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P2X3 receptor channels expressed in sensory neurons are activated by extracellular ATP and serve important roles in nociception and sensory hypersensitization, making them attractive therapeutic targets. Although several P2X3 structures are known, it is unclear how physiologically abundant Ca 2+ -ATP and Mg 2+ -ATP activate the receptor, or how divalent cations regulate channel function. We used structural, computational and functional approaches to show that a crucial acidic chamber near the nucleotide-binding pocket in human P2X3 receptors accommodates divalent ions in two distinct modes in the absence and presence of nucleotide. The unusual engagement between the receptor, divalent ion and the γ-phosphate of ATP enables channel activation by ATP-divalent complex, cooperatively stabilizes the nucleotide on the receptor to slow ATP unbinding and recovery from desensitization, a key mechanism for limiting channel activity. These findings reveal how P2X3 receptors recognize and are activated by divalent-bound ATP, aiding future physiological investigations and drug development., Competing Interests: ML, YW, RB, FM, SS, MH No competing interests declared, JF, KS Reviewing editor, eLife

The identity and forms of activating ligands for ion channels are fundamental to their physiological roles in rapid electrical signaling. P2X receptor channels are ATP-activated cation channels that serve important roles in sensory signaling and inflammation, yet the active forms of the nucleotide are unknown. In physiological solutions, ATP is ionized and primarily found in complex with Mg(2+). Here we investigated the active forms of ATP and found that the action of MgATP(2-) and ATP(4-) differs between subtypes of P2X receptors. The slowly desensitizing P2X2 receptor can be activated by free ATP, but MgATP(2-) promotes opening with very low efficacy. In contrast, both free ATP and MgATP(2-) robustly open the rapidly desensitizing P2X3 subtype. A further distinction between these two subtypes is the ability of Mg(2+) to regulate P2X3 through a distinct allosteric mechanism. Importantly, heteromeric P2X2/3 channels present in sensory neurons exhibit a hybrid phenotype, characterized by robust activation by MgATP(2-) and weak regulation by Mg(2+). These results reveal the existence of two classes of homomeric P2X receptors with differential sensitivity to MgATP(2-) and regulation by Mg(2+), and demonstrate that both restraining mechanisms can be disengaged in heteromeric channels to form fast and sensitive ATP signaling pathways in sensory neurons.