Abstract
This study examines how site-specific binding to 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 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 GABAAR 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 GABAARs.
Highlights
Neurosteroids (NS) are endogenous modulators of brain development and function and are important mediators of mood [1,2,3,4]
In addition to the canonical site at the interface between the transmembrane domain (TMD) of adjacent subunits [11,23,24,27], we identified NS binding sites within the α-helical bundles of both the α1 and β3 subunits of α1β3 GABAA receptor (GABAAR) [11]. 3α5αP binds to all three sites [11]; mutagenesis of these sites suggests that the intersubunit and α1 intrasubunit sites, but not the β3 intrasubunit site, contribute to 3α5αP PAM activity [11]
We examined a series of NS analogues with different stereochemistries or substituents in the 3- and 17-positions: 3β5αP, KK148, and KK150 for their ability to potentiate GABA-elicited currents and enhance orthosteric agonist ([3H]muscimol) binding. 3β5αP is the 3β-epimer of 3α5αP
Summary
Neurosteroids (NS) are endogenous modulators of brain development and function and are important mediators of mood [1,2,3,4]. NS can either activate or inhibit GABAARs. Positive allosteric modulatory NS (PAM-NS) such as allopregnanolone (3α5αP) potentiate the effect of GABA on GABAAR currents at low concentrations and directly activate the receptors at higher concentrations [5,10,11,12]. Negative allosteric modulatory NS (NAM-NS), such as epi-allopregnanolone (3β5αP) or pregnenolone sulfate (PS) inhibit GABAAR currents [13,14,15,16,17]. In addition to enhancing channel opening, PAM-NS increase the affinity of the GABAAR for orthosteric ligand binding, an effect thought to be mechanistically linked to channel gating [11,18]
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