While γ-aminobutyric acid (GABA) is the main inhibitory neurotransmitter, suitable tools to measure its concentration in living cells with high spatiotemporal resolution are missing. Herein, we describe the first ratiometric fluorescent sensor for GABA, dubbed GABA-Snifit, which senses GABA with high specificity and spatiotemporal resolution on the surface of living mammalian cells. GABA-Snifit is a semisynthetic fusion protein containing the GABA(B) receptor, SNAP- and CLIP-tag, a synthetic fluorophore and a fluorescent GABA(B) receptor antagonist. When assembled on cell surfaces, GABA-Snifit displays a GABA-dependent fluorescence emission spectrum in the range of 500-700 nm that permits sensing micromolar to millimolar GABA concentrations. The ratiometric change of the sensor on living cells is 1.8. Furthermore, GABA-Snifit can be utilized to quantify the relative binding affinities of GABA(B) receptor agonists, antagonists and the effect of allosteric modulators. These properties make GABA-Snifit a valuable tool to investigate the role of GABA and GABA(B) in biological systems.
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