Reactive Oxygen Species Potentiate the P2X2 Receptor Activity through Intracellular Cys430

P2X receptor channels (P2XRs) are allosterically modulated by several compounds, mainly acting at the ectodomain of the receptor. Like copper, mercury, a metal that induces oxidative stress in cells, also stimulates the activity of P2X(2)R and inhibits the activity of P2X(4)R. However, the mercury modulation is not related to the extracellular residues critical for copper modulation. To identify the site(s) for mercury action, we generated two chimeras using the full size P2X(2) subunit, termed P2X(2a), and a splice variant lacking a 69 residue segment in the C terminal, termed P2X(2b), as the donors for intracellular and transmembrane segments and the P2X(4) subunit as the donor for ectodomain segment of chimeras. The potentiating effect of mercury on ATP-induced current was preserved in Xenopus oocytes expressing P2X(4/2a) chimera but was absent in oocytes expressing P2X(4/2b) chimera. Site-directed mutagenesis experiments revealed that the Cys(430) residue mediates effects of mercury on the P2X(2a)R activity. Because mercury could act as an oxidative stress inducer, we also tested whether hydrogen peroxide (H2O2) and mitochondrial stress inducers myxothiazol and rotenone mimicked mercury effects. These experiments, done in both oocytes and human embryonic kidney HEK293 cells, revealed that these compounds potentiated the ATP-evoked P2X(2a)R and P2X(4/2a)R currents but not P2X(2b)R and P2X(2a)-C430A and P2X(2a)-C430S mutant currents, whereas antioxidants dithiothreitrol and N-acetylcysteine prevented the H2O2 potentiation. Alkylation of Cys(430) residue with methylmethane-thiosulfonate also abolished the mercury and H2O2 potentiation. Altogether, these results are consistent with the hypothesis that the Cys(430) residue is an intracellular P2X(2a)R redox sensor.