Construction of a Fluorescent Screening System of Allosteric Modulators for the GABA_A Receptor Using a Turn-On Probe

γ-Aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the central nervous system. The fast inhibitory actions of GABA are mainly mediated by GABA_A receptors (GABA_ARs), which are widely recognized as clinically relevant drug targets. However, it remains difficult to create screening systems for drug candidates that act on GABA_ARs because of the existence of multiple ligand-binding sites and the delicate pentameric structures of GABA_ARs. We here developed the first turn-on fluorescent imaging probe for GABA_ARs, which can be used to quantitatively evaluate ligand-receptor interactions under live cell conditions. Using noncovalent labeling of GABA_ARs with this turn-on probe, a new imaging-based ligand assay system, which allows discovery of positive allosteric modulators (PAMs) for the GABA_AR, was successfully constructed. Our system is applicable to high-throughput ligand screening, and we discovered new small molecules that function as PAMs for GABA_ARs. These results highlight the power of the use of a turn-on fluorescent probe to screen drugs for complicated membrane proteins that cannot be addressed by conventional methods.