Probing GABAA receptors with inhibitory neurosteroids

Sandra Seljeset,Damian P Bright,Philip Thomas,Trevor G Smart

doi:10.1016/j.neuropharm.2018.02.008

Sandra Seljeset, Damian P Bright + Show 2 more

Open Access

https://doi.org/10.1016/j.neuropharm.2018.02.008

Copy DOI

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”.

Highlights

Introduction1. Introduction g-aminobutyric acid type A receptors (GABAARs) are key proteins in the brain for maintaining control of neuronal excitation
To assess the inhibitory activity of pregnenolone sulphate (PS) at g-aminobutyric acid type A receptors (GABAARs), and to determine if the neurosteroid exhibits any receptor subtype selectivity, recombinant receptors incorporating a1-6 subunits with b2 and/or g2L/d were systematically expressed in HEK cells and studied using whole-cell electrophysiology
Given the GABA activation-dependence of PS inhibition, we examined if PS can bind within the GABA channel by employing a competition protocol with picrotoxin (PTX), an antagonist that is considered to operate as an open-channel blocker of GABAARs and other members of the pentameric ligand-gated ion channel family (Erkkila et al, 2008; Hibbs and Gouaux, 2011; Krishek et al, 1996a)

Summary

Introduction

1. Introduction g-aminobutyric acid type A receptors (GABAARs) are key proteins in the brain for maintaining control of neuronal excitation. Introduction g-aminobutyric acid type A receptors (GABAARs) are key proteins in the brain for maintaining control of neuronal excitation They are pentamers composed of three types of receptor subunits selected from: a1-6, b1-3, g1-3, d, ε, q and p (Fritschy and Panzanelli, 2014; Sigel and Steinmann, 2012; Smart, 2015). We have used chimeric receptors to probe the essential structural elements of the receptor subunits that contribute towards PS inhibition These approaches have allowed a direct comparison between the activities of PS at different GABAAR subtypes, and provided an indication as to whether modulation is more likely to be important for the activation of synaptic or extrasynaptic GABAARs

Objectives

Methods

Results

Conclusion