The Mechanism of Enantioselective Neurosteroid Actions on GABAA Receptors

Abstract

The neurosteroid allopregnanolone (ALLO) and pregnanolone (PREG), are equally effective positive allosteric modulators (PAMs) of GABA_A receptors. Interestingly, the PAM effects of ALLO are strongly enantioselective, whereas those of PREG are not. This study was aimed at determining the basis for this difference in enantioselectivity. The oocyte electrophysiology studies showed that <i>ent</i>-ALLO potentiates GABA-elicited currents in α₁β₃ GABA_A receptors with lower potency and efficacy than ALLO, PREG or <i>ent</i>-PREG. The small PAM effect of <i>ent</i>-ALLO was prevented by the α₁(Q242L) mutation in the intersubunit neurosteroid binding site between the β₃ and α₁ subunits. Consistent with this result, neurosteroid analogue photolabeling with mass spectrometric readout, showed that <i>ent</i>-ALLO binds weakly to the β₃-α₁ intersubunit binding site in comparison to ALLO, PREG and <i>ent</i>-PREG. Rigid body docking predicted that <i>ent</i>-ALLO binds in the intersubunit site with a preferred orientation 180° different than ALLO, PREG or <i>ent</i>-PREG, potentially explaining its weak binding and effect. Photolabeling studies did not identify differences between ALLO and <i>ent</i>-ALLO binding to the α₁ or β₃ intrasubunit binding sites that also mediate neurosteroid modulation of GABA_A receptors. The results demonstrate that differential binding of <i>ent</i>-ALLO and <i>ent</i>-PREG to the β₃-α₁ intersubunit site accounts for the difference in enantioselectivity between ALLO and PREG.