Steroid modulation of the chloride ionophore in rat brain: Structure-activity requirements, regional dependence and mechanism of action

Further in vitro studies of steroids active at the γ-aminobutyric acid(A) (GABA(A)) receptor regulated Cl^- channel labeled by [³⁵S]-t-butylbicyclophosphorothionate ([³⁵S]TBPS) reveal additional structural requirements necessary for activity. Evaluation of selected steroids for activity against TBPS-induced convulsions show similar requirements for activity. Interestingly, steroids (e.g., 5α-pregnan-3α,20α-diol) were identified that have high potency but limited efficacy as modulators of [³⁵S]TBPS binding. These characteristics are reminiscent of the clinically useful benzodiazepines (BZs) such as clonazepam. However, interactions between the prototypical anesthetic-barbiturate, sodium pentobarbital, and steroids active at the Cl^- channel suggest that they do not share a common site of action as allosteric modulators of [³⁵S]TBPS and BZ receptor binding. The most potent steroid evaluated, 5α-pregnan-3α-ol-20-one, modulates [³⁵S]TBPS binding at low concentrations (IC₅₀ ~ 17 nM) in a regionally dependent manner. All [³⁵S]TBPS binding sites appear to be functionally coupled to a steroid 'modulatory site.' Because several of the active steroids are metabolites of progesterone, their ability to inhibit the binding of [³H]promegestrone to the cytosolic progestin receptor in rat uterus was evaluated. Those steroids showing potent activity at the GABA(A) receptor-Cl^- ionophore were inactive at the intracellular progestin receptor. Such specificity coupled with their high potency provide additional support for the hypothesis that some of these steroids may be involved in the homeostatic regulation of brain excitability via the GABA(A)-BZ receptor complex.