Structural insights into opposing actions of neurosteroids on GABAA receptors

Abstract

Abstractγ-Aminobutyric acid type A (GABAA) receptors mediate fast inhibitory signaling in the brain and are targets of numerous drugs and endogenous neurosteroids. A subset of neurosteroids are GABAA receptor positive allosteric modulators; one of these, allopregnanolone, is the only drug approved specifically for treating postpartum depression. There is a consensus emerging from structural, physiological and photolabeling studies as to where positive modulators bind, but how they potentiate GABA activation remains unclear. Other neurosteroids are negative modulators of GABAA receptors, but their binding sites remain debated. Here we present structures of a synaptic GABAA receptor bound to allopregnanolone and two inhibitory sulfated neurosteroids. Allopregnanolone binds at the receptor-bilayer interface, in the consensus potentiator site. In contrast, inhibitory neurosteroids bind in the pore. MD simulations and electrophysiology support a mechanism by which allopregnanolone potentiates channel activity and suggest the dominant mechanism for sulfated neurosteroid inhibition is through pore block.

Divergent mechanisms of steroid inhibition in the human ρ1 GABAAreceptor

“…Although our results cannot entirely exclude transient occupancy of additional sites, they highlight the capacity of different steroids to modulate GABA A receptors via structurally distinct, largely exclusive mechanisms. Whereas the mechanism of PS inhibition we report here for ρ1 largely resembles that proposed for the canonical α1β2γ2 GABA A receptor 10 , the site of E2 inhibition appears specific to ρ subtypes.…”

“…The PS pose in ρ1-EM overlapped that in a recently reported complex with the canonical α1β2γ2 subtype, including the orientation of the sulfate group 10 (Figure 3G). A pore-block mechanism has similarly been proposed in the canonical subtype, supported by mutations in the inner pore that suppress inhibition 16 and disrupt PS stability in MD simulations 10 .…”

“…Accordingly, we modeled PS with the sulfate up for all further analyses. We observed no other steroidal densities in the PS dataset, including in the E2 site or canonical-subtype allopregnanolone site 10 .…”

“…We identified a single predominant conformation to an overall resolution of 2.5 Å with C5 symmetry (Figures S1, S2, S3A, Table 1). Although E2 has a similar backbone to neurosteroids like allopregnanolone, the intersubunit transmembrane site previously shown to bind allopregnanolone in α1β2ɣ2 10 only contained tubular densities in the ρ1-EM/E2 complex, similar to those observed in apo ρ1-EM 8 and likely corresponding to phospholipid tails (Figure S3C). Instead, we observed a density corresponding in size and shape to E2 at the ECD-TMD interface of each pair of adjacent subunits (Figures 1C, 1D).…”

“…Interestingly, a number of endogenous neuroactive steroids have been found to modulate GABA A receptors, including ρ1 9 . A site for steroid potentiation at the transmembrane subunit interface, facing the inner membrane leaflet, has been described in some detail; notably allopregnanolone, which is synthesized from progesterone locally in the brain, was recently resolved by cryo-EM at this site between β and α subunits in α1β2ɣ2 (so-called canonical) GABA A receptors 10,11 . The therapeutic relevance of such agents has received increasing attention with the effectiveness of allopregnanolone, and its synthetic derivative zuranolone, in treating post-partum depression 12 .…”