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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.

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Department of Biochemistry, Brandeis University, Waltham, MA.
Department of Biochemistry, Brandeis University, Waltham, MA
Department of Physiology and Biophysics, University of Colorado Anschutz Medical Campus, Aurora, CO.
Howard Hughes Medical Institute, Chevy Chase, MD.


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.

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