A High-Throughput Functional Assay for Characterization of γ-Aminobutyric AcidA Channel Modulators Using Cryopreserved Transiently Transfected Cells

The ionotropic γ-aminobutyric acid (GABA)A receptors are an important family of drug targets for a variety of neurological and psychiatric disorders. Selective modulation of certain subtypes of the receptor could lead to novel or improved therapies. However, the discovery of subtype-selective compounds has been hampered by the lack of a high-throughput functional assay and the difficulty in establishing stable cell lines expressing GABAA receptor in a proper subunit composition. To meet drug discovery need we developed a fluorescent imaging plate reader (FLIPR)-based membrane potential assay with sufficient robustness and reproducibility for use in a high-throughput format. Two major subtypes of GABAA receptor were used: GABAA1 and GABAA2, which are composed of (α1)2(β2)2γ2 and (α1)2(β3)2γ2, respectively. We expressed the receptors by transiently co-transfecting cells with the three subunit DNAs in separate constructs, and by controlling the ratio of the DNA amount for each subunit transfected we forced t...