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The Journal of Neuroscience, September 30, 2009, 29(39):12284-12291; doi:10.1523/JNEUROSCI.2096-09.2009

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Cellular/Molecular
Reactive Oxygen Species Potentiate the P2X₂ Receptor Activity through Intracellular Cys⁴³⁰

Claudio Coddou,^1,2 Juan F. Codocedo,¹ Shuo Li,² Juan G. Lillo,¹ Claudio Acuña-Castillo,³ Paulina Bull,¹ Stanko S. Stojilkovic,² and J. Pablo Huidobro-Toro¹

¹Centro Regulación Celular y Patología Prof. J. V. Luco, Instituto Milenio de Biología Fundamental y Aplicada, Departamentos de Fisiología, Genética Molecular y Microbiología, Facultad de Ciencias Biológicas, Pontificia Universidad Católica de Chile, Santiago, Chile, ²Section on Cellular Signaling, Program in Developmental Neuroscience, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892, and ³Departamento de Biología, Facultad de Química y Biología, Universidad de Santiago de Chile, Santiago, Chile

Correspondence should be addressed to Dr. J. Pablo Huidobro-Toro, Departamento de Fisiología, Facultad de Ciencias Biológicas, Pontificia Universidad Católica de Chile, Alameda 340, Casilla 114-D, PC 6513677, Santiago, Chile. Email: jphuid{at}bio.puc.cl

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 P2X₂R and inhibits the activity of P2X₄R. 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 P2X₂ 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 P2X₄ 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 Cys⁴³⁰ residue mediates effects of mercury on the P2X_2aR 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_2aR and P2X_4/2aR currents but not P2X_2bR and P2X_2a–C430A and P2X_2a–C430S mutant currents, whereas antioxidants dithiothreitrol and N-acetylcysteine prevented the H₂O₂ potentiation. Alkylation of Cys⁴³⁰ residue with methylmethane-thiosulfonate also abolished the mercury and H₂O₂ potentiation. Altogether, these results are consistent with the hypothesis that the Cys⁴³⁰ residue is an intracellular P2X_2aR redox sensor.

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Received May 4, 2009; revised June 26, 2009; accepted July 5, 2009.

Correspondence should be addressed to Dr. J. Pablo Huidobro-Toro, Departamento de Fisiología, Facultad de Ciencias Biológicas, Pontificia Universidad Católica de Chile, Alameda 340, Casilla 114-D, PC 6513677, Santiago, Chile. Email: jphuid{at}bio.puc.cl

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