In-vivo measurement of the extracellular ATP concentration by bio-luminescence: The pmeLUC probe

Extracellular ATP (eATP) serves as a crucial signaling molecule in diverse physiological and pathological processes, including neurotransmission, inflammation, and cancer. Despite its importance, accurate measuring eATP dynamics in vivo has remained a significant technical challenge. Traditional methods, such as soluble luciferase systems, fluorescent probes, microelectrode biosensors, and high-performance liquid chromatography (HPLC), exhibit limitations in spatial resolution, tissue permeability, and real-time monitoring. Fluorescent probes offer high spatial resolution but are hindered by poor tissue penetration and the need for excitation light. Microelectrode biosensors provide localized detection but require invasive procedures, while HPLC, though highly sensitive, is restricted to ex vivo applications.To address these limitations, the plasma-membrane-targeted luciferase (pmeLUC) probe was developed. This bioluminescent system allows real-time, quantitative monitoring of eATP levels in living cells and animal models without the need for external excitation light. The pmeLUC is anchored on the outer surface of the plasma membrane, positioning its catalytic site extracellularly for direct eATP sensing. Its high sensitivity, tissue permeability, and adaptability for both in vitro and in vivo studies have enabled significant advancements in understanding eATP dynamics across different pathological contexts, including tumor microenvironments, immune responses, and brain injury models. Furthermore, the creation of pmeLUC-transgenic mice and of AAV-mediated delivery systems, has expanded the applications of this tool for longitudinal and systemic monitoring of eATP in living organisms. This review highlights the rationale behind choosing pmeLUC over other methodologies, emphasizing its superior capabilities in overcoming existing technical barriers and advancing eATP research in both basic and translational sciences.