Characterization of steroid interactions with γ-aminobutyric acid receptor-gated chloride ion channels: Evidence for multiple steroid recognition sites

The potentiation of γ-aminobutyric acid (GABA) receptor-mediated ³⁶Cl^- uptake by various steroids has been characterized in rat cerebral cortical synaptoneurosomes. Several of these steroids, including 3α-hydroxy-5α-pregnan-20-one (3α-OH-DHP) and 3α,21-dihydroxy-5α-pregnan-20-one (THDOC), increase the potency of muscimol to stimulate ³⁶Cl^- uptake in a concentration-dependent and stereospecific manner. Concentration-response curves for 3α-OH-DHP, THDOC, 3α-hydroxy-pregn-4-en-20-one, and pentobarbital enhancement of muscimol-stimulated ³⁶Cl^- uptake are biphasic, with Hill coefficients significantly less than 1.0. Computer-modeling (ALLFIT analysis) of these curves suggests that these steroids and pentobarbital interact with multiple binding sites on GABA_A receptor(s). In contrast, the concentration-response curve for THDOC 21-mesylate is monophasic, with a smaller maximal response, and yields a Hill coefficients of 1.0. In addition to modulating GABA receptor-mediated ³⁶CP uptake, THDOC enhanced the ability of the benzodiazepine clonazepam to potentiate muscimol-stimulated ³⁶Cl^- uptake. The central benzodiazepine antagonist Ro15-1788 failed to inhibit THDOC-induced potentiation of muscimol-stimulated ³⁶Cl^- uptake, although it has been previously reported to inhibit some of the behavioral actions of THDOC. In contrast to the A ring-reduced metabolites and analogues of progesterone and deoxycorticosterone, glucocorticoids had no effect on muscimol-stimulated ³⁶Cl^- uptake in cerebral cortical synaptoneurosomes at concentrations between 20 nM and 5 μM.