Effect of neuroactive steroids on [3H]flumazenil binding to the GABAA receptor complex in vitro.

Modulation of benzodiazepine receptor ligand binding to the GABAA receptor complex by the neuroactive steroids 3 alpha-hydroxy-dihydroprogesterone (3 alpha-OH-DHP) and 3 alpha-hydroxycorticosterone (3 alpha- THDOC) was assessed in an in vitro binding assay with the benzodiazepine antagonist [3H]flumazenil using rat cortical membranes. Neuroactive steroids, pentobarbital, GABA and bicuculline did not significantly affect flumazenil binding. However, the addition of neuroactive steroids significantly decreased the Ki of benzodiazepine agonists, including alprazolam, diazepam and clonazepam, indicating an increase in agonist affinity. Only the addition of 3 beta-OH-DHP, an inactive stereoisomer had no effect on the Ki of these agonists. The binding of the benzodiazepine inverse agonist FG 7142 was not significantly affected by these steroids, but the addition of GABA significantly increased the Ki of FG 7142 indicating a decrease in inverse agonist affinity. High concentrations of GABA or bicuculline were able to occlude the 3 alpha-THDOC mediated decrease in alprasolam Ki, indicating a GABA dependent mechanism of binding enhancement. An advantage of using [3H]flumazenil is that neither the Ki nor the Bmax change in the presence of allosteric site modulators, permitting the simple and direct assessment of alterations in benzodiazepine ligand affinity for the GABAA receptor complex by neuroactive steroids.