Zemkova Hana, Yan Zonghe, Liang Zhaodong, Jelinkova Irena, Tomic Melanija, Stojilkovic Stanko S
Section on Cellular Signaling, NICHD, National Institutes of Health, Bethesda, Maryland 20892-4510, USA.
J Neurochem. 2007 Aug;102(4):1139-50. doi: 10.1111/j.1471-4159.2007.04616.x.
The localization of ATP binding site(s) at P2X receptors and the molecular rearrangements associated with opening and closing of channels are still not well understood. At P2X(4) receptor, substitution of the K67, F185, K190, F230, R278, D280, R295, and K313 ectodomain residues with alanine generated low or non-responsive mutants, whereas the F294A mutant was functional. The loss of receptor function was also observed in K67R, R295K, and K313R mutants, but not in F185W, K190R, F230W, R278K, and D280E mutants. To examine whether the loss of function reflects decreased sensitivity of mutants for ATP, we treated cells with ivermectin, an antiparasitic agent that enhances responsiveness of P2X(4)R. In the presence of ivermectin, all low or non-responsive mutants responded to ATP in a dose-dependent manner, with the EC(50) values for ATP of about 1, 2, 4, 20, 60, 125, 270, 420, 1000 and 2300 micromol/L at D280A, R278A, F185A, K190A, R295K, K313R, R295A, K313A, K67A and K67R mutants, respectively. These results indicate that lysines 67 and 313 and arginine 295 play a critical role in forming the proper three-dimensional structure of P2X(4)R for agonist binding and/or channel gating.
P2X受体上ATP结合位点的定位以及与通道开闭相关的分子重排仍未完全明确。在P2X(4)受体中，将胞外结构域的K67、F185、K190、F230、R278、D280、R295和K313残基替换为丙氨酸会产生低反应性或无反应性的突变体，而F294A突变体具有功能。在K67R、R295K和K313R突变体中也观察到受体功能丧失，但在F185W、K190R、F230W、R278K和D280E突变体中未观察到。为了研究功能丧失是否反映突变体对ATP的敏感性降低，我们用伊维菌素处理细胞，伊维菌素是一种抗寄生虫剂，可增强P2X(4)R的反应性。在伊维菌素存在的情况下，所有低反应性或无反应性的突变体均以剂量依赖性方式对ATP作出反应，在D280A、R278A、F185A、K190A、R295K、K313R、R295A、K313A、K67A和K67R突变体中，ATP的EC(50)值分别约为1、2、4、20、60、125、270、420、1000和2300 μmol/L。这些结果表明，赖氨酸67和313以及精氨酸295在形成P2X(4)R的合适三维结构以进行激动剂结合和/或通道门控方面起着关键作用。
