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A forty-kilodalton protein of the inner membrane is the mitochondrial calcium uniporter

Abstract

Mitochondrial Ca2+ homeostasis has a key role in the regulation of aerobic metabolism and cell survival1, but the molecular identity of the Ca2+ channel, the mitochondrial calcium uniporter2, is still unknown. Here we have identified in silico a protein (named MCU) that shares tissue distribution with MICU1 (also known as CBARA1), a recently characterized uniporter regulator3, is present in organisms in which mitochondrial Ca2+ uptake was demonstrated and whose sequence includes two transmembrane domains. Short interfering RNA (siRNA) silencing of MCU in HeLa cells markedly reduced mitochondrial Ca2+ uptake. MCU overexpression doubled the matrix Ca2+ concentration increase evoked by inositol 1,4,5-trisphosphate-generating agonists, thus significantly buffering the cytosolic elevation. The purified MCU protein showed channel activity in planar lipid bilayers, with electrophysiological properties and inhibitor sensitivity of the uniporter. A mutant MCU, in which two negatively charged residues of the putative pore-forming region were replaced, had no channel activity and reduced agonist-dependent matrix Ca2+ concentration transients when overexpressed in HeLa cells. Overall, these data demonstrate that the 40-kDa protein identified is the channel responsible for ruthenium-red-sensitive mitochondrial Ca2+ uptake, thus providing a molecular basis for this process of utmost physiological and pathological relevance.

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Figure 1: MCU includes two highly conserved transmembrane domains and is ubiquitously expressed in mammals, similarly to its putative regulator MICU1.
Figure 2: MCU silencing strongly inhibits mitochondrial Ca 2+ uptake without causing morphological rearrangement or changes in the electrochemical gradient.
Figure 3: MCU overexpression increases mitochondrial Ca 2+ accumulation in intact and permeabilized cells, buffers cytosolic [Ca 2+ ] rises, and sensitizes to apoptotic stimuli; GFP-tagged MCU demonstrates mitochondrial localization and indicates a putative membrane topology.
Figure 4: Purified MCU shows channel activity in lipid bilayers, whereas MCUD260Q,E263Q shows no channel activity and reduces [Ca2+]mt transients in cells.

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Acknowledgements

We thank M. Zoratti for help in the analysis of electrophysiological data, G. Merli for carrying out some of the experiments and P. Bernardi, T. Pozzan and L. Cendron for helpful discussions and for the N33-D1cpV expression plasmid (T. Pozzan ). This research was supported by grants from the Italian Ministry of Education, University and Research, European Commission (FP7 “MyoAGE”, no. 223576), National Institutes of Health (Grant #1P01AG025532-01A1), Cariparo Foundation (Padua), the Italian Association for Cancer Research (AIRC) and Telethon-Italy (GPP1005).

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Contributions

D.D.S. performed bioinformatic analysis, Ca2+ measurements and morphological analysis of organelles. A.R. performed molecular biology and gene expression analysis. D.D.S. and A.R. contributed equally to the study. E.T. expressed and purified the protein in heterologous systems; I.S. performed and analysed the electrophysiology experiments; R.R. discussed the results and wrote the paper.

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Correspondence to Rosario Rizzuto.

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De Stefani, D., Raffaello, A., Teardo, E. et al. A forty-kilodalton protein of the inner membrane is the mitochondrial calcium uniporter. Nature 476, 336–340 (2011). https://doi.org/10.1038/nature10230

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