ABSTRACT
This study examines how site-specific binding to the three identified neurosteroid binding sites in the α1β3 GABAA receptor (GABAAR) 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 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 GABAAR 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 GABAARs.
Footnotes
Yusuke Sugasawa sugasaway{at}wustl.edu
Wayland W. L. Cheng wayland.cheng{at}wustl.edu
John R. Bracamontes jbracamontes{at}wustl.edu
Zi-Wei Chen chenziwei{at}wustl.edu
Lei Wang leiwang{at}wustl.edu
Allison L. Germann germanna{at}wustl.edu
Spencer R. Pierce spencerp{at}wustl.edu
Thomas C. Senneff tcsenneff{at}wustl.edu
Kathiresan Krishnan krishnan{at}wustl.edu
David E. Reichert reichertd{at}wustl.edu
Douglas F. Covey dcovey{at}wustl.edu
Gustav Akk akk{at}wustl.edu