Garg Vivek, Kirichok Yuriy Y
Department of Physiology, University of California San Francisco, San Francisco, CA, USA.
Methods Mol Biol. 2019;1925:75-86. doi: 10.1007/978-1-4939-9018-4_7.
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 Ca 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.
在信号转导事件中，当胞质钙浓度升高至高于50 - 100 nM的静息水平时，线粒体可积累大量的钙。这种钙摄取主要由位于线粒体内膜的一种称为线粒体钙单向转运体（MCU）的大分子蛋白组装体介导。2004年，我们首次应用膜片钳技术记录线粒体内膜的钙电流，并明确证明MCU是一种高度选择性的钙通道。本章描述了如何应用膜片钳技术记录线粒体内膜的钙单向转运体电流、从心脏组织中分离线粒体以及使用法国压榨机制备线粒体膜间腔。我们还讨论了膜片钳技术与其他测定线粒体单向转运体活性方法相比的优势，以及将膜片钳技术应用于如此小的亚细胞器时的重要注意事项。通过对浴液和吸管溶液成分进行微小改变，相同的方法可用于研究线粒体内膜的任何其他电流（例如，H或Cl）。
