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Imaging and analysis of subplasmalemmal Ca^<2+> dynamics by FRET

通过 FRET 对质膜下 Ca^<2> 动力学进行成像和分析

基本信息

批准号：
15590724
负责人：
ISSHIKI Masashi
金额：
$ 2.24万
依托单位：
The University of Tokyo
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2003
资助国家：
日本
起止时间：
2003 至 2004
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-15590724/
关键词：
endothelium calcium signal cameleon FRET nitric oxide (NO)imaging PLC カベオラ

项目摘要

Subplasmalemmal Ca^<2+>, dynamically equilibrated with extracellular Ca^<2+>, affects numerous signaling molecules, effectors, and events within this restricted space. We demonstrated the presence of a novel Ca^<2+> wave propagating beneath the plasma membrane in response to acute elevation of extracellular[Ca^<2+>], by targeting a Ca^<2+> sensor, cameleon, to the endothelial plasmalemma. These subcortical waves, spatially distinct from classical cytosolic Ca^<2+> waves, originated in localized regions and propagated throughout the subplasmalemma. Translocation of an expressed GFP fused with a PH domain of PLCδ from the plasma membrane to the cytosol accompanied these subcortical waves, and U73122 attenuated not only the GFP-PHδ translocation, but also the peak amplitude of the subcortical Ca^<2+> waves ; this finding suggests the involvement of local IP_3 production through PLC-mediated PIP_2 hydrolysis in the initiation of these waves. Changes in NO production as well as PKCβ-GFP translocation from the cytosol to the plasma membrane, but not of GFP-PLA_2 to perinuclear endomembranes, were associated with the subplasmalernrnal Ca^<2+> changes. Thus, extracellular Ca^<2+> maintains the basal PLC activity of the plasma membrane, is involved in the initiation of compartmentalized subcortical Ca^<2+> waves, and regulates CA^<2+>-dependent signaling molecules residing in or translocated to the plasma membrane.

质膜下的Ca^2+与细胞外的Ca^2+动态平衡，影响着这一有限空间内的众多信号分子、效应物和事件。通过将Ca^2+传感器cameleon靶向内皮细胞质膜，我们证明了一种新的Ca^2+波在质膜下传播，以响应细胞外[Ca^2+]的急性升高。这些皮层下波在空间上不同于经典的胞质Ca^2+波，起源于局部区域，并在质膜下传播。与PLCδ的PH结构域融合的表达的GFP从质膜易位到胞质溶胶伴随着这些皮层下波，U 73122不仅减弱了GFP-PHδ易位，而且减弱了皮层下Ca^2+波的峰值幅度;这一发现表明通过PLC介导的PIP_2水解产生的局部IP_3参与了这些波的起始。NO产生的变化以及PKCβ-GFP从胞液向质膜的转位，而GFP-PLA_2向核周内膜的转位与质膜下Ca^<2+>的变化无关。因此，细胞外Ca^2+维持质膜的基础PLC活性，参与皮层下Ca^2+波的启动，并调节驻留在质膜中或转移到质膜的CA^2+依赖性信号分子。