Tarasov Andrei I, Rutter Guy A
Oxford Centre for Diabetes, Endocrinology and Metabolism, University of Oxford, Oxford, United Kingdom.
Section of Cell Biology, Department of Medicine, Imperial College London, London, United Kingdom.
Methods Enzymol. 2014;542:289-311. doi: 10.1016/B978-0-12-416618-9.00015-7.
ATP is not only recognized as the universal energy "currency" in most cells but also plays a less well-known role as an intracellular and extracellular messenger. Here, we review novel approaches for measuring free ATP (or ATP/ADP ratios) in living mammalian cells by using genetically encoded sensors. We also discuss the key technical aspects of routine real-time ATP/ADP monitoring using as a model one of the last-generation fluorescent probes, a fusion protein commonly known as "Perceval." Finally, we present detailed guidelines for the simultaneous measurement of cytosolic ATP/ADP ratios and Ca(2+) concentrations alongside electrical parameters in individual pancreatic β cells, in which energy metabolism is tightly linked to plasma membrane excitability to control the secretion of insulin. With appropriate variations, this approach can be adapted to the study of cytosolic ATP/ADP ratios and Ca(2+) concentrations in malignant cells, two important aspects of oncometabolism.
ATP不仅被认为是大多数细胞中的通用能量“货币”，还作为细胞内和细胞外信使发挥着鲜为人知的作用。在此，我们综述了利用基因编码传感器测量活的哺乳动物细胞中游离ATP（或ATP/ADP比率）的新方法。我们还讨论了使用一种最新一代荧光探针（一种通常称为“Perceval”的融合蛋白）作为模型进行常规实时ATP/ADP监测的关键技术方面。最后，我们给出了在单个胰腺β细胞中同时测量胞质ATP/ADP比率和Ca(2+)浓度以及电参数的详细指南，在胰腺β细胞中能量代谢与质膜兴奋性紧密相连以控制胰岛素分泌。经过适当修改，这种方法可适用于研究恶性细胞中的胞质ATP/ADP比率和Ca(2+)浓度，这是肿瘤代谢的两个重要方面。
