Development of a Robust Mammalian Cell-based Assay for Studying Recombinant α4 ß1/3 δ GABAA Receptor Subtypes.

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 ß1/3 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 ß1/3 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 ß1/3 subunits, at optimized ratios, we have established a well-defined system for expressing α4 ß1/3 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 ß1/3 δ and binary α4 ß1/3 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 ß1/3 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.

Metabotropic glutamate receptor 2 and corticotrophin-releasing factor receptor-1 gene expression is differently regulated by BDNF in rat primary cortical neurons.

Brain-derived neurotrophic factor (BDNF) is important for neuronal survival and plasticity. Incorporation of matured receptor proteins is an integral part of synapse formation. However, whether BDNF increases synthesis and integration of receptors in functional synapses directly is unclear. We are particularly interested in the regulation of the 5-hydroxytryptamine receptor 2A (5-HT(2A)R). This receptor form a functional complex with the metabotropic glutamate receptor 2 (mGluR2) and is recruited to the cell membrane by the corticotrophin-releasing factor receptor 1 (CRF-R1). The effect of BDNF on gene expression for all these receptors, as well as a number of immediate-early genes, was pharmacologically characterized in primary neurons from rat frontal cortex. BDNF increased CRF-R1 mRNA levels up to fivefold, whereas mGluR2 mRNA levels were proportionally downregulated. No effect on 5-HT(2A)R mRNA was seen. The effects were dose-dependent with half-maximal effective concentrations (EC(50)) around 1 ng/ml. After 24 h of incubation with BDNF, CRF-R1 mRNA levels had returned to baseline levels, whereas mGluR2 mRNA levels remained low. A significant reduction of all three receptor transcripts was observed after neuronal depolarization produced by high potassium. This study emphasizes the role of BDNF as an important regulator of receptor compositions in the synapse and provides further evidence that BDNF directly regulates important drug targets involved in cognition and mood.