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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Acknowledgments
This work is supported by NIH RO1 grants GM118939 and GM107710 to Y.Y.K. and an AHA Scientist Development Grant to V.G.
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Garg, V., Kirichok, Y.Y. (2019). Patch-Clamp Analysis of the Mitochondrial Calcium Uniporter. In: Raffaello, A., Vecellio Reane, D. (eds) Calcium Signalling. Methods in Molecular Biology, vol 1925. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9018-4_7
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DOI: https://doi.org/10.1007/978-1-4939-9018-4_7
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