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Development of a Robust Mammalian Cell-based Assay for Studying Recombinant α4 β1/3 δ GABAA Receptor Subtypes.

Abstract：

:δ-Containing GABAA 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 GABAA receptors. Inconveniently, the recombinant expression of especially α4 β1/3 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 α4 β1/3 δ receptors, we have engineered and validated a HEK293 Flp-In™ cell line stably expressing the human GABAA δ-subunit. Upon co-transfection of α4 and β1/3 subunits, at optimized ratios, we have established a well-defined system for expressing α4 β1/3 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 α4 β1/3 δ and binary α4 β1/3 receptors could be distinguished based on potency and kinetic profiles but not efficacy. As expected, DS2 was able to potentiate only δ-containing receptors, whereas Zn2+ had an inhibitory effect only at binary receptors. By contrast, the hitherto reported δ-selective compounds, AA29504 and 3-OH-2'MeO6MF, 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 α4 and β1/3 subunits into a δ-subunit stable HEK293 Flp-In™ cell line, portrays a simple 96-well format assay as a useful supplement to electrophysiological recordings on δ-containing GABAA receptors.

journal_name

Basic Clin Pharmacol Toxicol

journal_title

Basic & clinical pharmacology & toxicology

authors

Falk-Petersen CB,Søgaard R,Madsen KL,Klein AB,Frølund B,Wellendorph P

doi

10.1111/bcpt.12778

subject

Has Abstract

pub_date

2017-08-01 00:00:00

pages

119-129

issue

eissn

1742-7835

issn

1742-7843

journal_volume

121

pub_type

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Development of a Robust Mammalian Cell-based Assay for Studying Recombinant α4 β1/3 δ GABAA Receptor Subtypes.