Yunnan Center for Disease Prevention and Control, Kunming, China.
Acta Biochim Biophys Sin (Shanghai). 2011 Oct;43(10):822-30. doi: 10.1093/abbs/gmr075. Epub 2011 Aug 30.
Mitochondrial calcium plays a crucial role in mitochondrial metabolism, cell calcium handling, and cell death. However, some mechanisms concerning mitochondrial calcium regulation are still unknown, especially how mitochondrial calcium couples with cytosolic calcium. In this work, we constructed a novel mitochondrial calcium fluorescent indicator (mito-GCaMP2) by genetic manipulation. Mito-GCaMP2 was imported into mitochondria with high efficiency and the fluorescent signals co-localized with that of tetramethyl rhodamine methyl ester, a mitochondrial membrane potential indicator. The mitochondrial inhibitors specifically decreased the signals of mito-GCaMP2. The apparent K(d) of mito-GCaMP2 was 195.0 nmol/L at pH 8.0 in adult rat cardiomyocytes. Furthermore, we observed that mito-GCaMP2 preferred the alkaline pH surrounding of mitochondria. In HeLa cells, we found that mitochondrial calcium (Ca(2+)) responded to the changes of cytosolic calcium (Ca(2+)) induced by histamine or thapasigargin. Moreover, external Ca(2+) (100 μmol/L) directly induced an increase of Ca(2+) in permeabilized HeLa cells. However, in rat cardiomyocytes Ca(2+) did not respond to cytosolic calcium transients stimulated by electric pacing or caffeine. In permeabilized cardiomyocytes, 600 nmol/L free Ca(2+) repeatedly increased the fluorescent signals of mito-GCaMP2, which excluded the possibility that mito-GCaMP2 lost its function in cardiomyocytes mitochondria. These results showed that the response of mitochondrial calcium is diverse in different cell lineages and suggested that mitochondria in cardiomyocytes may have a special defense mechanism to control calcium flux.
线粒体钙在线粒体代谢、细胞钙处理和细胞死亡中起着至关重要的作用。然而，一些关于线粒体钙调节的机制仍不清楚，特别是线粒体钙如何与细胞质钙偶联。在这项工作中，我们通过遗传操作构建了一种新型的线粒体钙荧光指示剂（mito-GCaMP2）。mito-GCaMP2 高效导入线粒体，荧光信号与线粒体膜电位指示剂四甲基罗丹明甲酯（tetramethyl rhodamine methyl ester）共定位。线粒体抑制剂特异性降低了 mito-GCaMP2 的信号。在 pH8.0 下，成年大鼠心肌细胞中 mito-GCaMP2 的表观 Kd 为 195.0nmol/L。此外，我们观察到 mito-GCaMP2 更喜欢线粒体周围的碱性 pH 环境。在 HeLa 细胞中，我们发现线粒体钙 (Ca(2+)) 对组胺或 thapasigargin 引起的细胞质钙 (Ca(2+)) 变化有反应。此外，外源性 Ca(2+)（100μmol/L）直接诱导通透化的 HeLa 细胞中 Ca(2+) 增加。然而，在大鼠心肌细胞中，Ca(2+) 对电起搏或咖啡因刺激的细胞质钙瞬变没有反应。在通透化的心肌细胞中，600nmol/L 的游离 Ca(2+) 反复增加 mito-GCaMP2 的荧光信号，排除了 mito-GCaMP2 在心肌细胞线粒体中丧失功能的可能性。这些结果表明，线粒体钙的反应在不同的细胞系中是多样的，并表明心肌细胞中的线粒体可能具有特殊的防御机制来控制钙流。
