Differential dependence of store-operated and excitation-coupled Ca2+ entry in skeletal muscle on STIM1 and Orai1
Differential dependence of store-operated and excitation-coupled Ca2+ entry in skeletal muscle on STIM1 and Orai1
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DOI：
10.1113/jphysiol.2008.160481
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发表时间：
2008-10-15
影响因子：
5.5
通讯作者：
Dirksen, Robert T.
中科院分区：
文献类型：
--
作者：
Lyfenko, Alla D.;Dirksen, Robert T.
在不可兴奋的细胞中，激动剂诱导的细胞内钙库枯竭通过称为库操作的钙内流(SOCE)的过程来触发钙内流。在T淋巴细胞中，基质相互作用分子1(STIM1)作为库内钙感受器，Orai1作为库耗尽后由STIM1激活的钙离子通透性SOCE通道。骨骼肌存在两条功能不同的钙离子进入途径，一条由钙储备库耗竭(SOCE)激活，另一条由不需要储存库耗竭的持续/重复去极化激活(兴奋性耦合钙离子进入，ECCE)。然而，STIM1和Orai1在协调骨骼肌SOCE和ECCE活性中的作用以及这两条钙进入途径是代表不同的分子实体还是同一通道复合体的两种不同激活机制尚不清楚。在这里，我们使用siRNA介导的STIM1基因敲除、显性负性Orai1和渗透缺陷Orai1来解决这些问题，以确定STIM1和Orai1在骨骼肌管中存储操作和兴奋耦合钙离子进入中的作用。SOCE和ECCE活性通过细胞内Ca~(2+)测定和Mn2+猝灭试验进行定量。我们发现，STIM1 siRNA使STIM1蛋白减少了90%以上，并使SOCE活性丧失，而siRNA抗性hSTIM1的表达则完全恢复了SOCE活性。SOCE也被显性负性Orai1(E106Q)所取消，并被渗透缺陷Orai1(E190Q)的表达显著减少。相反，ECCE不受STIM1基因敲除、E106Q表达或E190Q表达的影响。这些结果首次证明，骨骼肌中的SOCE既需要STIM1也需要Orai1，并且SOCE和ECCE代表两个不同的分子实体。
In non-excitable cells, agonist-induced depletion of intracellular Ca2+ stores triggers Ca2+ influx via a process termed store-operated Ca2+ entry (SOCE). In T-lymphocytes, stromal interaction molecule 1 (STIM1) acts as the intra-store Ca2+ sensor and Orai1 functions as the Ca2+-permeable SOCE channel activated by STIM1 following store depletion. Two functionally distinct Ca2+ entry pathways exist in skeletal muscle; one activated by store depletion (SOCE) and a second by sustained/repetitive depolarization that does not require store depletion (excitation-coupled Ca2+ entry, ECCE). However, the role of STIM1 and Orai1 in coordinating SOCE and ECCE activity in skeletal muscle and whether these two Ca2+ entry pathways represent distinct molecular entities or two different activation mechanisms of the same channel complex is unknown. Here we address these issues using siRNA-mediated STIM1 knockdown, dominant-negative Orai1, and permeation-defective Orai1 to determine the role of STIM1 and Orai1 in store-operated and excitation-coupled Ca2+ entry in skeletal myotubes. SOCE and ECCE activity were quantified from both intracellular Ca2+ measurements and Mn2+ quench assays. We found that STIM1 siRNA reduced STIM1 protein by more than 90% and abolished SOCE activity, while expression of siRNA-resistant hSTIM1 fully restored SOCE. SOCE was also abolished by dominant-negative Orai1 (E106Q) and markedly reduced by expression of a permeation-defective Orai1 (E190Q). In contrast, ECCE was unaffected by STIM1 knockdown, E106Q expression or E190Q expression. These results are the first to demonstrate that SOCE in skeletal muscle requires both STIM1 and Orai1 and that SOCE and ECCE represent two distinct molecular entities.