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

质下Ca^<2+>与细胞外Ca^<2+>动态平衡，影响这个有限空间内的许多信号分子、效应物和事件。我们通过将Ca^<2+>传感器（cameleon）定位到内皮质膜上，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。这些皮质下波在空间上不同于经典的细胞质Ca^<2+>波，起源于局部区域并在质膜下传播。与PH结构域PLCδ融合的表达GFP从质膜向胞质溶胶的移位伴随着这些皮质下波，U73122不仅减弱了GFP- phδ的移位，而且减弱了皮质下Ca^<2+>波的峰值振幅；这一发现表明，通过plc介导的PIP_2水解，局部IP_3的产生参与了这些波的开始。NO生成的变化以及PKCβ-GFP从细胞质溶胶向质膜的转运，而不是GFP-PLA_2向核周内膜的转运，与质下细胞Ca^<2+>的变化有关。因此，细胞外Ca^<2+>维持了质膜的基本PLC活性，参与了区隔化皮质下Ca^<2+>波的启动，并调节了驻留在质膜或转运到质膜的Ca^<2+>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.