Cryo-EM elucidation of stoichiometric plasticity, asymmetric ligand recognition and allosteric coupling in human P2X2/3 heterotrimeric channels

P2X receptors are trimeric ATP-gated ion channels that assemble as homo- or heterotrimers, with heteromeric forms exhibiting intrinsic asymmetry that influences function. Here, we report four high-resolution cryo–EM structures of human P2X2/3 heterotrimers representing distinct functional states, including ATP-bound assemblies (P2X332 and P2X223), the apo form, and a ligand/ATP-bound closed conformation. The three ATP-binding sites show asymmetric recognition of MgATP²⁻ and ATP⁴⁻, and channel activation requires occupancy of only two MgATP²⁻ molecules. Gefapixant binds a single allosteric site and selectively inhibits MgATP²⁻, but not ATP⁴⁻, binding, indicating orthosteric–allosteric coupling within the heterotrimer. Structural features of the transmembrane domain define ion permeation, particularly for Ca²⁺. Despite asymmetric ligand interactions, gating remains largely symmetric, with minor differences in desensitization. These findings provide a structural framework linking asymmetry to coordinated channel function and open avenues for subtype-selective therapeutic intervention.