Site-specific effects of neurosteroids on GABA_A receptor activation and desensitization

This study examines how site-specific binding to three identified neurosteroid-binding sites in the α₁β₃ GABA_A receptor (GABA_AR) contributes to neurosteroid allosteric modulation. We found that the potentiating neurosteroid, allopregnanolone, but not its inhibitory 3β-epimer epi-allopregnanolone, binds to the canonical β₃(+)-α₁(-) intersubunit site that mediates receptor activation by neurosteroids. In contrast, both allopregnanolone and epi-allopregnanolone bind to intrasubunit sites in the β₃ subunit, promoting receptor desensitization and the α₁ subunit promoting effects that vary between neurosteroids. Two neurosteroid analogues with diazirine moieties replacing the 3-hydroxyl (KK148 and KK150) bind to all three sites, but do not potentiate GABA_AR currents. KK148 is a desensitizing agent, whereas KK150 is devoid of allosteric activity. These compounds provide potential chemical scaffolds for 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_ARs.