Molecular determinants of Na+/Ca2+ exchange (NCX1) inhibition by SEA0400
Molecular determinants of Na+/Ca2+ exchange (NCX1) inhibition by SEA0400
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DOI：
10.1074/jbc.m310491200
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发表时间：
2004-02-27
影响因子：
4.8
通讯作者：
Katsuragi, T
中科院分区：
文献类型：
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作者：
Iwamoto, T;Kita, S;Katsuragi, T
SEA0400是一种有效的选择性Na+/Ca2+交换（NCX）抑制剂。我们在ncx转染的成纤维细胞中评估了SEA0400对Na-i（+）依赖性Ca-45（2+）摄取和全细胞Na+/Ca2（+）交换电流的抑制作用。与NCX2、NCX3和NCKX2相比，SEA0400优先抑制NCX1对Ca-45（2+）的摄取。SEA0400也选择性地阻断NCX1转染物的向外交换电流。我们通过NCX1/NCX3嵌合分析在NCX1分子中寻找可能形成SEA0400受体的区域。结果表明，NCX1和NCX3之间的药物反应差异主要是由第一个细胞内环和第5个跨膜段引起的。进一步的定点突变表明，ph -213的多重突变显著降低了对SEA0400的敏感性，而不影响对KB-R7943的敏感性。我们还发现Gly-833-to-Cys突变（在α -2重复序列内）大大降低了SEA0400的抑制作用，但出乎意料的是，具有NCKX2 α -2重复序列的NCX1嵌合体具有正常的药物敏感性。此外，具有突变交换剂抑制肽区域的交换剂，表现为无法检测到或加速Na+依赖性失活，分别显着降低对SEA0400的敏感性或超敏性。为了验证NCX抑制剂的有效性，我们在猪肾小管细胞缺氧损伤模型中检测了SEA0400的肾保护作用。SEA0400在表达野生型NCX1的管状细胞中保护缺氧/再氧诱导的细胞损伤，而在表达SEA0400不敏感突变体的细胞中没有保护作用。这些结果表明，ph -213、Gly-833和消除Na+依赖性失活的残基是SEA0400抑制NCX的关键决定因素，它们的突变体对于验证SEA0400抑制NCX的药理意义非常有用。
SEA0400 is a potent and selective Na+/Ca2+ exchanger (NCX) inhibitor. We evaluated the inhibitory effects of SEA0400 on Na-i(+)-dependent Ca-45(2+) uptake and whole-cell Na+/Ca2(+) exchange currents in NCX-transfected fibroblasts. SEA0400 preferentially inhibited Ca-45(2+) uptake by NCX1 compared with inhibitions by NCX2, NCX3, and NCKX2. SEA0400 also selectively blocked outward exchange currents from NCX1 transfectants. We searched for regions that may form the SEA0400 receptor in the NCX1 molecule by NCX1/NCX3 chimeric analysis. The results suggest that the first intracellular loop and the fifth transmembrane segment are mostly responsible for the differential drug responses between NCX1 and NCX3. Further site-directed mutagenesis revealed that multiple mutations at Phe-213 markedly reduced sensitivity to SEA0400 without affecting that to KB-R7943. We also found that Gly-833-to-Cys mutation (within the alpha-2 repeat) greatly reduced the inhibition by SEA0400, but unexpectedly the NCX1 chimera with an alpha-2 repeat from NCKX2 possessed normal drug sensitivity. In addition, exchangers with mutated exchanger inhibitory peptide regions, which display either undetectable or accelerated Na+-dependent inactivation, had a markedly reduced sensitivity or hypersensitivity to SEA0400, respectively. To verify the efficacy of the NCX inhibitor, we examined the renoprotective effect of SEA0400 in a hypoxic injury model using porcine renal tubular cells. SEA0400 protected against hypoxia/reoxygenation-induced cell damage in tubular cells expressing wild-type NCX1 but not in cells expressing SEA0400-insensitive mutants. These results suggest that Phe-213, Gly-833, and residues that eliminate Na+-dependent inactivation are critical determinants for the inhibition by SEA0400, and their mutants are very useful for checking the pharmacological importance of NCX inhibition by SEA0400.