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Patch-Clamp Analysis of the Mitochondrial Calcium Uniporter

  • Vivek Garg
  • Yuriy Y. KirichokEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1925)

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.

Key words

Mitochondria Patch clamp Calcium channel Uniporter MCU 

Notes

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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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Department of PhysiologyUniversity of California San FranciscoSan FranciscoUSA

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