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J Gen Physiol. 2018 Jul 2;150(7):1035-1043. doi: 10.1085/jgp.201812015. Epub 2018 Jun 11.

Electrical recordings of the mitochondrial calcium uniporter in Xenopus oocytes.

Author information

1
Department of Biochemistry, Brandeis University, Waltham, MA.
2
Department of Biochemistry, Brandeis University, Waltham, MA mftsai@brandeis.edu.
3
Department of Physiology and Biophysics, University of Colorado Anschutz Medical Campus, Aurora, CO.
4
Howard Hughes Medical Institute, Chevy Chase, MD.

Abstract

The mitochondrial calcium uniporter is a multisubunit Ca2+ channel that mediates mitochondrial Ca2+ uptake, a cellular process crucial for the regulation of oxidative phosphorylation, intracellular Ca2+ signaling, and apoptosis. In the last few years, genes encoding uniporter proteins have been identified, but a lack of efficient tools for electrophysiological recordings has hindered quantitative analysis required to determine functional mechanisms of this channel complex. Here, we redirected Ca2+-conducting subunits (MCU and EMRE) of the human uniporter to the plasma membrane of Xenopus oocytes. Two-electrode voltage clamp reveals inwardly rectifying Ca2+ currents blocked by a potent inhibitor, Ru360 (half maximal inhibitory concentration, ~4 nM), with a divalent cation conductivity of Ca2+ > Sr2+ > Ba2+, Mn2+, and Mg2+ Patch clamp recordings further reveal macroscopic and single-channel Ca2+ currents sensitive to Ru360. These electrical phenomena were abolished by mutations that perturb MCU-EMRE interactions or disrupt a Ca2+-binding site in the pore. Altogether, this work establishes a robust method that enables deep mechanistic scrutiny of the uniporter using classical strategies in ion channel electrophysiology.

PMID:
29891485
PMCID:
PMC6028504
DOI:
10.1085/jgp.201812015
[Indexed for MEDLINE]
Free PMC Article

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