Neurosteroid Binding and Actions on GABAA Receptors.

Neurosteroids positively modulate GABA_A receptor (GABA_AR) channel activity by binding to a transmembrane domain intersubunit site. Using photo-affinity labeling and an ELIC-α₁GABA_AR chimera, we investigated the impact of mutations within the intersubunit site on neurosteroid binding. These mutations reduce neither photolabeling within the intersubunit site nor competitive prevention of labeling by allopregnanolone. Instead, these mutations change the orientation of neurosteroid photolabeling. The data indicate that mutations at Gln242 or Trp246 that eliminate neurosteroid effects do not eliminate neurosteroid binding within the intersubunit site, but significantly alter the preferred orientation of the neurosteroid within the site. The interactions formed by Gln242 and Trp246 within this pocket play a vital role in determining the orientation of the neurosteroid. We also examined how site-specific binding to three identified neurosteroid-binding sites in the α₁β₃GABA_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 bind to all three sites, but do not potentiate GABA_AR currents. One is a desensitizing agent, whereas the other is devoid of allosteric activity. 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_AR.