Development of a Hybrid-Type Fluorescent GABA Sensor through Structure-Guided Efficient Screening

γ-Aminobutyric acid (GABA) is a major inhibitory neurotransmitter in the brain, and visualizing its fast synaptic dynamics is crucial for understanding neural function. However, detecting these transient signals requires sensors with both high spatiotemporal resolution and robust performance under physiological conditions. Here, we report the development of a novel hybrid-type fluorescent GABA sensor generated by site-specifically labeling a fluorescent dye onto a GABA-binding protein. For efficiently identifying the optimal labeling site, we devised a structure-guided screening strategy based on a principle derived from our comprehensive analysis of a homologous glutamate-binding protein: that optimal labeling sites are primarily concentrated at the edge of the cleft regions. Applying this principle to an AI-predicted structure of the GABA-binding protein led to the identification of our lead sensor, Pf622 G183C-JF585, which exhibits a large dynamic range (>300%), high selectivity for GABA with little response to other neurotransmitters, fast kinetics, and excellent stability within the physiological pH range. Importantly, Pf622 G183C-JF585 successfully detected endogenous GABA released from cultured neurons upon electrical stimulation, demonstrating its utility for detecting the transient release of GABA. This new high-performance sensor, therefore, represents a powerful tool for the spatiotemporal analysis of GABAergic neurotransmission.