Bcl-2-mediated alterations in endoplasmic reticulum Ca2+ analyzed with an improved genetically encoded fluorescent sensor
Bcl-2-mediated alterations in endoplasmic reticulum Ca2+ analyzed with an improved genetically encoded fluorescent sensor
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DOI：
10.1073/pnas.0408030101
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发表时间：
2004-12-14
影响因子：
11.1
通讯作者：
Tsien, RY
中科院分区：
文献类型：
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作者：
Palmer, AE;Jin, C;Tsien, RY
内质网（ER）作为Ca2+的细胞仓库，从内质网释放的Ca2+在许多关键的信号反应中起作用，包括胞吐、收缩、代谢、转录调节、受精和凋亡。鉴于内质网所起的核心作用，我们对这些信号传导过程的理解可以通过在单个细胞中直接成像[Ca2+]（ER）的能力大大增强。我们通过重新设计钙调蛋白和钙调蛋白结合肽的结合界面，创造了一种基因编码的Ca2+指示剂。该传感器改善了反应动力学和理想的K-d成像内质网Ca2+，不再受到大量过量的天然钙调素的干扰。重要的是，它提供了一个显著的改进比所有以前的方法监测[Ca2+](ER)，并已用于直接表明，在MCF-7乳腺癌细胞中，抗凋亡蛋白B细胞淋巴瘤2 (Bcl-2) (i)降低[Ca2+]（ER）通过增加Ca2+泄漏在静息条件下和（ii）改变Ca2+振荡由ATP诱导。绿茶化合物表没食子儿茶素没食子酸酯对Bcl-2的急性抑制导致[Ca2+]（ER）的增加，这是由于抑制了Bcl-2介导的Ca2+泄漏。
The endoplasmic reticulum (ER) serves as a cellular storehouse for Ca2+, and Ca2+ released from the ER plays a role in a host of critical signaling reactions, including exocytosis, contraction, metabolism, regulation of transcription, fertilization, and apoptosis. Given the central role played by the ER, our understanding of these signaling processes could be greatly enhanced by the ability to image [Ca2+](ER) directly in individual cells. We created a genetically encoded Ca2+ indicator by redesigning the binding interface of calmodulin and a calmodulin-binding peptide. The sensor has improved reaction kinetics and a K-d ideal for imaging Ca2+ in the ER and is no longer perturbed by large excesses of native calmodulin. Importantly, it provides a significant improvement over all previous methods for monitoring [Ca2+](ER) and has been used to directly show that, in MCF-7 breast cancer cells, the antiapoptotic protein B cell lymphoma 2 (Bcl-2) (i) lowers [Ca2+](ER) by increasing Ca2+ leakage under resting conditions and (ii) alters Ca2+ oscillations induced by ATP, and that acute inhibition of Bcl-2 by the green tea compound epigallocatechin gallate results in an increase in [Ca2+](ER) due to inhibition of Bcl-2-mediated Ca2+ leakage.