Institute of Chemical Sciences and Engineering (ISIC), Institute of Bioengineering, NCCR in Chemical Biology, Ecole Polytechnique Fédérale de Lausanne (EPFL), 1015 Lausanne, Switzerland.
J Am Chem Soc. 2012 Nov 21;134(46):19026-34. doi: 10.1021/ja306320s. Epub 2012 Nov 5.
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.
虽然γ-氨基丁酸（GABA）是主要的抑制性神经递质，但缺乏能够以高时空分辨率在活细胞中测量其浓度的合适工具。本文描述了第一个用于 GABA 的比率荧光传感器，称为 GABA-Snifit，它可以在活哺乳动物细胞表面上以高特异性和时空分辨率感应 GABA。GABA-Snifit 是一种半合成融合蛋白，包含 GABA(B)受体、SNAP 和 CLIP 标签、合成荧光团和荧光 GABA(B)受体拮抗剂。当组装在细胞表面上时，GABA-Snifit 在 500-700nm 的范围内显示出 GABA 依赖性荧光发射光谱，允许感应微摩尔至毫摩尔 GABA 浓度。传感器在活细胞上的比率变化为 1.8。此外，GABA-Snifit 可用于定量测定 GABA(B)受体激动剂、拮抗剂和变构调节剂的相对结合亲和力及其作用。这些特性使 GABA-Snifit 成为研究 GABA 和 GABA(B)在生物系统中作用的有价值工具。
