Visualization of inositol 1,4,5-trisphosphate receptor type III with green fluorescent protein in living cells
Visualization of inositol 1,4,5-trisphosphate receptor type III with green fluorescent protein in living cells
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DOI：
10.1054/ceca.2001.0262
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发表时间：
2002-02-01
期刊：
影响因子：
4
通讯作者：
Tojyo, Y
中科院分区：
文献类型：
--
作者：
Morita, T;Tanimura, A;Tojyo, Y
肌醇 1,4,5-三磷酸受体 (IP3R) 是一种细胞内 Ca2+ 通道，可将 Ca2+ 释放到细胞质中，其亚细胞分布被认为对 Ca2+ 信号传导具有显着影响。我们构建了包含与 GFP 连接的全长大鼠 3 型 IP3R (GFP-IP(3)R3) 的质粒载体，用于在哺乳动物细胞中表达。 Western blot分析显示表达的融合蛋白同时含有GFP和全长3型IP3R。荧光共聚焦显微镜显示，即使在用皂苷进行细胞透化后，GFP-IP(3)R3 的荧光也分布到网状网络结构中。我们进一步使用细胞内膜的重要荧光标记 DiOC(6) 可视化细胞内膜，并提供证据表明 GFP-IP(3)R3 的分布与内质网的分布重叠。我们的结果表明 GFP-IP(3)R3 可用于可视化活细胞中的 IP3R，并为后续的突变和功能研究铺平道路。 (C) 2002 Elsevier Science Ltd. 保留所有权利。
Inositol 1,4,5-trisphosphate receptor (IP3R) is an intracellular Ca2+ channel that releases Ca2+ into the cytosol and its subcellular distribution is believed to have significant effects on Ca2+ signalling. We constructed a plasmid vector containing full-length rat type 3 IP3R linked to GFP (GFP-IP(3)R3) for expression in mammalian cells. Western blot analyses revealed that the expressed fusion protein contained both GFP and full-length type 3 IP3R. Fluorescence confocal microscopy showed that the fluorescence of GFP-IP(3)R3 was distributed to reticular network structures, even after cell permeabilization with saponin. We further visualized intracellular membranes with DiOC(6), a vital fluorescent marker for intracellular membranes, and provide evidence that the distribution of GFP-IP(3)R3 overlaps with the distribution of the endoplasmic reticulum. Our results indicate that GFP-IP(3)R3 can be used to visualize IP3R in living cells, and pave the way for subsequent mutational and functional studies. (C) 2002 Elsevier Science Ltd. All rights reserved.