Modulation of the GABAA receptor by depressant barbiturates and pregnane steroids

1 The modulation of the γ-aminobutyric acidA (GABAA) receptor by reduced metabolites of progesterone and deoxycorticosterone has been compared with that produced by depressant barbiturates in: (a) voltage-clamp recordings from bovine enzymatically isolated chromaffin cells in cell culture, and (b) an assay of the specific binding of [3H]-muscimol to a preparation of porcine brain membranes. 2 The progesterone metabolites 5α- and 5β-pregnan-3α-ol-20-one (≥30 nm) reversibly and dosedependently enhanced the amplitude of membrane currents elicited by locally applied GABA (100 μm), and over the concentration range 30 nM-100 μm stimulated the binding of [3H]-muscimol. In contrast, 5α- and 5β-pregnan-3β-ol-20-one (30 nM-100 μm) had little effect in either assay, indicating a marked stereoselectivity of steroid action. 3 Scatchard analysis of the ligand binding data suggested an apparent increase in the number, rather than the affinity, of detectable [3H]-muscimol binding sites as the principle action of the active steroid isomers. 4 GABA-evoked currents were also potentiated by androsterone (1 μm) and the deoxycorticosterone metabolite 5α-pregnane-3α,21-diol-20-one (100 nm). 5 Secobarbitone (10–100 μm), pentobarbitone (10–300 μm) and phenobarbitone (100–500 μm) reversibly and dose-dependently potentiated the amplitude of GABA-evoked currents in the absence of any change in their reversal potential. 6 At relatively high concentrations (≥ 30 μm) secobarbitone and pentobarbitone directly elicited a membrane current. It is concluded that such currents result from GABAA receptor-channel activation since they share a common reversal potential with GABA-evoked responses (≥0mV), are reversibly antagonized by bicuculline (3 μm), and potentiated by either diazepam (1 μm) or 5β-pregnan-3α-ol-20-one (500 nm). 7 Secobarbitone (1 μm-1 mm) dose-dependently enhanced the binding of [3H]-muscimol. In common with the active steroids, an increase in the apparent number of binding sites was responsible for this effect. 8 A saturating concentration (1 mm) of secobarbitone in the ligand binding assay did not suppress the degree of enhancement of control binding produced by 5β-pregnan-3α-ol-20-one (30 nM-100 μm). Similarly the steroid, at a concentration of 100 μm, did not influence the enhancement of [3H]-muscimol binding by secobarbitone (1 μm-1 mm). In all combinations of concentrations tested, the effects of secobarbitone and 5β-pregnan-3α-ol-20-one on [3H]-muscimol binding were additive. 9 In conjunction with previously published observations, the present data indicate close similarities in the GABA-mimetic and potentiating actions of barbiturates and steroids. However, the results obtained with combinations of steroids and barbiturates in the ligand binding assay appear inconsistent with the two classes of compound interacting with a common site to modulate the GABAA receptor activity.