Arnaudeau S, Kelley W L, Walsh J V, Demaurex N
Department of Physiology, University of Geneva, 1211 Geneva 4, Switzerland.
J Biol Chem. 2001 Aug 3;276(31):29430-9. doi: 10.1074/jbc.M103274200. Epub 2001 May 17.
To study Ca(2+) fluxes between mitochondria and the endoplasmic reticulum (ER), we used "cameleon" indicators targeted to the cytosol, the ER lumen, and the mitochondrial matrix. High affinity mitochondrial probes saturated in approximately 20% of mitochondria during histamine stimulation of HeLa cells, whereas a low affinity probe reported averaged peak values of 106 +/- 5 microm, indicating that Ca(2+) transients reach high levels in a fraction of mitochondria. In concurrent ER measurements, Ca(2+) averaged 371 +/- 21 microm at rest and decreased to 133 +/- 14 microm and 59 +/- 5 microm upon stimulation with histamine and thapsigargin, respectively, indicating that substantial ER refilling occur during agonist stimulation. A larger ER depletion was observed when mitochondrial Ca(2+) uptake was prevented by oligomycin and rotenone or when Ca(2+) efflux from mitochondria was blocked by CGP 37157, indicating that some of the Ca(2+) taken up by mitochondria is re-used for ER refilling. Accordingly, ER regions close to mitochondria released less Ca(2+) than ER regions lacking mitochondria. The ER heterogeneity was abolished by thapsigargin, oligomycin/rotenone, or CGP 37157, indicating that mitochondrial Ca(2+) uptake locally modulate ER refilling. These observations indicate that some mitochondria are very close to the sites of Ca(2+) release and recycle a substantial portion of the captured Ca(2+) back to vicinal ER domains. The distance between the two organelles thus determines both the amplitude of mitochondrial Ca(2+) signals and the filling state of neighboring ER regions.
为了研究线粒体与内质网（ER）之间的Ca(2+)通量，我们使用了靶向细胞质、内质网腔和线粒体基质的“cameleon”指示剂。在组胺刺激HeLa细胞的过程中，高亲和力的线粒体探针在约20%的线粒体中达到饱和，而低亲和力探针报告的平均峰值为106±5微摩尔，表明Ca(2+)瞬变在一部分线粒体中达到高水平。在内质网的同步测量中，静止时[Ca(2+)]内质网平均为371±21微摩尔，在用组胺和毒胡萝卜素刺激后分别降至133±14微摩尔和59±5微摩尔，表明在激动剂刺激期间内质网会大量重新填充。当用寡霉素和鱼藤酮阻止线粒体Ca(2+)摄取或用CGP 37157阻断线粒体Ca(2+)外流时，观察到内质网的耗竭更大，表明线粒体摄取的一些Ca(2+)被重新用于内质网重新填充。因此，靠近线粒体的内质网区域释放的Ca(2+)比没有线粒体的内质网区域少。毒胡萝卜素、寡霉素/鱼藤酮或CGP 37157消除了内质网的异质性，表明线粒体Ca(2+)摄取局部调节内质网重新填充。这些观察结果表明，一些线粒体非常靠近Ca(2+)释放位点，并将捕获的Ca(2+)的很大一部分再循环回邻近的内质网结构域。因此，这两个细胞器之间的距离决定了线粒体Ca(2+)信号的幅度和邻近内质网区域的填充状态。
