Stim1 and Orai1 mediate CRAC currents and store-operated calcium entry important for endothelial cell proliferation.
Stim1 and Orai1 mediate CRAC currents and store-operated calcium entry important for endothelial cell proliferation.
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DOI：
10.1161/01.res.0000338496.95579.56
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发表时间：
2008-11-21
影响因子：
20.1
通讯作者：
Trebak M
中科院分区：
文献类型：
--
作者：
Abdullaev IF;Bisaillon JM;Potier M;Gonzalez JC;Motiani RK;Trebak M
最近在钙库操纵的钙离子进入（SOCE）途径中的突破已经将Stim 1鉴定为内质网（ER）Ca 2+传感器，并且将Orai 1鉴定为造血细胞中表达的高度Ca 2+选择性CRAC通道的孔形成亚基。然而，以前的研究表明，内皮细胞（EC）SOCE是由非选择性经典瞬时受体电位（TRPC）通道家族，TRPC 1或TRPC 4介导的。在这里，我们表明，被动存储耗尽毒胡萝卜素或受体激活凝血酶或血管内皮生长因子（VEGF）激活相同的途径，在初级EC与经典的SOCE药理学功能。EC具有典型的Ca 2+释放激活的Ca 2+电流（ICRAC），尽管幅度很小。使用一个机动，放大电流在二价的自由浴解决方案，我们表明，EC CRAC具有类似的特性，从RBL细胞记录，即一个类似的时间过程的激活，敏感性2-氨基乙氧基二苯基硼酸盐（2-APB）和低浓度的镧系元素，和大Na+电流显示典型的去电位。Stim 1或Orai 1的RNA沉默基本上消除了EC中的SOCE和ICRAC，这分别由Stim 1或Orai 1的异位表达所拯救。令人惊讶的是，TRPC 1或TRPC 4蛋白的敲低对SOCE和ICRAC没有影响。Stim 1在EC中的异位表达使它们的ICRAC增加到与RBL细胞中的ICRAC相当的大小。敲低Stim 1、Stim 2或Orai 1蛋白均能抑制EC增殖，并使细胞周期阻滞于S期和G2/M期，但Orai 1蛋白的敲低效果优于Stim蛋白。这些结果首次确立了Stim 1/Orai 1在内皮SOCE通路中的需求。
Recent breakthroughs in the store-operated Calcium (Ca2+) entry (SOCE) pathway have identified Stim1 as the endoplasmic reticulum (ER) Ca2+ sensor and Orai1 as the pore forming subunit of the highly Ca2+ selective CRAC channel expressed in hematopoietic cells. Previous studies however, have suggested that endothelial cell (EC) SOCE is mediated by the non-selective Canonical Transient Receptor Potential (TRPC) channel family, TRPC1 or TRPC4. Here we show that passive store depletion by thapsigargin or receptor activation by either thrombin or the vascular endothelial growth factor (VEGF) activates the same pathway in primary EC with classical SOCE pharmacological features. EC possess the archetypical Ca2+ release-activated Ca2+ current (ICRAC), albeit of a very small amplitude. Using a maneuver that amplifies currents in divalent free bath solutions, we show that EC CRAC has similar characteristics to that recorded from RBL cells, namely a similar time course of activation, sensitivity to 2-Aminoethoxydiphenyl borate (2-APB) and low concentrations of lanthanides, and large Na+ currents displaying the typical depotentiation. RNA silencing of either Stim1 or Orai1 essentially abolished SOCE and ICRAC in EC which were rescued by ectopic expression of either Stim1 or Orai1, respectively. Surprisingly, knockdown of either TRPC1 or TRPC4 proteins had no effect on SOCE and ICRAC. Ectopic expression of Stim1 in EC increased their ICRAC to a size comparable to that in RBL cells. Knockdown of Stim1, Stim2 or Orai1 inhibited EC proliferation and caused cell cycle arrest at S and G2/M phase, although Orai1 knockdown was more efficient than that of Stim proteins. These results are first to establish the requirement of Stim1/Orai1 in the endothelial SOCE pathway.