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

Abstract

Listen

Translate article

Modulation of benzodiazepine receptor ligand binding to the GABA A receptor complex by the neuroactive steroids 3α-hydroxy-dihydroprogesterone (3α-OH-DHP) and 3α-hydroxycorticosterone (3α-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 K i of benzodiazepine agonists, including alprazolam, diazepam and clonazepam, indicating an increase in agonist affinity. Only the addition of 3β-OH-DHP, an inactive stereoisomer had no effect on the K i 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 K i of FG 7142 indicating a decrease in inverse agonist affinity. High concentrations of GABA or bicuculline were able to occlude the 3α-THDOC mediated decrease in alprazolam K i, indicating a GABA dependent mechanism of binding enhancement. An advantage of using [ 3H]flumazenil is that neither the K d nor the B max change in the presence of allosteric site modulators, permitting the simple and direct assessment of alterations in benzodiazepine ligand affinity for the GABA A receptor complex by neuroactive steroids.