Functional stoichiometry of the unitary calcium-release-activated calcium channel
Functional stoichiometry of the unitary calcium-release-activated calcium channel
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单一钙释放激活钙通道的功能化学计量
DOI：
10.1073/pnas.0806499105
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发表时间：
2008-09-09
影响因子：
11.1
通讯作者：
Chen, Liangyi
中科院分区：
文献类型：
--
作者：
Ji, Wei;Xu, Pingyong;Chen, Liangyi
两种蛋白质，内质网中的STIM 1和质膜中的Orai 1，是激活细胞表面的Ca 2+释放激活的Ca 2+（CRAC）通道所必需的。这些蛋白质如何相互作用以组装功能性CRAC通道仍然不确定。在这里，我们确定需要多少Orai 1和STIM 1分子才能形成功能性CRAC通道。我们设计了几个基因表达的荧光Orai1串联多聚体和荧光，组成型活性STIM1突变体。串联多聚体组装成CRAC通道，如整流内向电流和细胞质钙升高所示。CRAC通道在全内反射显微镜中可视化为荧光斑点。使用单分子成像技术，可以观察单个荧光团的光漂白，并计算漂白步骤作为每个CRAC通道复合物的化学计量的量度。我们的结论是，在一个活跃的CRAC通道的亚基化学计量是四个Orai 1分子和两个STIM 1分子。荧光共振能量转移实验也表明，四个Orai1亚基形成组装通道。从单个荧光团的荧光强度，我们可以估计我们的转染的HEK293细胞具有几乎400，000个CRAC通道，并且当细胞内Ca2+储存耗尽时，通道聚集在含有100，300个通道的聚集体中，放大了局部Ca2+进入。
Two proteins, STIM1 in the endoplasmic reticulum and Orai1 in the plasma membrane, are required for the activation of Ca2+ release-activated Ca2+ (CRAC) channels at the cell surface. How these proteins interact to assemble functional CRAC channels has remained uncertain. Here, we determine how many Orai1 and STIM1 molecules are required to form a functional CRAC channel. We engineered several genetically expressed fluorescent Orai1 tandem multimers and a fluorescent, constitutively active STIM1 mutant. The tandem multimers assembled into CRAC channels, as seen by rectifying inward currents and by cytoplasmic calcium elevations. CRAC channels were visualized as fluorescent puncta in total internal reflection microscopy. With single-molecule imaging techniques, it was possible to observe photo-bleaching of individual fluorophores and to count the steps of bleaching as a measure of the stoichiometry of each CRAC channel complex. We conclude that the subunit stoichiometry in an active CRAC channel is four Orai1 molecules and two STIM1 molecules. Fluorescence resonance energy transfer experiments also showed that four Orai1 subunits form the assembled channel. From the fluorescence intensity of single fluorophores, we could estimate that our transfected HEK293 cells had almost 400,000 CRAC channels and that, when intracellular Ca2+ stores were depleted, the channels clustered in aggregates containing ≈1,300 channels, amplifying the local Ca2+ entry.