Coddou Claudio, Morales Bernardo, González Jorge, Grauso Marta, Gordillo Felipe, Bull Paulina, Rassendren Francois, Huidobro-Toro J Pablo
Centro de Regulación Celular y Patología J. V. Luco, Instituto Milenio Biología Fundamental y Aplicada, MIFAB, Departamentos de Fisiología y Genética Molecular y Microbiología, Pontificia Universidad Católica de Chile, Santiago 1, Chile.
J Biol Chem. 2003 Sep 19;278(38):36777-85. doi: 10.1074/jbc.M305177200. Epub 2003 Jun 20.
To elucidate the role of extracellular histidines in the modulation of the rat P2X4 receptor by trace metals, we generated single, double, and triple histidine mutants for residues 140, 241, and 286, replacing them with alanines. cDNAs for the wild-type and receptor mutants were expressed in Xenopus laevis oocytes and in human embryonic kidney 293 cells and examined by the two electrode and patch clamp techniques, respectively. Whereas copper inhibited concentration-dependently the ATP-gated currents in the wild-type and in the single or double H241A and H286A receptor mutants, all receptors containing H140A were insensitive to copper in both cell systems. The characteristic bell-shaped concentration-response curve of zinc observed in the wild-type receptor became sigmoid in both oocytes and human embryonic kidney cells expressing the H140A mutant; in these mutants, the zinc potentiation was 2.5-4-fold larger than in the wild-type. Results with the H140T and H140R mutants further support the importance of a histidine residue at this position. We conclude that His-140 is critical for the action of copper, indicating that this histidine residue, but not His-241 or His-286, forms part of the inhibitory allosteric metal-binding site of the P2X4 receptor, which is distinct from the putative zinc facilitator binding site.
为阐明细胞外组氨酸在痕量金属对大鼠P2X4受体调节中的作用，我们构建了140、241和286位残基的单、双、三组氨酸突变体，将它们替换为丙氨酸。野生型和受体突变体的cDNA分别在非洲爪蟾卵母细胞和人胚肾293细胞中表达，并分别通过双电极和膜片钳技术进行检测。野生型以及单或双H241A和H286A受体突变体中，铜浓度依赖性地抑制ATP门控电流，而在两个细胞系统中，所有含H140A的受体对铜均不敏感。野生型受体中观察到的锌特征性钟形浓度-反应曲线，在表达H140A突变体的卵母细胞和人胚肾细胞中均变为S形；在这些突变体中，锌增强作用比野生型大2.5至4倍。H140T和Hl40R突变体的结果进一步支持了该位置组氨酸残基的重要性。我们得出结论，His-140对铜的作用至关重要，表明该组氨酸残基而非His-241或His-286构成了P2X4受体抑制性变构金属结合位点的一部分，该位点与假定的锌促进剂结合位点不同。
