Coddou Claudio, Codocedo Juan F, Li Shuo, Lillo Juan G, Acuña-Castillo Claudio, Bull Paulina, Stojilkovic Stanko S, Huidobro-Toro J Pablo
Departamentos de Fisiología, Facultad de Ciencias Biológicas, Centro Regulación Celular y Patología Prof J V Luco, Instituto Milenio de Biología Fundamental y Aplicada, Pontificia Universidad Católica de Chile, Santiago, Chile.
J Neurosci. 2009 Sep 30;29(39):12284-91. doi: 10.1523/JNEUROSCI.2096-09.2009.
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 (H(2)O(2)) 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 H(2)O(2) potentiation. Alkylation of Cys(430) residue with methylmethane-thiosulfonate also abolished the mercury and H(2)O(2) potentiation. Altogether, these results are consistent with the hypothesis that the Cys(430) residue is an intracellular P2X(2a)R redox sensor.
P2X受体通道（P2XRs）受到多种化合物的变构调节，这些化合物主要作用于受体的胞外结构域。与铜一样，汞这种能在细胞中诱导氧化应激的金属，也能刺激P2X(2)R的活性并抑制P2X(4)R的活性。然而，汞的调节作用与对铜调节至关重要的细胞外残基无关。为了确定汞的作用位点，我们构建了两个嵌合体，使用全长P2X(2)亚基（称为P2X(2a)）和C末端缺少69个残基片段的剪接变体（称为P2X(2b)）作为胞内和跨膜片段的供体，以及P2X(4)亚基作为嵌合体胞外结构域片段的供体。汞对ATP诱导电流的增强作用在表达P2X(4/2a)嵌合体的非洲爪蟾卵母细胞中得以保留，但在表达P2X(4/2b)嵌合体的卵母细胞中则不存在。定点突变实验表明，半胱氨酸（Cys）430残基介导了汞对P2X(2a)R活性的影响。由于汞可作为氧化应激诱导剂，我们还测试了过氧化氢（H(2)O(2)）以及线粒体应激诱导剂粘噻唑啉和鱼藤酮是否模拟了汞的作用。这些在卵母细胞和人胚肾HEK293细胞中进行的实验表明，这些化合物增强了ATP诱发的P2X(2a)R和P2X(4/2a)R电流，但对P2X(2b)R、P2X(2a)-C430A和P2X(2a)-C430S突变体电流没有增强作用，而抗氧化剂二硫苏糖醇和N-乙酰半胱氨酸则阻止了H(2)O(2)的增强作用。用甲硫代磺酸甲酯对Cys(430)残基进行烷基化也消除了汞和H(2)O(2)的增强作用。总之，这些结果与Cys(430)残基是胞内P2X(2a)R氧化还原传感器这一假设一致。
