Endogenous forms of ATP-ATP4- and MgATP2--orchestrate distinct pathophysiological signaling via biased activation of P2X3 receptors.

P2X receptors, a family of ATP-activated ion channels, encompass subtypes P2X1-7, which are expressed in both homo- and heterotrimeric forms across various tissues. These receptors play crucial roles in pathophysiological processes such as synaptic transmission, nociception, cough, and taste perception. Extracellular ATP exists as both MgATP2- and ATP4-, with P2X3 responding to both. The evolutionary rationale for two nearly identical ligands and their distinct signaling potential remains unclear. While previous structural studies suggest a uniform ATP recognition mechanism for two endogenous ATP forms, we propose that MgATP2- and ATP4- activate P2X3 through distinct mechanisms, leading to differential physiological and pathological outcomes. Using mutagenesis, voltage-clamp fluorometry, and small molecule interventions, we identify divergent interactions of ATP4- and MgATP2- with P2X3, despite binding to the same orthosteric pocket. In P2rx3D158A/D158A transgenic mice, which selectively impair MgATP2- activation, we find that MgATP2- modulates ammonia-induced cough frequency without affecting complete Freund's adjuvant-induced inflammatory pain or sweet taste preference. P2rx3-/- mice show deficits in all three responses. The allosteric inhibitor aurintricarboxylic acid selectively modulates ATP4- and MgATP2- effects, resulting in distinct antitussive and analgesic outcomes in vivo. These findings uncover a mechanism of P2X3 activation by its endogenous ligands, diverging from previous structural models and resembling the biased activation mechanisms observed in G-protein-coupled receptors, offering insights for P2X3-targeted therapeutics.