Site-specific effects of neurosteroids on GABA _A receptor activation and desensitization

This study examines how site-specific binding to the three identified neurosteroid binding sites in the α ₁ β ₃ GABA _A receptor (GABA _A R) contributes to neurosteroid allosteric modulation. We found that the potentiating neurosteroid, allopregnanolone, but not its inhibitory 3β-epimer epi-allopregnanolone, binds to the canonical β ₃ (+)–α ₁ (-) intersubunit site that mediates receptor activation by neurosteroids. In contrast, both allopregnanolone and epi-allopregnanolone bind to intrasubunit sites in the β ₃ subunit, promoting receptor desensitization and the α ₁ subunit promoting ligand-specific effects. Two neurosteroid analogues with diazirine moieties replacing the 3-hydroxyl (KK148 and KK150) bind to all three sites, but do not potentiate GABA _A R currents. KK148 is a desensitizing agent, whereas KK150 is devoid of allosteric activity. These compounds provide potential chemical scaffolds for site-specific and general neurosteroid antagonists. Collectively, these data show that differential occupancy and efficacy at three discrete neurosteroid binding sites determine whether a neurosteroid has potentiating, inhibitory, or competitive antagonist activity on GABA _A Rs.