Adenosine 5’ triphosphate (ATP) is a ubiquitous extracellular signaling messenger. Here, we describe a method for in-vivo imaging of extracellular ATP with high spatiotemporal resolution. We prepared a comprehensive set of cysteine-substitution mutants of ATP-binding protein, Bacillus FoF1-ATP synthase ε subunit, labeled with small-molecule fluorophores at the introduced cysteine residue. Screening revealed that the Cy3-labeled glutamine-105 mutant (Q105C-Cy3; designated ATPOS) shows a large fluorescence change in the presence of ATP, with submicromolar affinity, pH-independence, and high selectivity for ATP over ATP metabolites and other nucleotides. To enable in-vivo validation, we introduced BoNT/C-Hc for binding to neuronal plasma membrane and Alexa Fluor 488 for ratiometric measurement. The resulting ATPOS complex binds to neurons in cerebral cortex of living mice, and clearly visualized a concentrically propagating wave of extracellular ATP release in response to electrical stimulation. ATPOS should be useful to probe the extracellular ATP dynamics of diverse biological processes in vivo.

中文翻译：

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使用混合型荧光传感器对大脑中细胞外 ATP 进行实时体内成像


腺苷 5' 三磷酸 (ATP) 是一种普遍存在的细胞外信号传导信使。在这里，我们描述了一种具有高时空分辨率的细胞外 ATP 体内成像方法。我们制备了一套全面的 ATP 结合蛋白、芽孢杆菌 FoF1-ATP 合酶 ε 亚基的半胱氨酸取代突变体，并在引入的半胱氨酸残基处用小分子荧光团进行标记。筛选显示，Cy3 标记的谷氨酰胺-105 突变体（Q105C-Cy3；命名为 ATPOS）在 ATP 存在下表现出较大的荧光变化，具有亚微摩尔亲和力、pH 无关性以及对 ATP 相对于 ATP 代谢物和其他核苷酸的高选择性。为了实现体内验证，我们引入了用于与神经元质膜结合的 BoNT/C-Hc 和用于比率测量的 Alexa Fluor 488。由此产生的 ATPOS 复合物与活体小鼠大脑皮层的神经元结合，并清晰地显示出响应电刺激的细胞外 ATP 释放的同心传播波。 ATPOS 应该有助于探测体内不同生物过程的细胞外 ATP 动态。