title: Voltage- and [ATP]-dependent gating of the P2X(2) ATP receptor channel.
abstract: P2X receptors are ligand-gated cation channels activated by extracellular adenosine triphosphate (ATP). Nonetheless, P2X(2) 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 conductance-voltage 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 P2X(2) is determined in a complex way that involves both membrane voltage and ATP binding. The activation phase was similarly recorded in HEK293 cells expressing P2X(2) 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 (TM) helix, 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, whereas a G344P mutant retained an activation phase that was slower than the wild type (WT). Using glycine-scanning mutagenesis in the background of G344A, we could recover the activation phase by introducing a glycine residue into the middle of second 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 P2X(2)-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].
authors.affiliation: Division of Biophysics and Neurobiology, Department of Molecular Physiology, National Institute for Physiological Sciences, Aichi, Japan. fujiwara@phys2.med.osaka-u.ac.jp
authors.forename: Yuichiro
authors.initials: Y
authors.lastname: Fujiwara
authors.forename: Batu
authors.initials: B
authors.lastname: Keceli
authors.forename: Koichi
authors.initials: K
authors.lastname: Nakajo
authors.forename: Yoshihiro
authors.initials: Y
authors.lastname: Kubo
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D002478:Cells, Cultured; D005998:Glycine / Q000235:genetics / Q000378:metabolism; D006801:Humans; D015640:Ion Channel Gating; D007700:Kinetics; D009154:Mutation; D009865:Oocytes / Q000378:metabolism; D018408:Patch-Clamp Techniques; D018048:Receptors, Purinergic P2 / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D058476:Receptors, Purinergic P2X2; D014982:Xenopus laevis / Q000378:metabolism
chemical_list: C546401:P2RX2 protein, human; D018048:Receptors, Purinergic P2; D058476:Receptors, Purinergic P2X2; D000255:Adenosine Triphosphate; D005998:Glycine