P2X receptors are ligand-gated cation channels activated by extracellular ATP. Nonetheless, P2X2 channel currents observed during the steady state after ATP application are known to exhibit voltage-dependence; there is a gradual increase in the inward current upon hyperpolarization. We used a Xenopus oocyte expression system and two-electrode voltage clamp to analyze this “activation” phase quantitatively. We characterized the G-V relationship in the presence of various [ATP], and observed that it shifted toward more depolarized potentials with increases in [ATP]. By analyzing the rate constants for the channel's transition between a closed and an open state, we showed that the gating of P2X2 is determined in a complex way that involves both membrane voltage and ATP binding. The activation phase was similarly recorded in HEK293 cells expressing P2X2 even by inside-out patch clamp after intensive perfusion, excluding a possibility that the gating is due to block/unblock by endogenous blocker(s) of oocytes. We investigated its structural basis by substituting a glycine residue (G344) in the second transmembrane helix (2nd-TM), which may provide a kink that could mediate “gating.” We found that, instead of a gradual increase, the inward current through the G344A mutant increased instantaneously upon hyperpolarization, while, a G344P mutant retained an "activation" phase that was slower than the wild-type. Using glycine-scanning mutagenesis in the background of G344A, we could recover the “activation” phase by introducing a glycine residue into the middle of 2nd-TM. These results demonstrate that the flexibility of G344 contributes to the voltage dependent “gating”. Finally we assumed a three-state model consisting of a fast ATP-binding step and a following gating step and estimated the rate constants for the latter in P2X2-WT. We then executed simulation analyses using the calculated rate constants and successfully reproduced the results observed experimentally, voltage-dependent activation that is accelerated by increases in [ATP].
