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