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Development and Characterization of a Fluorescent Protein‐Based Biosensor for the Detection of Extracellular ATP

Extracellular adenosine 5′‐triphosphate (ATP) is a fundamental component of purinergic signaling that contributes to wide ranging physiological processes and pathophysiological conditions. New methods that enable direct measurement of extracellular ATP dynamics with high spatial and temporal resolution are expected to advance our understanding of purinergic signaling mechanisms in healthy and pathological states. Several distinct classes of fluorescent protein‐based biosensors have emerged as powerful molecular tools that report real‐time intracellular ATP dynamics with single‐cell and subcellular resolution, but there are no reports of leveraging such technology for the detection of extracellular ATP. Therefore, the focus of this study was to develop and characterize a series of fluorescent protein‐based biosensors for the detection of extracellular ATP. Our strategy was to anchor several candidate ATP biosensors to the plasma membrane and to position the biosensor in the extracellular space, allowing for the detection of extracellular ATP. We present the extensive characterization of one such cell surface‐tethered candidate biosensor. Live‐cell fluorescence microscopy was used to verify biosensor expression and localization at the cell surface and to measure sensor response to exogenous application of ATP when expressed by cell types including Neuro2A and HEK293 cells. Furthermore, we present progress using this sensor to measure extracellular ATP dynamics that are modulated by cellular processes, including ligand‐stimulated cellular release of ATP and degradation of ATP by ectonucleotidase activity. In conclusion, we are developing a genetically‐encoded fluorescent protein‐based biosensor that currently demonstrates the ability to measure dynamic changes in extracellular ATP levels in live‐cell proof‐of‐concept studies.