Probing GABAA receptors with inhibitory neurosteroids

Abstract

γ-aminobutyric acid type A receptors (GABAARs) are important components of the central nervous system and they are functionally tasked with controlling neuronal excitability. These receptors are subject to post-translational modification and also to modulation by endogenous regulators, such as the neurosteroids. These modulators can either potentiate or inhibit GABAAR function. Whilst the former class of neurosteroids are considered to bind to and act from the transmembrane domain of the receptor, the domains that are important for the inhibitory neurosteroids remain less clear. In this study, we systematically compare a panel of recombinant synaptic-type and extrasynaptic-type GABAARs expressed in heterologous cell systems for their sensitivity to inhibition by the classic inhibitory neurosteroid, pregnenolone sulphate. Generally, peak GABA current responses were inhibited less compared to steady-state currents, implicating the desensitised state in inhibition. Moreover, pregnenolone sulphate inhibition increased with GABA concentration, but showed minimal voltage dependence. There was no strong dependence of inhibition on receptor subunit composition, the exception being the ρ1 receptor, which is markedly less sensitive. By using competition experiments with pregnenolone sulphate and the GABA channel blocker picrotoxinin, discrete binding sites are proposed. Furthermore, by assessing inhibition using site-directed mutagenesis and receptor chimeras comprising α, β or γ subunits with ρ1 subunits, the receptor transmembrane domains are strongly implicated in mediating inhibition and most likely the binding location for pregnenolone sulphate in GABAARs.This article is part of the “Special Issue Dedicated to Norman G. Bowery”.

Inhibitable photolabeling by neurosteroid diazirine analog in the β3-Subunit of human hetereopentameric type A GABA receptors

“…For 3α,5α-steroids: THDOC 3, alphaxalone 4, 6-Azi-OAP 6and 21-pTFDBzox-AP itself (16), 3α,5β-steroid (pregnanolone, 1) and 6-azi-OP 5, the agreement between IC 50 and EC 50 was very good. Furthermore, pregnenolone sulfate, a 3β-steroid that inhibits GABA currents, 31 neither bound to the 21-pTFDBzox-AP site nor enhanced desensitization. Considering that the binding studies were carried out in much more dilute suspensions than the photolabeling studies, we regard the pharmacological evidence establishing that photoincorporation was occurring in the site that was responsible for enhancing desensitization to be strong.…”

Site-specific effects of neurosteroids on GABAA receptor activation and desensitization

“…Steroids with a sulfate rather than a hydroxyl at the 3-carbon are also GABA A R NAMs thought to act at sites distinct from GABA A R PAMs (5,13,14,17,32). The precise location of this site is unclear, but crystallographic studies have demonstrated a possible binding site between TM3 and TM4 on the intracellular end of the α-subunit TMD (17,24).…”

“…While 3β-OH NS and PS both inhibit GABA A Rs, they likely act via interactions with distinct sites (5,13,14,16,17,23,24).…”

Biosynthesis and signalling functions of central and peripheral nervous system neurosteroids in health and disease

“…The site of inhibitory neurosteroid binding to GABA A R is unknown. However, it is thought to be outside the pore, and distinct from the binding sites of both potentiating neurosteroids and picrotoxinin (GABA A R open channel blocker) binding [115]. The evidence for this is that inhibition is not voltage-dependent, as would be expected for an open channel blocker, the inhibitory effects of pregnenolone sulfate and picrotoxin are additive, and, unlike potentiating neurosteroids, pregnenolone sulfate on the inner membrane leaflet cannot affect GABA A R. Neurosteroid mediated GABA A R inhibition is more effective at higher GABA concentrations, suggesting that the binding site is exposed by receptor activation [116].…”