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 alpha1 beta2 gamma2L and alpha1beta2 receptors, mutants alpha1V256S beta2 gamma2L and alpha1beta2A252Sgamma2L 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 alpha1beta2 receptor, the corresponding potencies of PS remained unchanged in comparison with the alpha1 beta2 gamma2L receptor. A homologous mutation of the residue at 2' position closest to the cytoplasmic end of the M(2) helix to serine on both alpha1 and beta2 subunit, alpha1V256S and beta2A252S, abolished the inhibition of GABA(A) receptor by PS. In comparison with the wild type alpha1beta2gamma2L receptor, mutants alpha1V256S beta2 gamma2L and alpha1beta2 A252S gamma2L receptors did not affect the Zn(2+) inhibition. Furthermore, a significant increase in GABA potency was observed in the mutant alpha1V256S beta2 gamma2L receptor (P < 0.05), but not the mutant alpha1beta2 A252S gamma2L receptor compared with the wild type receptor.
Conclusions: Pregnenolone sulphate was a gamma2-subunit independent inhibitor in the GABA(A) receptor, whereas the Zn(2+) antagonism was profoundly influenced by the gamma2-subunit. It is likely that the 2' residue closest to the N-terminus of the protein at M(2) helix on both alpha1 and beta2 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.