Knock-down of the type 3 ryanodine receptor impairs sustained Ca2+ signaling via the T cell receptor/CD3 complex
Knock-down of the type 3 ryanodine receptor impairs sustained Ca2+ signaling via the T cell receptor/CD3 complex
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DOI：
10.1074/jbc.m209061200
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发表时间：
2002-12-27
影响因子：
4.8
通讯作者：
Guse, AH
中科院分区：
文献类型：
--
作者：
Schwarzmann, N;Kunerth, S;Guse, AH
在 Jurkat T 细胞中，3 型兰尼碱受体 (RyR) 通过稳定整合表达 3 型兰尼碱受体反义 RNA 的质粒而被敲低。通过基因组 DNA 的 PCR、逆转录酶 PCR 的反义 RNA 表达以及 Western blot 的有效降低 3 型兰尼碱受体蛋白的表达，证明了反义质粒在各个克隆中的稳定整合。选定的克隆成功用于分析 T 细胞受体/CD3 复合物介导的 Ca2+ 信号传导。 3 型 RyR 表达的减少导致 (i) 持续相中的 Ca2+ 信号传导显着降低，以及 (ii) 透化细胞中对环 ADP-核糖的反应显着受损，但对 D-肌醇 1,4,5-三磷酸没有反应。首次通过共聚焦 Ca2+ 成像直接可视化 3 型 RyR 在持续 Ca2+ 信号传导中的作用，将其视为对亚细胞 Ca2+ 信号的数量和强度的重要贡献。这些数据表明 3 型兰尼碱受体对于 T 细胞的持续 Ca2+ 反应至关重要。
In Jurkat T cells, the type 3 ryanodine receptor (RyR) was knocked-down by stable integration of plasmid expressing type 3 ryanodine receptor antisense RNA. Stable integration of the antisense plasmid in individual clones was demonstrated by PCR of genomic DNA, expression of antisense RNA by reverse transcriptase PCR, and efficiently reduced expression of type 3 ryanodine receptor protein by Western blot. Selected clones were successfully used to analyze T cell receptor/CD3 complex-mediated Ca2+ signaling. Reduced expression of the type 3 RyR resulted in (i) significantly decreased Ca2+ signaling in the sustained phase and (ii) in permeabilized cells in a significantly impaired response toward cyclic ADP-ribose but not to D-myo-inositol 1,4,5-trisphosphate. For the first time, the role of the type 3 RyR in sustained Ca2+ signaling was directly visualized by confocal Ca2+ imaging as a significant contribution to the number and the magnitude of subcellular Ca2+ signals. These data suggest that the type 3 ryanodine receptor is essential in the sustained Ca2+ response in T cells.