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

亚质膜上的钙离子与细胞外钙离子动态平衡，影响着这个受限空间内的众多信号分子、效应器和事件。我们通过将钙离子传感器Cameleon定位于内皮细胞质膜，证明了在细胞外[Ca^&lt；2+&gt；]急剧升高的情况下，在质膜下传播的一种新的钙波。这些皮质下波在空间上不同于经典的胞浆Ca^&lt；2+&gt；波，起源于局部区域，并在整个亚质膜中传播。表达的绿色荧光蛋白与PLCδ的PH结构域从质膜到胞浆的转位伴随着这些皮层下波，U73122不仅减弱了绿色荧光蛋白-PHδ的转位，而且还减弱了皮质下钙波的峰值幅度；这一发现表明，通过PLC介导的PIP2水解，局部IP_3的产生参与了这些波的启动。NO的产生和PKCβ-GFP从胞浆到质膜的移位，而不是GFP-PLA2到核周内膜的移位，与亚质网钙离子和钙离子的变化有关。因此，胞外Ca~(2+)&gt；维持质膜的基础PLC活性，参与启动分区的皮质下Ca~(2+)波，并调节位于质膜内或移位到质膜上的CA^&lt；2+&gt；依赖的信号分子。

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.