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 pEC50 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.