Yellow Fluorescent Protein-Based Assay to Measure GABA _A Channel Activation and Allosteric Modulation in CHO-K1 Cells

Abstract

The γ-aminobutyric acid A (GABA _A) ion channels are important drug targets for treatment of neurological and psychiatric disorders. Finding GABA _A channel subtype selective allosteric modulators could lead to new improved treatments. However, the progress in this area has been obstructed by the challenging task of developing functional assays to support screening efforts and the generation of cells expressing functional GABA _A ion channels with the desired subtype composition. To address these challenges, we developed a yellow fluorescent protein (YFP)-based assay to be able to study allosteric modulation of the GABA _A ion channel using cryopreserved, transiently transfected, assay-ready cells. We show for the first time how the MaxCyte STX electroporation instrument can be used to generate CHO-K1 cells expressing functional GABA _A α2β3γ2 along with a halide sensing YFP-H148Q/I152L (YFP-GABA _A2 cells). As a basis for a cell-based assay capable of detecting allosteric modulators, experiments with antagonist, ion channel blocker and modulators were used to verify GABA _A subunit composition and functionality. We found that the I ⁻ concentration used in the YFP assay affected both basal quench of YFP and potency of GABA. For the first time the assay was used to study modulation of GABA with 7 known modulators where statistical analysis showed that the assay can distinguish modulatory pEC ₅₀ differences of 0.15. In conclusion, the YFP assay proved to be a robust, reproducible and inexpensive assay. These data provide evidence that the assay is suitable for high throughput screening (HTS) and could be used to discover novel modulators acting on GABA _A ion channels.