The neuroactive steroid 3 alpha-hydroxy-5 beta-pregnan-20-one is a two-component modulator of ligand binding to the GABAA receptor

Neuroactive steroids allosterically inhibit [35S]t-butylbicyclophosphorothionate ([35S]TBPS) and enhance [3H]flunitrazepam binding to the GABAA receptor complex. In the presence of 5 microM GABA, 3 alpha-hydroxy-5 beta-pregnan-20-one (3 alpha, 5 beta-P) inhibits [35S]TBPS binding with high- (IC50 21-32 nM) and low- (IC50 24-63 microM) affinity components in bovine cortical, cerebellar, and hippocampal membranes. The percentage of high-affinity sites ranges from 53% in cortex to 65% in cerebellum and hippocampus. However, 3 alpha, 5 beta-P is a single-site inhibitor in thalamus (IC50 43 nM). In the absence of GABA, similar affinities for the high- and low-affinity components were detected, although the percentages of high-affinity sites were reduced. Similarly, 3 alpha, 5 beta-P enhances [3H]flunitrazepam binding with high- (EC50 44-58 nM) and low- (EC50 2-13 microM) affinity components which account for 71-77% and 23-29% of the sites, respectively, in cortex, cerebellum and hippocampus. 3 alpha, 5 beta-P is a single-site enhancer in thalamus (EC50 80 nM). In contrast to 3 alpha,5 beta-P, 3 alpha-hydroxy-5 alpha-pregnan-20-one (3 alpha,5 alpha-P) is a single site modulator of [35S]TBPS and [3H]flunitrazepam binding in all regions examined. These data provide pharmacological evidence consistent with receptor heterogeneity for neuroactive steroids.