Mutations of the Ca2+-sensing Stromal Interaction Molecule STIM1 Regulate Ca2+ Influx by Altered Oligomerization of STIM1 and by Destabilization of the Ca2+ Channel Orai1
Mutations of the Ca2+-sensing Stromal Interaction Molecule STIM1 Regulate Ca2+ Influx by Altered Oligomerization of STIM1 and by Destabilization of the Ca2+ Channel Orai1
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DOI：
10.1074/jbc.m112.417246
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发表时间：
2013-01-18
影响因子：
4.8
通讯作者：
Niemeyer, Barbara A.
中科院分区：
文献类型：
--
作者：
Kilch, Tatiana;Alansary, Dalia;Niemeyer, Barbara A.
内质网Ca 2+浓度的下降触发其Ca 2+传感器蛋白基质相互作用分子1（STIM 1）寡聚化并在内质网-质膜连接处积聚，在那里它激活Orai 1通道，提供钙库操作的Ca 2+进入。为了阐明STIM 1的N-糖基化位点的功能意义，我们创建了天冬酰胺-131和天冬酰胺-171的不同突变。STIM 1 NN/DQ导致功能的强增益。膜片钳，全内反射荧光（TIRF）显微镜，和荧光恢复后光漂白（FRAP）的分析表明，STIM 1 DQ突变体的表达增加了活性Orai 1通道的数量和速率的STIM 1易位到内质网质膜连接的电流潜伏期减少。令人惊讶的是，STIM 1 DQ的共表达减少了Orai 1蛋白，改变了STIM 1：Orai 1的化学计量。我们描述了一种新的数学工具来描绘改变STIM 1或Orai 1扩散参数的化学计量变化的影响。该突变体揭示了一种新的机制，即“超活性”STIM 1 DQ导致改变的寡聚化速率常数和Orai 1的降解，并改变激活剂（STIM 1）与效应物（Orai 1）的化学计量比，从而改变Ca 2+稳态。
A drop of endoplasmic reticulum Ca2+ concentration triggers its Ca2+ ssensor protein stromal interaction molecule 1 (STIM1) to oligomerize and accumulate within endoplasmic reticulum-plasma membrane junctions where it activates Orai1 channels, providing store-operated Ca2+ entry. To elucidate the functional significance of N-glycosylation sites of STIM1, we created different mutations of asparagine-131 and asparagine-171. STIM1 NN/DQ resulted in a strong gain of function. Patch clamp, Total Internal Reflection Fluorescent (TIRF) microscopy, and fluorescence recovery after photobleaching (FRAP) analyses revealed that expression of STIM1 DQ mutants increases the number of active Orai1 channels and the rate of STIM1 translocation to endoplasmic reticulum-plasma membrane junctions with a decrease in current latency. Surprisingly, co-expression of STIM1 DQ decreased Orai1 protein, altering the STIM1: Orai1 stoichiometry. We describe a novel mathematical tool to delineate the effects of altered STIM1 or Orai1 diffusion parameters from stoichiometrical changes. The mutant uncovers a novel mechanism whereby "superactive" STIM1 DQ leads to altered oligomerization rate constants and to degradation of Orai1 with a change in stoichiometry of activator (STIM1) to effector (Orai1) ratio leading to altered Ca2+ homeostasis.