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Development of a Robust Mammalian Cell‐based Assay for Studying Recombinant α 4 β 1/3 δ GABA A Receptor Subtypes
Author(s) -
FalkPetersen Christina B.,
Søgaard Rikke,
Madsen Kenneth L.,
Klein Anders B.,
Frølund Bente,
Wellendorph Petrine
Publication year - 2017
Publication title -
basic and clinical pharmacology and toxicology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.805
H-Index - 90
eISSN - 1742-7843
pISSN - 1742-7835
DOI - 10.1111/bcpt.12778
Subject(s) - receptor , hek 293 cells , recombinant dna , gabaa receptor , transfection , cell culture , chemistry , biology , patch clamp , microbiology and biotechnology , pharmacology , biochemistry , gene , genetics
δ‐Containing GABA A receptors are located extrasynaptically and mediate tonic inhibition. Their involvement in brain physiology positions them as interesting drug targets. There is thus a continued interest in establishing reliable recombinant expression systems for δ‐containing GABA A receptors. Inconveniently, the recombinant expression of especially α 4 β 1/3 δ receptors has been found to be notoriously difficult, resulting in mixed receptor populations and/or stoichiometries and differential pharmacology depending on the expression system used. With the aim of developing a facile and robust 96‐well format cell‐based assay for extrasynaptic α 4 β 1/3 δ receptors, we have engineered and validated a HEK 293 Flp‐In™ cell line stably expressing the human GABA A δ‐subunit. Upon co‐transfection of α 4 and β 1/3 subunits, at optimized ratios, we have established a well‐defined system for expressing α 4 β 1/3 δ receptors and used the fluorescence‐based FLIPR Membrane Potential ( FMP ) assay to evaluate their pharmacology. Using the known reference compounds GABA and THIP , ternary α 4 β 1/3 δ and binary α 4 β 1/3 receptors could be distinguished based on potency and kinetic profiles but not efficacy. As expected, DS 2 was able to potentiate only δ‐containing receptors, whereas Zn 2+ had an inhibitory effect only at binary receptors. By contrast, the hitherto reported δ‐selective compounds, AA 29504 and 3‐ OH ‐2'MeO6 MF , were non‐selective. The expression system was further validated using patch clamp electrophysiology, in which the superagonism of THIP was confirmed. The established FMP assay set‐up, based on transient expression of human α 4 and β 1/3 subunits into a δ‐subunit stable HEK 293 Flp‐In™ cell line, portrays a simple 96‐well format assay as a useful supplement to electrophysiological recordings on δ‐containing GABA A receptors.