Bayesian inference of functional asymmetry in the homotrimeric ligand-gated ion channel P2X2

The trimeric ATP-gated receptor P2X₂ offers a minimalist scaffold for dissecting subunit-coupled allosteric activation. Despite closed and open structures, the physical origin of the transient 'flip' intermediate and P2X₂'s negative cooperativity remain unresolved. Rapid patch-clamp kinetics parsed by Bayesian inference (MacroIR) and supported by atomistic simulations reveal that P2X₂ activates through a sequential, asymmetric coupling mechanism. ATP binding to any inter subunit pocket selectively reduces the rotational barrier on one of the two framing subunits, triggering partial activation of the receptor while the other subunit is minimally affected. This rotation then raises the barrier for the next ATP-binding event, accounting for the receptor's negative cooperativity. Under ligand-free conditions, heightened rotation barriers trap the channel in its closed conformation and quantitatively reproduce the spontaneous current fluctuations we record. These results overturn the canonical view of symmetric, concerted gating in P2X₂ and explains the classical flip state as an obligatory structural intermediate.