TY - JOUR
T1 - Development of a Robust Mammalian Cell-based Assay for Studying Recombinant α4 β1/3 δ GABAA Receptor Subtypes
AU - Falk-Petersen, Christina B
AU - Søgaard, Rikke
AU - Madsen, Kenneth L
AU - Klein, Anders B
AU - Frølund, Bente
AU - Wellendorph, Petrine
N1 - © 2017 Nordic Association for the Publication of BCPT (former Nordic Pharmacological Society).
PY - 2017/8
Y1 - 2017/8
N2 - 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 Zn(2+) 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.
AB - 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 Zn(2+) 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.
U2 - 10.1111/bcpt.12778
DO - 10.1111/bcpt.12778
M3 - Journal article
C2 - 28299900
SN - 1742-7835
VL - 121
SP - 119
EP - 129
JO - Basic and Clinical Pharmacology and Toxicology
JF - Basic and Clinical Pharmacology and Toxicology
IS - 2
ER -