Optimizing the Expression of Recombinant αβγ GABA A Receptors in HEK293 Cells for High-Throughput Screening

Abstract

Despite being important clinical targets, it is not straightforward to reliably express recombinant trimeric αβγ GABA-A receptors (GABA_ARs) for high-throughput screening. This study therefore sought to devise a simple and reliable means of transiently expressing α1β1γ1 and α1β1γ2 GABA_ARs in HEK293 cells. Expression efficiencies resulting from 5 different transfection strategies were assessed by flow cytometry and pharmacological analysis using an anion-sensitive yellow fluorescent protein-based assay. PolyFect™ and Effectene™, employed according to the manufacturers' instructions, conferred the strongest and most reliable expression of trimeric αβγ GABA_ARs. Functional analysis via the yellow fluorescent protein assay revealed dramatic differences in the pharmacological properties of γ1- and γ2-containing receptors, consistent with previous electrophysiological characterizations. The authors conclude that this method of expressing and screening recombinant GABA_ARs provides an effective means of discovering novel GABA_AR modulators for use as therapeutic lead compounds and pharmacological probes. (Journal of Biomolecular Screening 2009:86-91)

Keywords

chloride channel drug discovery yellow fluorescent protein inhibitory neurotransmission

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