Stoichiometric requirements for trapping and gating of Ca2+ release-activated Ca2+ (CRAC) channels by stromal interaction molecule 1 (STIM1)
Stoichiometric requirements for trapping and gating of Ca2+ release-activated Ca2+ (CRAC) channels by stromal interaction molecule 1 (STIM1)
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DOI：
10.1073/pnas.1101664108
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发表时间：
2011-08-09
影响因子：
11.1
通讯作者：
Lewis, Richard S.
中科院分区：
文献类型：
--
作者：
Hoover, Paul J.;Lewis, Richard S.
内质网(ER)、钙传感器、基质相互作用分子1(STIM1)和钙释放激活的钙通道蛋白Orai1之间的物理相互作用决定了细胞内钙离子的进入。最近的研究支持一种扩散陷阱机制，在这种机制中，内质网钙离子耗竭导致STIM1聚集在ER-质膜(PM)连接处，在那里它与Orai1结合，捕获并激活覆盖的PM中的移动CRAC通道为了确定CRAC通道捕获和激活的化学计量要求，我们在HEK细胞中以不同的比例表达了mCherry-STIM1和Orai1-GFP，并量化了存储耗尽后ER-PM连接处的CRAC电流(I-CRAC)激活和STIM1：ORAI1比率。通过竞争有限数量的STIM1，高水平的Orai1将连接STIM1：ORAI1的比率降低到0.3-0.6的下限，表明一到两个STIM1的结合足以使四聚体CRAC通道固定在ER-PM连接处。在表达恒定数量的STIM1的细胞中，CRAC电流是Orai1表达的高度非线性钟形函数，通道捕获的最小化学计量比未能引起显著的激活。峰电流出现的比率类似于2 STIM1：ORAI1，这表明最大的CRAC通道活动需要每个通道结合8个STIM1。Orai1的进一步增加导致通道活性和快速钙依赖失活平行下降。数据可以用一个模型很好地描述，在这个模型中，STIM1以负协同作用与Orai1结合，而由于STIM1稳定开放状态，通道以正协同作用开放。
Store-operated Ca2+ entry depends critically on physical interactions of the endoplasmic reticulum (ER) Ca2+ sensor stromal interaction molecule 1 (STIM1) and the Ca2+ release-activated Ca2+ (CRAC) channel protein Orai1. Recent studies support a diffusion-trap mechanism in which ER Ca2+ depletion causes STIM1 to accumulate at ER-plasma membrane (PM) junctions, where it binds to Orai1, trapping and activating mobile CRAC channels in the overlying PM. To determine the stoichiometric requirements for CRAC channel trapping and activation, we expressed mCherry-STIM1 and Orai1-GFP at varying ratios in HEK cells and quantified CRAC current (I-CRAC) activation and the STIM1:Orai1 ratio at ER-PM junctions after store depletion. By competing for a limited amount of STIM1, high levels of Orai1 reduced the junctional STIM1:Orai1 ratio to a lower limit of 0.3-0.6, indicating that binding of one to two STIM1s is sufficient to immobilize the tetrameric CRAC channel at ER-PM junctions. In cells expressing a constant amount of STIM1, CRAC current was a highly nonlinear bell-shaped function of Orai1 expression and the minimum stoichiometry for channel trapping failed to evoke significant activation. Peak current occurred at a ratio of similar to 2 STIM1:Orai1, suggesting that maximal CRAC channel activity requires binding of eight STIM1s to each channel. Further increases in Orai1 caused channel activity and fast Ca2+-dependent inactivation to decline in parallel. The data are well described by a model in which STIM1 binds to Orai1 with negative cooperativity and channels open with positive cooperativity as a result of stabilization of the open state by STIM1.