Efficient expression of concatenated α1β2δ and α1β3δ GABAA receptors, their pharmacology and stoichiometry. (27th February 2021)

• Record Type: Journal Article
• Authors: Liao, Vivian Wan Yu; Chebib, Mary; Ahring, Philip Kiær
• Is Part Of: British journal of pharmacology. Volume 178:Number 7(2021)
• Page Start: 1556
• Page End: 1573
• Publication Date: 2021-02-27
• Subjects: allopregnanolone -- DS2 -- ethanol -- etomidate -- GABA -- receptor -- stoichiometry; Pharmacology -- Periodicals; Chemotherapy -- Periodicals; Drug Therapy -- Periodicals; Pharmacology -- Periodicals; 615.1
• DOI: 10.1111/bph.15380
• Languages: English
• ISSNs: 0007-1188

Abstract

Abstract : Background and Purpose: GABAA receptors containing δ‐subunits 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 published misconceptions and contradictory conclusions made regarding the pharmacology and stoichiometry of δ‐containing receptors. The aim of this study was to obtain robust homogenous expression of α1βδ receptors for in‐depth investigation. Experimental Approach: Novel δ‐containing pentameric concatenated constructs were designed. The resulting α1β2δ and α1β3δ GABAA receptor concatemers were investigated by two‐electrode voltage‐clamp electrophysiology using Xenopus laevis oocytes. Key Results: First, while homogenous α1βδ GABAA 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 optimised constructs that give unidirectional assembly of concatemers, we found that the δ subunit cannot directly participate in 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 X. 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, for example, neurosteroids. Conclusion and Implications: Our data answer important outstanding questions regarding the pharmacology and stoichiometry of α1δ‐containing GABAA receptors and pave the way for future analysis and drug discovery efforts.