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

The ionotropic gamma-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 GABA(A) 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 GABA(A) receptor were used: GABA(A1) and GABA(A2), which are composed of (alpha 1)(2)(beta 2)(2)gamma 2 and (alpha 1)(2)(beta 3)(2)gamma 2, 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 the cells to express GABA(A) receptors in a pharmacologically relevant form. A large batch of transfected human embryonic kidney 293 cells were cryopreserved and used to screen and evaluate GABA(A) modulators. In these cells, agonist activation of GABA(A) receptor resulted in Cl- efflux and membrane depolarization, which was detected by FLIPR as an increase in fluorescence signal. Based on our characterization of several known GABA(A) modulators and a test set of compounds known to bind to the GABA(A) benzodiazepine site, we have demonstrated the validity and utility of this assay for discovery of novel pharmacological agents acting at GABA(A) receptors.