title: Development of a Robust Mammalian Cell-based Assay for Studying Recombinant α4β1/3δ GABAAReceptor Subtypes.
abstract: δ-Containing GABAAreceptors 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 GABAAreceptors. Inconveniently, the recombinant expression of especially α4β1/3δ 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 α4β1/3δ receptors, we have engineered and validated a HEK293 Flp-In™ cell line stably expressing the human GABAAδ-subunit. Upon co-transfection of α4and β1/3subunits, at optimized ratios, we have established a well-defined system for expressing α4β1/3δ receptors and used the fluorescence-based FLIPR Membrane Potential (FMP) assay to evaluate their pharmacology. Using the known reference compounds GABA and THIP, ternary α4β1/3δ and binary α4β1/3receptors 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 α4and β1/3subunits 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 GABAAreceptors.
authors.affiliation: Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark.
authors.forename: Christina B
authors.initials: CB
authors.lastname: Falk-Petersen
authors.affiliation: Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark.
authors.forename: Rikke
authors.initials: R
authors.lastname: Søgaard
authors.affiliation: Molecular Neuropharmacology and Genetics Laboratory, Lundbeck Foundation Center for Biomembranes in Nanomedicine, Department of Neuroscience and Pharmacology, Faculty of Health and Medical Sciences, The Panum Institute, University of Copenhagen, Copenhagen, Denmark.
authors.forename: Kenneth L
authors.initials: KL
authors.lastname: Madsen
authors.affiliation: Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark.
authors.forename: Anders B
authors.initials: AB
authors.lastname: Klein
authors.affiliation: Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark.
authors.forename: Bente
authors.initials: B
authors.lastname: Frølund
authors.affiliation: Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark.
authors.forename: Petrine
authors.initials: P
authors.lastname: Wellendorph
mesh_terms: D005456:Fluorescent Dyes / Q000737:chemistry; D018755:GABA Agonists / Q000494:pharmacology; D057809:HEK293 Cells; D006801:Humans; D007150:Immunohistochemistry; D007700:Kinetics; D008564:Membrane Potentials / Q000187:drug effects; D018613:Microscopy, Confocal; D009154:Mutation; D009419:Nerve Tissue Proteins / Q000819:agonists / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D018408:Patch-Clamp Techniques; D011963:Receptors, GABA-A / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D015203:Reproducibility of Results; D015032:Zinc / Q000494:pharmacology; D005680:gamma-Aminobutyric Acid / Q000378:metabolism
chemical_list: D005456:Fluorescent Dyes; D018755:GABA Agonists; C546260:GABRA4 protein, human; C546264:GABRB1 protein, human; C546273:GABRB3 protein, human; C464500:GABRD protein, human; D009419:Nerve Tissue Proteins; D011963:Receptors, GABA-A; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D005680:gamma-Aminobutyric Acid; D015032:Zinc