Correlation of neuroactive steroid modulation of [ super(35)S]t-butylbicyclophosphorothionate and [ super(3)H]flunitrazepam binding and gamma -aminobutyric acid sub(A) receptor function

Neuroactive steroids, including endogenously occurring metabolites of progesterone and deoxycorticosterone as well as their synthetic derivatives, are positive allosteric modulators of the gamma -aminobutyric acid (GABA) sub(A) receptor complex. They inhibit the binding of [ super(35)S]t-butylbicyclophosphorothionate ([ super(35)S]TBPS), enhance the binding of [ super(3)H]flunitrazepam, and potentiate GABA-evoked chloride currents and agonist-stimulated super(36)Cl super(-) uptake. The structure-activity relationship for 31 neuroactive steroids and related compounds was explored by examining their relative ability to inhibit [ super(35)S]TBPS binding in rat brain cortical membranes. A free 3 alpha -hydroxy group is necessary for high potency inhibition. Whereas hydroxylation in the 21-position and subsequent esterification maintain activity, 11 alpha - or 12 alpha -hydroxylation greatly reduces activity. Introduction of a double bond between the 9- and 11-positions reduces potency, whereas a double bond in the 4-position reduces the maximal extent of inhibition. Comparing the activities of these neuroactive steroids and related compounds in the [ super(35)S]TBPS and [ super(3)H]flunitrazepam assays, there is a strong correlation between potency and magnitude of modulation, indicating that the neuroactive steroid binding site is similarly coupled to the TBPS and benzodiazepine sites in rat cortex. However, there is a weaker correlation between the degree of modulation in either binding assay and potentiation of muscimol-stimulated super(36)Cl super(-) uptake in rat cortical synaptoneurosomes. Using an electrophysiological approach, stronger correlations, were observed between the magnitude of modulation in the binding assays and potentiation of GABA-evoked chloride currents in Xenopus oocytes expressing human alpha 1 beta 1 gamma 2L receptor complexes. Thus, neuroactive steroid modulation of [ super(35)S]TBPS and [ super(3)H]flunitrazepam binding is predictive of functional activity at the GABA sub(A) receptor complex.