Patch-Clamp Analysis of the Mitochondrial Calcium Uniporter
Patch-Clamp Analysis of the Mitochondrial Calcium Uniporter
复制标题
DOI：
10.1007/978-1-4939-9018-4_7
复制
发表时间：
2019-01-01
期刊：
影响因子：
--
通讯作者：
Kirichok, Yuriy Y.
中科院分区：
文献类型：
--
作者：
Garg, Vivek;Kirichok, Yuriy Y.
在信号传导事件期间，当细胞溶质钙升高至高于50-100 nM的静息水平时，线粒体积累大量的钙。这种钙的摄取主要是由一种大分子蛋白质组装介导的，称为线粒体钙单向转运体（MCU），位于线粒体内膜中。2004年，我们首次应用膜片钳技术记录了线粒体内膜钙电流，明确证明MCU是一种高选择性的钙通道。本章介绍了如何应用膜片钳技术记录线粒体内膜的Ca 2+单向转运体电流，从心脏组织中分离线粒体，并使用French Press制备线粒体。我们还讨论了膜片钳技术的优势相比，其他方法确定线粒体单向转运体的活性和重要的考虑因素，应用膜片钳技术，这样一个小的亚细胞器。在浴和移液器溶液组成的微小变化的情况下，相同的方法可以应用于研究任何其他电流（例如，H+或Cl-）。
Mitochondria accumulate significant amounts of calcium when cytosolic calcium is elevated above the resting levels of 50-100 nM during signaling events. This calcium uptake is primarily mediated by a macromolecular protein assembly called mitochondrial calcium uniporter (MCU) that resides in the mitochondrial inner membrane. In 2004, we applied patch-clamp technique for the first time to record calcium currents from the mitochondrial inner membrane and proved unequivocally that MCU is a highly selective calcium channel. This chapter describes how patch-clamp technique can be applied to record the Ca2+ uniporter currents from the mitochondrial inner membrane, isolation of mitochondria from the heart tissue, and preparation of mitoplast using French Press. We also discuss advantages of patch-clamp technique as compared to other methods of determining mitochondrial uniporter activity and important considerations in applying patch-clamp technique to such a small subcellular organelle. With small variations in the bath and pipette solution composition, the same methodology can be applied to study any other currents (e.g., H+ or Cl-) from the mitochondrial inner membrane.