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

Abstract

Mitochondrial Ca(2+) homeostasis has a key role in the regulation of aerobic metabolism and cell survival, but the molecular identity of the Ca(2+) channel, the mitochondrial calcium uniporter, 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 regulator, is present in organisms in which mitochondrial Ca(2+) uptake was demonstrated and whose sequence includes two transmembrane domains. Short interfering RNA (siRNA) silencing of MCU in HeLa cells markedly reduced mitochondrial Ca(2+) uptake. MCU overexpression doubled the matrix Ca(2+) 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 Ca(2+) 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 Ca(2+) uptake, thus providing a molecular basis for this process of utmost physiological and pathological relevance.

Figure 1. MCU includes two highly conserved transmembrane domains and is ubiquitously expressed in mammals, similarly to its putative regulator MICU1

a, Alignment of the putative transmembrane domain and pore region of MCU proteins from 14 different species. b, Quantitative Real Time PCR analysis of mouse tissues. mRNA extraction and quantitative PCR as described in the methods section. Expression levels are normalized to skeletal muscle, and presented as means ± S.D. (n = 3).

Figure 2. MCU silencing strongly inhibits mitochondrial Ca²⁺ uptake without causing morphological rearrangement or changes in the electrochemical gradient

a, [Ca²⁺]_mt and b, [Ca²⁺]_cyt measurements in MCU-silenced cells. c, Fluorescence images of mtRFP- and erGFP-labeled mitochondria and ER, respectively. d and e, Mitochondrial number and volume, as deduced by calculating object number (d) and size (e). f, ER/mitochondria colocalization, estimated by Pearson's correlation coefficient. g, TMRM fluorescence measurements. h, [Ca²⁺]_mt measurements in permeabilized cells. In this and following figures, experiments are representative of >5 trials, conditions are in the methods section, and statistics in Supplementary Table 2. * p<0.05, ** p<0.001

Figure 3. MCU overexpression increases mitochondrial Ca²⁺ accumulation in intact and permeabilized cells, buffers cytosolic [Ca²⁺] rises, and sensitizes to apoptotic simuli; GFP-tagged MCU demonstrates mitochondrial localization and suggests a putative membrane topology

a, [Ca²⁺]_mt and b, [Ca²⁺]_cyt measurements in MCU-overexpressing cells. c, [Ca²⁺]_mt measurements in permeabilized cells. d, Cell viability upon apoptotic challenge. e, Subcellular fractionation of MCU-GFP- and mtGFP-expressing cells and Western blotting for GFP, matrix (HSP60) and OMM (VDAC1) markers. f, GFP proteinase K degradation and fluorescence quenching by Trypan blue in permeabilized cells. g, Schematic representation of the predicted MCU topology. RuR, Ruthenium Red. ** p<0.001.

Figure 4. The purified MCU protein exhibits channel activity in lipid bilayers, with the properties of the uniporter; MCU^D260Q,E263Q shows no channel activity and reduces [Ca²⁺]_mt transients in cells

a-c Electrophysiological recordings of purified MCU or MCU^D260Q,E263Q, produced in E. coli (a and b) or in vitro (c) and reconstituted in planar lipid bilayers. a, MCU traces at different applied voltages; b, MCU traces before and after addition of RuR or Gd³⁺; c, MCU^D260Q,E263Q traces. After 10 minutes recording with no current, MCU was added and channel activity was detected after 1-2 minutes (lower trace). d, [Ca²⁺]_mt measurements in MCU^D260Q,E263Q-expressing cells.