolone sulphate and Zn2+ inhibit recombinant rat GABAA receptor through different channel property

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Abstract
Aims:  We compared the antagonistic effects of state‐dependent gamma‐aminobutyric acid A (GABAA) receptor blockers picrotoxin, Zn2+ and pregnenolone sulphate (PS) on GABA‐ and pentobarbital‐activated currents in recombinant rat GABAA 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 GABAA 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 GABAA 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 GABAA receptors.

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