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  • Title: Patch-Clamp Analysis of the Mitochondrial Calcium Uniporter.
    Author: Garg V, Kirichok YY.
    Journal: Methods Mol Biol; 2019; 1925():75-86. PubMed ID: 30674018.
    Abstract:
    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.
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