The neurosteroid allopregnanolone (ALLO) and pregnanolone (PREG), are equally effective positive allosteric modulators (PAMs) of GABAA 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 ent-ALLO potentiates GABA-elicited currents in α1β3 GABAA receptors with lower potency and efficacy than ALLO, PREG or ent-PREG. The small PAM effect of ent-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 ent-ALLO binds weakly to the β3-α1 intersubunit binding site in comparison to ALLO, PREG and ent-PREG. Rigid body docking predicted that ent-ALLO binds in the intersubunit site with a preferred orientation 180° different than ALLO, PREG or ent-PREG, potentially explaining its weak binding and effect. Photolabeling studies did not identify differences between ALLO and ent-ALLO binding to the α1 or β3 intrasubunit binding sites that also mediate neurosteroid modulation of GABAA receptors. The results demonstrate that differential binding of ent-ALLO and ent-PREG to the β3-α1 intersubunit site accounts for the difference in enantioselectivity between ALLO and PREG.
Keywords: GABA-A receptors; enantiomers; neurosteroids; photolabeling.