Dissecting the facilitator and inhibitor allosteric metal sites of the P2X4 receptor channel -: Critical roles of Cys132 for zinc potentiation and Asp138 for copper inhibition
Dissecting the facilitator and inhibitor allosteric metal sites of the P2X4 receptor channel -: Critical roles of Cys132 for zinc potentiation and Asp138 for copper inhibition
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DOI：
10.1074/jbc.m706925200
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发表时间：
2007-12-21
影响因子：
4.8
通讯作者：
Huidobro-Toro, J. Pablo
中科院分区：
文献类型：
--
作者：
Coddou, Claudio;Acuna-Castillo, Claudio;Huidobro-Toro, J. Pablo
锌和铜是中枢神经系统中配体门控离子通道的非典型调节剂。我们试图确定参与微量金属相互作用的大鼠P2X(4)受体的氨基酸，特别是在His(140)的直线性附近，这个残基以前被认为是铜诱导的抑制ATP诱发电流的关键。对于丙氨酸，定点突变取代了位于Thr(123)和Thr(146)之间胞外结构域区的明显氨基酸。在非洲爪哇卵母细胞中表达野生型和受体突变体的cDNA，并用双电极技术进行检测。Cys(132)，而不是Cys(126)，对锌诱导的受体活性的增强是至关重要的，但对铜诱导的抑制则不是。锌以浓度依赖的方式抑制C132a突变体的ATP门控电流。同样，Asp(138)，而不是Asp(131)对铜和锌的抑制是关键的；此外，突变体D138A对锌的增强反应是野生型受体的20倍。天冬氨酸(129)、天冬氨酸(Asp131)和苏氨酸(133)在金属调控中起次要作用。我们的结论是，P2X4受体的这个区域有一个口袋，可以与两个不同的促进和抑制金属变构位点进行微量金属配位。此外，Cys132似乎并不完全作为结构受体通道折叠基序参与，而是作为锌调节的配体，突出了痕量金属在神经元兴奋性中的作用。
Zinc and copper are atypical modulators of ligand-gated ionic channels in the central nervous system. We sought to identify the amino acids of the rat P2X(4) receptor involved in trace metal interaction, specifically in the immediate linear vicinity of His(140), a residue previously identified as being critical for copper-induced inhibition of the ATP-evoked currents. Site-directed mutagenesis replaced conspicuous amino acids located within the extracellular domain region between Thr(123) and Thr(146) for alanines. cDNAs for the wild-type and the receptor mutants were expressed in Xenopus laevis oocytes and examined by the two-electrode technique. Cys(132), but not Cys(126), proved crucial for zinc-induced potentiation of the receptor activity, but not for copper-induced inhibition. Zinc inhibited in a concentration-dependent manner the ATP-gated currents of the C132A mutant. Likewise, Asp(138), but not Asp(131) was critical for copper and zinc inhibition; moreover, mutant D138A was 20-fold more reactive to zinc potentiation than wild-type receptors. Asp(129), Asp131, and Thr(133) had minor roles in metal modulation. We conclude that this region of the P2X4 receptor has a pocket for trace metal coordination with two distinct and separate facilitator and inhibitor metal allosteric sites. In addition, Cys132 does not seem to participate exclusively as a structural receptor channel folding motif but plays a role as a ligand for zinc modulation highlighting the role of trace metals in neuronal excitability.