Central to the role of the mitochondrion in cellular metabolism is its ability to control the fluxes of the key signalling ion, Ca2+. This is done by a highly selective ion channel known as the mitochondrial calcium uniporter. The molecular nature of this channel has remained elusive, but now two groups report the identification of a 40-kilodalton protein in the inner membrane of mitochondria as the active channel of the uniporter. This protein contains two transmembrane domains and exhibits calcium-channel activity in vitro and in vivo. 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.

Here we define the molecular nature of the mitochondrial permeability transition pore (PTP), a key effector of cell death. The PTP is regulated by matrix cyclophilin D (CyPD), which also binds the lateral stalk of the F _O F ₁ ATP synthase. We show that CyPD binds the oligomycin sensitivity-conferring protein subunit of the enzyme at the same site as the ATP synthase inhibitor benzodiazepine 423 (Bz-423), that Bz-423 sensitizes the PTP to Ca ²⁺ like CyPD itself, and that decreasing oligomycin sensitivity-conferring protein expression by RNAi increases the sensitivity of the PTP to Ca ²⁺ . Purified dimers of the ATP synthase, which did not contain voltage-dependent anion channel or adenine nucleotide translocator, were reconstituted into lipid bilayers. In the presence of Ca ²⁺ , addition of Bz-423 triggered opening of a channel with currents that were typical of the mitochondrial megachannel, which is the PTP electrophysiological equivalent. Channel openings were inhibited by the ATP synthase inhibitor AMP-PNP (γ-imino ATP, a nonhydrolyzable ATP analog) and Mg ²⁺ /ADP. These results indicate that the PTP forms from dimers of the ATP synthase.

Mitochondrial calcium accumulation was recently shown to depend on a complex composed of an inner-membrane channel (MCU and MCUb) and regulatory subunits (MICU1, MCUR1, and EMRE). A fundamental property of MCU is low activity at resting cytosolic Ca(2+) concentrations, preventing deleterious Ca(2+) cycling and organelle overload. Here we demonstrate that these properties are ensured by a regulatory heterodimer composed of two proteins with opposite effects, MICU1 and MICU2, which, both in purified lipid bilayers and in intact cells, stimulate and inhibit MCU activity, respectively. Both MICU1 and MICU2 are regulated by calcium through their EF-hand domains, thus accounting for the sigmoidal response of MCU to [Ca(2+)] in situ and allowing tight physiological control. At low [Ca(2+)], the dominant effect of MICU2 largely shuts down MCU activity; at higher [Ca(2+)], the stimulatory effect of MICU1 allows the prompt response of mitochondria to Ca(2+) signals generated in the cytoplasm.

Mitochondrial calcium uniporter (MCU) channel is responsible for Ruthenium Red‐sensitive mitochondrial calcium uptake. Here, we demonstrate MCU oligomerization by immunoprecipitation and Förster resonance energy transfer (FRET) and characterize a novel protein (MCUb) with two predicted transmembrane domains, 50% sequence similarity and a different expression profile from MCU. Based on computational modelling, MCUb includes critical amino‐acid substitutions in the pore region and indeed MCUb does not form a calcium‐permeable channel in planar lipid bilayers. In HeLa cells, MCUb is inserted into the oligomer and exerts a dominant‐negative effect, reducing the [Ca2+]mt increases evoked by agonist stimulation. Accordingly, in vitro co‐expression of MCUb with MCU drastically reduces the probability of observing channel activity in planar lipid bilayer experiments. These data unveil the structural complexity of MCU and demonstrate a novel regulatory mechanism, based on the inclusion of dominant‐negative subunits in a multimeric channel, that underlies the fine control of the physiologically and pathologically relevant process of mitochondrial calcium homeostasis.

The field of mitochondrial ion channels has recently seen substantial progress, including the molecular identification of some of the channels. An integrative approach using genetics, electrophysiology, pharmacology, and cell biology to clarify the roles of these channels has thus become possible. It is by now clear that many of these channels are important for energy supply by the mitochondria and have a major impact on the fate of the entire cell as well. The purpose of this review is to provide an up-to-date overview of the electrophysiological properties, molecular identity, and pathophysiological functions of the mitochondrial ion channels studied so far and to highlight possible therapeutic perspectives based on current information.