Lorca Ramón A, Coddou Claudio, Gazitúa M Consuelo, Bull Paulina, Arredondo Cristián, Huidobro-Toro J Pablo
Departmentos de Fisiología, Centro de Regulacion Celular y Patologia J.V. Luco, Instituto MIFAB, Faculty de Ciencias biologicas, Pontifica Universidad Catolica de Chile, Santiago, Chile.
J Neurochem. 2005 Oct;95(2):499-512. doi: 10.1111/j.1471-4159.2005.03387.x.
To assess the mechanism of P2X2 receptor modulation by transition metals, the cDNA for the wild-type receptor was injected to Xenopus laevis oocytes and examined 48-72 h later by the two-electrode voltage-clamp technique. Copper was the most potent of the trace metals examined; at 10 microm it evoked a 25-fold potentiation of the 10 microm ATP-gated currents. Zinc, nickel or mercury required 10-fold larger concentrations to cause comparable potentiations, while palladium, cobalt or cadmium averaged only 12- and 3-fold potentiations, respectively. Platinum was inactive. The non-additive effect of copper and zinc at 10-100 microm suggests a common site of action; these metals also shifted to the left the ATP concentration-response curves. To define residues necessary for trace metal modulation, alanines were singly substituted for each of the nine histidines in the extracellular domain of the rat P2X2 receptor. The H120A and H213A mutants were resistant to the modulator action of copper, zinc and other metals with the exception of mercury. Mutant H192A showed a reduction but not an abrogation of the copper or zinc potentiation. H245A showed less affinity for copper while this mutant flattened the zinc-induced potentiation. Mutant H319A reduced the copper but not the zinc-induced potentiation. In contrast, mutants H125A, H146A, H152A and H174A conserved the wild-type receptor sensitivity to trace metal modulation. We propose that His120, His192, His213 and His245 form part of a common allosteric metal-binding site of the P2X2 receptor, which for the specific coordination of copper, but not zinc, additionally involves His319.
为评估过渡金属对P2X2受体的调节机制，将野生型受体的cDNA注射到非洲爪蟾卵母细胞中，并在48 - 72小时后通过双电极电压钳技术进行检测。在所检测的痕量金属中，铜的作用最为显著；在10微摩尔浓度时，它能使10微摩尔ATP门控电流增强25倍。锌、镍或汞需要10倍以上的浓度才能产生类似的增强作用，而钯、钴或镉分别平均只能使电流增强12倍和3倍。铂则无活性。10 - 100微摩尔浓度下铜和锌的非相加效应表明它们有共同的作用位点；这些金属还使ATP浓度 - 反应曲线向左移动。为确定痕量金属调节所需的残基，将丙氨酸逐个取代大鼠P2X2受体胞外结构域中的九个组氨酸。H120A和H213A突变体对铜、锌和其他金属（汞除外）的调节作用具有抗性。H192A突变体显示铜或锌增强作用有所降低，但未完全消除。H245A对铜的亲和力降低，而该突变体使锌诱导的增强作用趋于平缓。H319A突变体降低了铜诱导的增强作用，但对锌诱导的增强作用无影响。相反，H125A、H146A、H152A和H174A突变体保留了野生型受体对痕量金属调节的敏感性。我们提出，His120、His192、His213和His245构成了P2X2受体共同变构金属结合位点的一部分，对于铜（而非锌）的特异性配位，还涉及His319。
