Regulated assembly and neurosteroid modulation constrain GABA_Areceptor pharmacologyin vivo

Abstract

Type A GABA receptors (GABAARs) are the principal inhibitory receptors in the brain and the target of a wide range of clinical agents, including anesthetics, sedatives, hypnotics, and antidepressants. However, our understanding of GABAAR pharmacology has been hindered by the vast number of pentameric assemblies that can be derived from a total 19 different subunits and the lack of structural knowledge of clinically relevant receptors. Here, we isolate native murine GABAAR assemblies containing the widely expressed α1 subunit, and elucidate their structures in complex with drugs used to treat insomnia (zolpidem and flurazepam) and postpartum depression (the neurosteroid allopregnanolone). Using cryo-EM analysis and single-molecule photobleaching experiments, we uncover only three structural populations in the brain: the canonical α1β2γ2 receptor containing two α1 subunits and two unanticipated assemblies containing one α1 and either an α2, α3 or α5 subunit. Both of the noncanonical assemblies feature a more compact arrangement between the transmembrane and extracellular domains. Interestingly, allopregnanolone is bound at the transmembrane α/β subunit interface, even when not added to the sample, revealing an important role for endogenous neurosteroids in modulating native GABAARs. Together with structurally engaged lipids, neurosteroids produce global conformational changes throughout the receptor that modify both the pore diameter and binding environments for GABA and insomnia medications. Together, our data reveal that GABAAR assembly is a strictly regulated process that yields a small number of structurally distinct complexes, defining a structural landscape from which subtype-specific drugs can be developed.

Identification of a binding site for bupropion inGloeobacter violaceusligand-gated ion channel

“…Previous work by Cheng et al on mutation I271W indicates this mutation does not affect the inhibition of allopregnanolone; however, I271W significantly reduces the inhibition of KK200, a neurosteroid analogue (16). The counterpart of this Ile residue in the M3 segment of GABA β2 TMD, residue L297, lines the binding pocket of allopregnanolone as recently reported by Gouaux and co-workers(19) (Fig. 2 E).…”

“…The corresponding residue to T214, residue V242 of the α1 subunit of murine GABA A receptors, has also been observed to line the binding pocket for neurosteroids (Fig. 2 E) (19). A nearby residue, W217 in GLIC, and its counterpart, residue W245 of the α1 subunit of GABA A receptors, also contributes to the neurosteroid binding pocket (16,19,21,23).…”

“…The W217 counterpart residue in GABA A receptors is W245 in M1 of the α1 subunit, and this residue W245 lines the binding sites of the neurosteroids (Fig. 2 E) (19,21,23). This conserved Trp residue in M1 thus contributes to the allosteric modulation either directly via its lipophilic side chain as observed by the change in pH 50 and/or via binding of lipophilic allosteric modulators like neurosteroids.…”

“…In GABA A receptors, the interfaces between M1 and M3 of adjacent subunits house binding sites for many clinically used drugs such as propofol, diazepam, and allopregnanolone (Fig. 2) (18,19,48). Based on the lipophilicity of bupropion and preliminary molecular docking screening for GLIC and antidepressants, residues T214 and W217 in M1 and residues F267 and I271 in M3 of the GLIC transmembrane domain were suggested to participate in bupropion modulation of GLIC (data not shown).…”

“…2 E) (19). A nearby residue, W217 in GLIC, and its counterpart, residue W245 of the α1 subunit of GABA A receptors, also contributes to the neurosteroid binding pocket (16,19,21,23). Taken together, bupropion when bound to this site in GLIC would extend from T214 to W217, yielding an overall comparable binding pocket to neurosteroids in pentameric ligand gated ion channels.…”