Methodological guidelines for P2X receptor assays and data interpretation

C. Gorecki, Dariusz and Adinolfi, Elena and Adriouch, Sahil and Coutinho-Silva, Robson and Engel, Tobias and Gölöncsér, Flóra and Haag, Friedrich and Illés, Péter and A. Jacobson, Kenneth and Koch-Nolte, Friedrich and E. Mansoor, Steven and Matute, Carlos and Melino, Gerry and Novak, Ivana and Pegoraro, Anna and Pelegrin, Pablo and Piacentini, Mauro and C. Robson, Simon and M. H. Rumney, Robin and Seman, Michel and Sluyter, Ronald and Sperlágh, Beáta and Tang, Yong and Tarantini, Mario and Ulrich, Henning and Vouret-Craviari, Valerie and Ye, Qing and G. Yegutkin, Gennady and Verkhratsky, Alexei (2026) Methodological guidelines for P2X receptor assays and data interpretation. CELL DEATH AND DISEASE. ISSN 2041-4889 (In Press)

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Abstract

P2X receptors (P2XR) are a family of seven cation channels gated by extracellular ATP (eATP). Activation of P2XRs results in diverse cellular responses, including cell signalling, proliferation, differentiation, and death - all critically important in multiple physiological and pathophysiological states. These receptors, therefore, represent therapeutic targets of considerable interest. However, P2XRs, while structurally related, exhibit highly divergent and context -dependent functions. Their spatiotemporal and functional complexity is evident by overlapping expression across multiple cell types that can shift dynamically during physiological processes or disease progression. Furthermore, P2XRs can assemble as homo - or hetero -trimers, with distinct functional properties. These factors complicate definitive identification of a given P2XR responsible for a specific pathophysiological effect. Receptor activity in vivo is transient because of receptor - specific mechanisms and follows eATP breakdown by ectonucleotidases. Any correlation of ATP release with receptor engagement, as assessed in vitro, often does not correspond with the in vivo dynamics. Translation from animal models to humans is complicated by the species -specific pharmacology of some P2XRs, confounded by many animal models in use not fully replicating human P2XR function and regulation in pathology. Furthermore, there are no clinical biomarkers to distinguish incomplete receptor blockade from lack of therapeutic effect. Thus, translation has been very limited. To identify and validate specific P2XR functionalities, future experimental designs should use approaches and assays that can reliably assess receptor involvement, while reducing methodologically flawed findings. We propose guidelines developed in consultation with the purinergic community for consistent and reliable research practices in P2XR studies. We dedicate this work to the memory of Francesco di Virgilio. He was not only an outstanding leader in purinergic signalling research but a mentor and friend to many of us, always inspiring a culture of data reliability and scientific innovation.

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