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September 23, 2025Open Access

An Improved Ratiometric FRET Biosensor with Higher Affinity for Extracellular ATP

ACAutumn Cholger JCJason M. Conley ECEvelyne Colomb

Key Points

The ECATS2 biosensor exhibits over three-fold increased affinity for extracellular ATP compared to its predecessor, enhancing detection capabilities.
This biosensor utilizes mutagenesis and optimized cell-surface display to significantly improve performance in real-time monitoring.
The study demonstrates that the sensor effectively detects extracellular ATP release during hypoosmotic stress in astrocyte cultures, highlighting its applicability.
These enhancements contribute to more precise tools for exploring extracellular ATP dynamics and purinergic signaling mechanisms.

Abstract

Adenosine triphosphate (ATP) is readily released into the extracellular space as an autocrine and paracrine purinergic signaling molecule. We originally reported a genetically encoded, fluorescent protein-based Förster Resonance Energy Transfer (FRET) biosensor that can detect micromolar levels of extracellular ATP. Through mutagenesis of the ATP binding site and optimization of cell-surface display, here we report the development of a second-generation biosensor called ECATS2 with greater than three-fold higher affinity for extracellular ATP. We found that the tether length between the FRET biosensor and the cell surface anchor is critical to optimization of its performance. Furthermore, we demonstrate that the improved sensor can detect extracellular ATP release upon hypoosmotic stress in cultured astrocytes. This new sensor contributes an improved tool for the ratiometric detection of extracellular ATP dynamics and purinergic signaling.

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