Calcium store refilling and STIM activation in STIM- and Orai-deficient cell lines.
Calcium store refilling and STIM activation in STIM- and Orai-deficient cell lines.
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STIM 和 Orai 缺陷细胞系中的钙储存补充和 STIM 激活。
DOI：
10.1007/s00424-018-2165-5
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发表时间：
2018-10
期刊：
影响因子：
--
通讯作者：
Wang Y
中科院分区：
文献类型：
--
作者：
Zheng S;Zhou L;Ma G;Zhang T;Liu J;Li J;Nguyen NT;Zhang X;Li W;Nwokonko R;Zhou Y;Zhao F;Liu J;Huang Y;Gill DL;Wang Y
通过STIM蛋白和奥赖通道的联合作用介导的钙库操纵的钙内流（SOCE）在不同的细胞类型中普遍存在。多个STIM和奥赖基因的存在使得难以分配每个STIM和奥赖同源物在介导Ca 2+信号中的特定作用。使用CRISPR/Cas9基因编辑工具，我们产生了STIM或所有三种奥赖同源物缺失的细胞，并直接监测了储存的Ca 2+和Ca 2+信号。我们发现，未受刺激的SOCE敲除细胞仍然保留野生型（wt）细胞的50~70%的ER Ca ~（2+）储备。短暂暴露后的存储排空条件下，急性再填充的ER Ca 2+商店被完全阻断在KO细胞。然而，在培养24小时后，储存物最终被重新填充。因此，SOCE对于ER Ca 2+的立即再填充是至关重要的，但对于长期ER Ca 2+稳态的维持是不利的。使用奥赖空背景三重Orai-KO细胞，我们检查了一系列截短的STIM 1变体的质膜易位性质。FRET分析表明，尽管STIM 1的PM束缚通过促进STIM 1在ER-PM连接中的寡聚化、迁移和积累来加速STIM 1的激活，但STIM 1的构象转换、寡聚化和聚集并不需要它。即使在ER-PM连接处没有明显的斑点形成，当在STIM KO细胞中表达时，STIM 11 -491和STIM 11 -666仍然可以拯救SOCE。因此，STIM分子的ER-PM捕获和聚集仅促进SOCE激活过程，但对于奥赖通道的激活不是必需的。本文的在线版本（10.1007/s 00424 -018-2165-5）包含补充材料，可供授权用户使用。
Mediated through the combined action of STIM proteins and Orai channels, store-operated Ca2+ entry (SOCE) functions ubiquitously among different cell types. The existence of multiple STIM and Orai genes has made it difficult to assign specific roles of each STIM and Orai homolog in mediating Ca2+ signals. Using CRISPR/Cas9 gene editing tools, we generated cells with both STIM or all three Orai homologs deleted and directly monitored store Ca2+ and Ca2+ signals. We found that unstimulated, SOCE null KO cells still retain 50~70% of ER Ca2+ stores of wildtype (wt) cells. After brief exposure to store-emptying conditions, acute refilling of ER Ca2+ stores was totally blocked in KO cells. However, after 24 h in culture, stores were eventually refilled. Thus, SOCE is critical for immediate refilling of ER Ca2+ but is dispensable for the maintenance of long-term ER Ca2+ homeostasis. Using the Orai null background triple Orai-KO cells, we examined the plasma membrane translocation properties of a series of truncated STIM1 variants. FRET analysis reveals that, even though PM tethering of STIM1 expedites the activation of STIM1 by facilitating its oligomerization, migration, and accumulation in ER-PM junctions, it is not required for the conformational switch, oligomerization, and clustering of STIM1. Even without overt puncta formation at ER-PM junctions, STIM11–491 and STIM11–666 could still rescue SOCE when expressed in STIM KO cells. Thus, ER-PM trapping and clustering of STIM molecules only facilitates the process of SOCE activation, but is not essential for the activation of Orai channels. The online version of this article (10.1007/s00424-018-2165-5) contains supplementary material, which is available to authorized users.
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影响因子：
64.5
作者：
Park CY;Hoover PJ;Mullins FM;Bachhawat P;Covington ED;Raunser S;Walz T;Garcia KC;Dolmetsch RE;Lewis RS
通讯作者：
Lewis RS
影响因子：
1.5
作者：
Korzeniowski MK;Baird B;Holowka D
通讯作者：
Holowka D
DOI：
10.1016/j.bbrc.2015.02.110
发表时间：
2015-04-24
影响因子：
3.1
作者：
Hogan, Patrick G.;Rao, Anjana
通讯作者：
Rao, Anjana
影响因子：
4.8
作者：
He, LP;Hewavitharana, T;Gill, DL
通讯作者：
Gill, DL
影响因子：
64.5
作者：
Palty, Raz;Raveh, Adi;Reuveny, Eitan
通讯作者：
Reuveny, Eitan