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Efficient expression of concatenated α1β2δ and α1β3δ GABA A receptors, their pharmacology and stoichiometry
Author(s) -
Liao Vivian Wan Yu,
Chebib Mary,
Ahring Philip Kiær
Publication year - 2021
Publication title -
british journal of pharmacology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.432
H-Index - 211
eISSN - 1476-5381
pISSN - 0007-1188
DOI - 10.1111/bph.15380
Subject(s) - gabaa receptor , receptor , concatemer , xenopus , homomeric , gabaa rho receptor , cys loop receptors , allosteric regulation , ion channel , protein subunit , biophysics , ligand gated ion channel , biology , neuroscience , microbiology and biotechnology , chemistry , biochemistry , acetylcholine receptor , genome , nicotinic acetylcholine receptor , gene
Background and Purpose GABA A 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 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 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 GABA A receptors and pave the way for future analysis and drug discovery efforts.