Efficient expression of concatenated α1β2δ and α1β3δ GABAA receptors, their pharmacology and stoichiometry

Abstract

Background and Purpose δ‐containing GABA_A receptors are notorious for being difficult to study in vitro due to heterogeneity of expressed receptor populations and low GABA‐evoked current amplitudes. Thus, there are some misconception and contradictory conclusions made regarding the pharmacology and stoichiometry of δ‐containing receptors in the literature. The aim of this study was therefore to obtain robust homogenous expression of α1βδ receptors and perform in‐depth investigations. Experimental Approach Novel δ‐containing pentameric concatenated constructs were designed and resulting α1β2δ and α1β3δ GABA_A receptor concatemers were investigated by two‐electrode voltage‐clamp electrophysiology using Xenopus laevis oocytes. Key Results First, while homogenous α1βδ GABA_A receptor pools could not be obtained by manipulating the ratio of injected cRNAs of free α1, β2/3 and δ subunits, concatenated pentameric α1β2δ and α1β3δ constructs resulted in robust expression levels of concatemers. Second, by using optimized constructs that give unidirectional assembly of concatemers, it was found that the δ subunit cannot directly participate to GABA binding and receptor activation. Hence, functional δ‐containing receptors are likely to all have a conventional 2α:2β:1δ stoichiometry arranged as βαβαδ when viewed counterclockwise from the extracellular side. Third, α1β2/3δ receptors were found to express efficiently in Xenopus laevis oocytes but have a low estimated open probability of ~0.5% upon GABA activation. Because of this, these receptors are uniquely susceptible to positive allosteric modulation by e.g. neurosteroids. Conclusion and Implications Our data answers important outstanding questions regarding the pharmacology and stoichiometry of α1δ‐containing GABA_A receptors and pave the way for future analysis and drug‐discovery efforts.