A Low Affinity GCaMP3 Variant (GCaMPer) for Imaging the Endoplasmic Reticulum Calcium Store.
A Low Affinity GCaMP3 Variant (GCaMPer) for Imaging the Endoplasmic Reticulum Calcium Store.
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DOI：
10.1371/journal.pone.0139273
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发表时间：
2015
期刊：
影响因子：
3.7
通讯作者：
Harvey BK
中科院分区：
文献类型：
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作者：
Henderson MJ;Baldwin HA;Werley CA;Boccardo S;Whitaker LR;Yan X;Holt GT;Schreiter ER;Looger LL;Cohen AE;Kim DS;Harvey BK
内质网钙稳态对细胞功能至关重要，在包括神经退行性疾病和心血管疾病在内的多种疾病中被破坏。由于内质网内有高浓度的钙，研究这种亚细胞区室需要针对这些条件进行优化的工具。为了开发用于该细胞器的单荧光基团基因编码钙指示剂，我们将GCaMP3的低亲和力变体定位于ER管腔（GCaMPer(10.19)）。构建了一组病毒载体，在人神经母细胞瘤细胞、大鼠原代皮层神经元和人诱导多能干细胞来源的心肌细胞中表达GCaMPer。我们观察了GCaMPer（10.19）荧光在内质网钙储存的药理学操作下的动态变化。此外，在心肌细胞自发跳动期间，观察到内质网周期性的钙外排。GCaMPer（10.19）在活细胞内质网钙成像和提供生理和病理状态下腔内钙动力学方面具有实用价值。
Endoplasmic reticulum calcium homeostasis is critical for cellular functions and is disrupted in diverse pathologies including neurodegeneration and cardiovascular disease. Owing to the high concentration of calcium within the ER, studying this subcellular compartment requires tools that are optimized for these conditions. To develop a single-fluorophore genetically encoded calcium indicator for this organelle, we targeted a low affinity variant of GCaMP3 to the ER lumen (GCaMPer (10.19)). A set of viral vectors was constructed to express GCaMPer in human neuroblastoma cells, rat primary cortical neurons, and human induced pluripotent stem cell-derived cardiomyocytes. We observed dynamic changes in GCaMPer (10.19) fluorescence in response to pharmacologic manipulations of the ER calcium store. Additionally, periodic calcium efflux from the ER was observed during spontaneous beating of cardiomyocytes. GCaMPer (10.19) has utility in imaging ER calcium in living cells and providing insight into luminal calcium dynamics under physiologic and pathologic states.