Marsault R, Murgia M, Pozzan T, Rizzuto R
Department of Biomedical Sciences and CNR, University of Padova, Italy.
EMBO J. 1997 Apr 1;16(7):1575-81. doi: 10.1093/emboj/16.7.1575.
Theoretical models and indirect experimental observations predict that Ca2+ concentrations at the inner surface of the plasma membrane may reach, upon stimulation, values much higher than those of the bulk cytosol. In the past few years, we have shown that the Ca2+-sensitive photoprotein aequorin can be intracellularly targeted and utilized for specifically monitoring the [Ca2+] of various organelles. In this work, we extend this approach to the study of the cytoplasmic rim beneath the plasma membrane. We have constructed a new aequorin chimera by fusing the photoprotein with SNAP-25, a neuronal protein which is recruited to the plasma membrane after the post-translational addition of a lipid anchor. The SNAP-25-aequorin chimera, expressed in the rat aortic smooth muscle cell line A7r5, appears correctly sorted as revealed by immunocytochemistry. Using this probe, we demonstrate that the mean [Ca2+] of this cytoplasmic region ([Ca2+]pm) can reach values >10-fold higher than those of the bulk cytosol ([Ca2+]c) upon activation of Ca2+ influx through plasma membrane channels. In unstimulated cells, the mean [Ca2+]pm appears also to be higher than the bulk cytosol, presumably reflecting the existence of microdomains of high [Ca2+].
理论模型和间接实验观察预测，在受到刺激时，质膜内表面的Ca2+浓度可能会达到远高于胞浆溶胶整体浓度的值。在过去几年中，我们已经表明，对Ca2+敏感的光蛋白水母发光蛋白可以在细胞内靶向，并用于特异性监测各种细胞器的[Ca2+]。在这项工作中，我们将这种方法扩展到质膜下方细胞质边缘的研究。我们通过将光蛋白与SNAP-25融合构建了一种新的水母发光蛋白嵌合体，SNAP-25是一种神经元蛋白，在翻译后添加脂质锚后被募集到质膜上。在大鼠主动脉平滑肌细胞系A7r5中表达的SNAP-25-水母发光蛋白嵌合体，经免疫细胞化学显示分类正确。使用该探针，我们证明，通过质膜通道激活Ca2+内流后，该细胞质区域的平均Ca2+可以达到比胞浆溶胶整体([Ca2+]c)高10倍以上的值。在未受刺激的细胞中，平均[Ca2+]pm似乎也高于胞浆溶胶整体，这可能反映了高[Ca2+]微区的存在。
