Abstract
P2X receptors are ligand-gated cation channels activated by extracellular ATP. The P2X2 channel current at the steady-state after ATP application is known to have voltage-dependence, i.e.–it shows inward rectification, and a gradual increase in the inward current is observed upon hyperpolarization. We analyzed this "activation" phase quantitatively under two-electrode voltage clamp using Xenopus oocytes expression system, and also approached its structural background by mutating a glycine residue (G344) in the 2nd-transmembrane helix (2nd-TM), a putative kink for the "gating". We observed that the inward current of G344A mutant increased instantaneously upon hyperpolarization without a gradual increase. On the contrary, G344P mutant showed a slower "activation" than that of WT. We also analyzed the conductance-voltage relationship by measuring the tail current, and observed that the half-maximal voltage of "activation" of G344P was shifted to the hyperpolarized potential in comparison with that of WT. The mutation did not affect the ATP dose-response relationships significantly. By the glycine scanning mutageneses on the G344A mutant background, we observed a recovery of the "activation" phase by introducing a glycine residue to the middle region of 2nd-TM. Taken together, we speculate that the flexibility of G344 in the 2nd-TM contributes to the voltage dependent "gating" of P2X2 channel, which could be caused by an intrinsic or extrinsic mechanism to the channel molecule. [J Physiol Sci. 2006;56 Suppl:S151]
