Pharmacological and Biophysical Characteristics of Picrotoxin-Resistant, δSubunit-Containing GABAA Receptors

Hong-Jin Shu,John Bracamontes,Steven Mennerick,Charles F Zorumski,Joe Henry Steinbach,Xinguo Lu

doi:10.3389/fnsyn.2021.763411

Hong-Jin Shu, John Bracamontes + Show 4 more

Open Access

https://doi.org/10.3389/fnsyn.2021.763411

Copy DOI

Abstract

GABAA receptors (GABAARs) play a crucial role in inhibition in the central nervous system. GABAARs containing the δ subunit mediate tonic inhibition, have distinctive pharmacological properties and are associated with disorders of the nervous system. To explore this receptor sub-class, we recently developed mice with δ-containing receptors rendered resistant to the common non-competitive antagonist picrotoxin (PTX). Resistance was achieved with a knock-in point mutation (T269Y; T6’Y) in the mouse genome. Here we characterize pharmacological and biophysical features of GABAARs containing the mutated subunit to contextualize results from the KI mice. Recombinant receptors containing δ T6’Y plus WT α4 and WT β2 subunits exhibited 3-fold lower EC50 values for GABA but not THIP. GABA EC50 values in native receptors containing the mutated subunit were in the low micromolar range, in contrast with some published results that have suggested nM sensitivity of recombinant receptors. Rectification properties of δ-containing GABAARs were similar to γ2-containing receptors. Receptors containing δ T6’Y had marginally weaker sensitivity to positive allosteric modulators, likely a secondary consequence of differing GABA sensitivity. Overexpression of δT6’Y in neurons resulted in robust PTX-insensitive IPSCs, suggesting that δ-containing receptors are readily recruited by synaptically released GABA. Overall, our results give context to the use of δ receptors with the T6’Y mutation to explore the roles of δ-containing receptors in inhibition.

Highlights

Activation of GABAA receptors (GABAARs) inhibits targets expressing the receptors and sculpts patterns of the activity responsible for thought, emotion, and virtually all brain functions
The comparable mutation in δ subunit-containing receptors has not been characterized in as much detail, we have shown that kinetics and agonist sensitivity are similar to wild type (WT) in native dentate granule cells (DGCs) (Sun et al, 2018, 2019)
We found that receptors with a mutated δ subunit exhibit ∼3-fold higher sensitivity to GABA, leading to marginally altered responsiveness to allosteric modulators

Summary

Introduction

Activation of GABAARs inhibits targets expressing the receptors and sculpts patterns of the activity responsible for thought, emotion, and virtually all brain functions. Δ-Containing receptors are thought to have high sensitivity to certain positive allosteric modulators, such as neurosteroids and ethanol (Mihalek et al, 1999; Spigelman et al, 2003; Stell et al, 2003; Wei et al, 2004; Glykys et al, 2007), our recent results questioned this selectivity (Lu et al, 2020). Features of both GABAAR subclasses make them attractive targets for therapeutics in different situations. An open question is whether receptors containing the mutant δ subunit are sufficiently different pharmacologically from WT δ-containing receptors to explain the unexpected results

Methods

Results

Conclusion