Abdullaev Iskandar F, Bisaillon Jonathan M, Potier Marie, Gonzalez Jose C, Motiani Rajender K, Trebak Mohamed
Cardiovascular Sciences, MC8, Albany Medical College, 47 New Scotland Avenue, MC-8, Albany, NY 12208, USA.
Circ Res. 2008 Nov 21;103(11):1289-99. doi: 10.1161/01.RES.0000338496.95579.56. Epub 2008 Oct 9.
Recent breakthroughs in the store-operated calcium (Ca(2+)) entry (SOCE) pathway have identified Stim1 as the endoplasmic reticulum Ca(2+) sensor and Orai1 as the pore forming subunit of the highly Ca(2+)-selective CRAC channel expressed in hematopoietic cells. Previous studies, however, have suggested that endothelial cell (EC) SOCE is mediated by the nonselective canonical transient receptor potential channel (TRPC) 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 activates the same pathway in primary ECs with classical SOCE pharmacological features. ECs possess the archetypical Ca(2+) release-activated Ca(2+) current (I(CRAC)), 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 rat basophilic leukemia cells, namely a similar time course of activation, sensitivity to 2-aminoethoxydiphenyl borate, and low concentrations of lanthanides, and large Na(+) currents displaying the typical depotentiation. RNA silencing of either Stim1 or Orai1 essentially abolished SOCE and I(CRAC) in ECs, 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 I(CRAC). Ectopic expression of Stim1 in ECs increased their I(CRAC) to a size comparable to that in rat basophilic leukemia 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 our knowledge to establish the requirement of Stim1/Orai1 in the endothelial SOCE pathway.
近年来，在储存式钙（Ca(2+)）内流（SOCE）途径方面取得的突破，已确定Stim1为内质网钙传感器，而Orai1为造血细胞中表达的高钙（Ca(2+)）选择性CRAC通道的孔形成亚基。然而，先前的研究表明，内皮细胞（EC）的SOCE是由非选择性的典型瞬时受体电位通道（TRPC）家族、TRPC1或TRPC4介导的。在此，我们表明，毒胡萝卜素引起的被动储存耗竭或凝血酶或血管内皮生长因子引起的受体激活，在具有经典SOCE药理学特征的原代EC中激活了相同的途径。EC具有典型的钙释放激活钙电流（I(CRAC)），尽管幅度非常小。通过一种在无二价离子的浴液中放大电流的操作，我们表明EC的CRAC与从大鼠嗜碱性白血病细胞记录到的具有相似特征，即相似的激活时间进程、对2-氨基乙氧基二苯硼酸盐的敏感性、对低浓度镧系元素的敏感性，以及显示典型去增强作用的大Na(+)电流。Stim1或Orai1的RNA沉默基本消除了EC中的SOCE和I(CRAC)，分别通过Stim1或Orai1的异位表达得以挽救。令人惊讶的是，TRPC1或TRPC4蛋白的敲低对SOCE和I(CRAC)没有影响。Stim1在EC中的异位表达将其I(CRAC)增加到与大鼠嗜碱性白血病细胞中相当的大小。Stim1、Stim2或Orai1的敲低抑制了EC增殖，并导致细胞周期停滞在S期和G2/M期，尽管Orai1敲低比Stim蛋白更有效。据我们所知，这些结果首次确立了Stim1/Orai1在内皮SOCE途径中的必要性。
