Construction of a Fluorescent Screening System of Allosteric Modulators for the GABAA 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 GABAA receptors (GABAARs), which are widely recognized as clinically relevant drug targets. However, it remains difficult to create screening systems for drug candidates that act on GABAARs because of the existence of multiple ligand-binding sites and the delicate pentameric structures of GABAARs. We here developed the first turn-on fluorescent imaging probe for GABAARs, which can be used to quantitatively evaluate ligand–receptor interactions under live cell conditions. Using noncovalent labeling of GABAARs with this turn-on probe, a new imaging-based ligand assay system, which allows discovery of positive allosteric modulators (PAMs) for the GABAAR, was successfully constructed. Our system is applicable to high-throughput ligand screening, and we discovered new small molecules that function as PAMs for GABAARs. 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.