MCU;mitochondrial Ca2+ uniporter;Ca2+ signaling;mitochondrial metabolism
Every cell type, in every tissue and at any evolutionary level, can communicate with the surrounding environment and with neighboring cells. Both intercellular and extracellular communication play fundamental roles in shaping cell behavior and driving cell fate decisions. Cell-to-cell and environmental signals are normally conveyed by distinct extracellular mediators (hydrophilic or hydrophobic compounds, mechanical, ionic, cell–cell interactions, etc.) that are normally perceived by cells through surface receptors. These receptors convey them into a limited number of intracellular molecules, which are referred to as ‘second messengers’, which, in turn, forward the message to intracellular effectors finally activating the ultimate cellular responses. Despite the plethora of different signals and stimuli that cells may receive, only a few molecules to date have been described as second messengers of intracellular communication. Among them, the most common and, definitively, the most extensively studied is Ca 2+ .
participate in the decoding of a vast range of stimuli and the variety of cellular components involved in the Ca 2+ signal transduction is extremely wide, including basically all kinds of components, organelles, and molecules [ 1 ] [ 2 ] [ 3 ] . The research studies on Ca 2+ second messenger started more than one hundred years ago, with the initial recognition of the role of Ca 2+ in muscle cell contraction [ 4 ] . Since then, the understanding of Ca 2+ signaling regulation and dynamics has progressively increased leading to the definition of the concept of Ca 2+ compartmentalization and to the demonstration of the existence of microdomains of local high Ca 2+ concentration [ 5 ] , which are crucial for the fine-tuning and correct triggering of the Ca 2+ -dependent cellular effects [ 1 ] . Mitochondria
play a fundamental
role in the orchestration of cellular Ca2+
signals. Indeed, mitochondria
are not a store of rapidly releasable
(such as the ER), but rather they efficiently accumulate Ca 2+
entry from the extracellular space or upon
release from ER
stores [ 6 ] . Upon
into mitochondria exerts a central function
the modulation of cell metabolism. Mitochondria host the enzymes and complexes of the TCA cycle, fatty acid oxidation (FAO), and oxidative phosphorylation (OXPHOS) thus representing the site of the major metabolic pathways and enzymes for cell energy supply, which deserved them the name of ‘cellular powerhouses’. Interestingly, Ca 2+ entry and oxidative activity are two strictly intertwined aspects of mitochondrial physiology. The increase of
the mitochondrial matrix
Ca 2+ level
stimulates both Ca2+
In addition, mitochondria also actively participate in the tuning of global Ca 2+ signals thanks to their ability to take up Ca 2+ during intracellular Ca 2+ elevation with
net result of buffering the cytosolic cation concentration thus modulating the overall cellular Ca 2+ response. This buffering capacity is due to two crucial characteristics of the mitochondria: (i) their strategic position in close contacts to the Ca 2+ release channels of the ER store [ 6 ] and the plasma membrane in immune cells [ 12 ] (ii) the presence on their inner membrane of highly selective and efficient machinery for taking up Ca 2+ , the MCU complex. Finally, the large buffering capacity of mitochondria can protect cells from Ca 2+ overload. Indeed, an excessive accumulation of the cation in the mitochondrial matrix triggers the permeability transition pore (PTP) opening, the release of pro-apoptotic factors, and finally, induction of programmed cell death [ 13 ] . Given the strong association between mitochondrial Ca 2+ overload and apoptosis induction, the maintenance of mitochondrial Ca 2+ homeostasis is thus a crucial aspect for ensuring cell survival [ 14 ] [ 15 ] .
Given the extreme relevance of mitochondria Ca 2+ signaling for cell physiology, the unveiling of the molecular factors mediating mitochondrial Ca 2+ entry and the mechanism(s) of their regulation has been one of the scientific challenges of recent years. In this review, we aim to summarize some of the milestone achievements in the history of mitochondrial Ca2+ research with a particular focus on the recent findings of the mitochondrial Ca2+ uniporter and its role in organ physiology. We will briefly describe the early studies leading to the demonstration of the Ca 2+ accumulation capacity of mitochondria, then we will go through the historical chronicle of the discoveries of the mitochondrial Ca 2+ uniporter genes and multiple regulators ( Figure 1 ) and we will conclude with an excursus on the physiological relevance of mitochondrial Ca 2+ uptake in the context of skeletal muscle tissue.
Figure 1. Timeline of the identification of MCU complex components.
The most relevant findings on the structure and composition of the MCU complex discovery are summarized and chronologically presented along a timeline covering the last 10 years. Schematic cartoons show the different components of the MCU complex according to the date of their discovery along the timeline.
Timeline of MCU Identification It is suggestive to recall that
notion of Ca2+ ions being relevant to organ physiology dates back to more than one century ago when the first report on the physiological action of Ca2+ ions appeared in 1883 [ 4 ] . At that time, Ringer described the effects of Ca 2+ addition to isolated frog hearts and demonstrated that the supplementation of Ca 2+ in the perfusion solution actively induces and sustains the contraction of the organ ex vivo [ 4 ]. This seminal observation revealed that Ca 2+ is a fundamental messenger within cells, a concept that then extended to virtually every cell type and physiological and pathological process, giving rise to a broad field of study commonly referred to as the field of intracellular Ca 2+ signaling. The intrinsic ability of contracting myocytes to operate ex vivo and to rapidly and effectively respond to environmental condition changes made them the ideal experimental system for the investigation of the role of Ca 2+ in organ and cell physiology and was extensively exploited by researchers in the following years. The original concept of the existence of intracellular compartments acting as Ca 2+ stores to accumulate the cation required to sustain muscle contraction has been later postulated and demonstrated in 1947 by Heilbrunn [ 16 ] . However, although surprising, the identification of the sarco/endoplasmic reticulum (SR/ER) as the principal cellular Ca 2+ store came only 20 years later. It was in the 1960′s, with the identification of Ca 2+ pumping machinery on intracellular membranes (in particular the calcium pump of the sarcoplasmic reticulum, better known as SERCA) by three independent scientists [ 17 ] [ 18 ] [ 19 ] [ 20 ] and the advent of new methodologies for the measurement of intracellular Ca 2+ concentration [ 21 ] that the ER and its specialized counterpart in muscle cells (the SR) were recognized the main cellular reservoir of Ca 2+ . Before
pioneering work of
on cardiac myocyte preparations from rat hearts firstly described
some subcellular compartments
at that time, as the entities actively accumulating Ca 2+ [ 22 ]
. Interestingly, these “sarcosomes”
did not consist of ER but, instead, they corresponded to
isolated mitochondria, to which the addition of Ca2+
caused the block of their oxidative phosphorylation activity. Thus, Ca2+
ions behaved as mitochondria
Despite that, Ca 2+ appeared to exert a peculiar inhibitory action on mitochondrial OXPHOS activity, which differed from the other irreversible uncouplers known at that period (dinitrophenol, dicoumarol, rotenone, antimycin A, azide, or cyanide), due to the reversibility of its action [ 23 ] . This assigned to Ca 2+ ions a functional role in mitochondria activity. After that, a series of land-marking works
in the early 60′s
experimentally revealed that energized mitochondria can actively take up Ca 2+ [ 24 ] [ 25 ] [ 26 ] . Interestingly, these results anticipated the clear demonstration of a driving force for Ca 2+ accumulation in mitochondria, i.e., the chemiosmotic theory , postulated and validated by Peter Mitchell
matrix. Shortly after, at the University
and his group made important discoveries on the Ca 2+ -dependent modulation of
three critical oxidative enzymes resident in mitochondria
Despite all these conceptual advancements, two major issues
were destined to puzzle the scientific community for decades. On one end, there is the apparent paradox between the physiological concentration of cytosolic Ca2+ , which was estimated in the submicromolar range [ 29 ] [ 30 ] [ 31 ], and the low affinity of the mitochondrial Ca2+ uptake , whose half-maximal rate ( Km) was measured in the order of several µM (reviewed in [ 32 ]) . On the other end, the fundamental question about the molecular identity of the IMM apparatus responsible for the entry of Ca2+ into mitochondria was still without an answer.
around 30 and 50 years, respectively, to find solutions to those two enigmas . The answer to the former came with the advent of innovative and sophisticated technologies allowing the assessment of Ca 2+ distribution at the sub-cellular and sub-organellar levels, including mitochondria. Indeed, thanks to the development of Ca2+ -sensitive genetic probes and recombinant fluorescent proteins targeted to specific intracellular microdomains [ 33 ] [ 34 ], it was possible to measure variations of Ca2+ concentration in defined and limited areas of the cell (such as the cytosolic face of plasmalemma [ 35 ] or the surface of outer mitochondrial membrane [ 36 ] , or the Golgi cisternae [ 37 ] ) as well as the relative positioning of the intracellular organelles. These studies were pivotal in the field of cell biology for two main reasons: i) they allowed the observation of the intrinsic heterogeneity of cellular Ca 2+ distribution, definitively demonstrating that large variations in Ca 2+ concentration are highly regionalized within the cell cytoplasm and allowing the direct measure of Ca 2+ levels in the mitochondria matrix as well as in the ER lumen; ii) they pinpointed the fact that organelles, including ER and mitochondria, are in close contact with each other through macromolecular structures involving proteins from both the compartments. In the case of ER-mitochondrial contacts, these structures are biochemically isolated as mitochondria-associated membranes (MAMs) and are formed by membrane channels, as the IP 3 R and VDAC, respectively, and adaptor proteins of both organelles, such as Grp75, mitofusins, PACS [ 38 ] . Upon cell stimulation, the massive release of Ca 2+ through the ER membrane clusters of IP 3 Rs generates microdomains of high Ca2+ concentration right at the mouth of the channel pores, exactly where mitochondria are located. This allows mitochondria to perceive a local cation concentration sufficient to meet the low affinity of the mitochondrial Ca2+ uptake machinery Another fundamental advancement in mitochondrial signaling occurred when scientists found the
answer to the second
the molecular identity of the mitochondrial Ca2+
machinery. The chronicle of MCU discoveries actually started in 2010, with the identification of the first gene required for the uptake of Ca 2+ by mitochondria, CBARA1 , coding for the mitochondrial Ca 2+ uptake 1 protein (MICU1) [ 40 ] , then followed by the identification of the mitochondrial channel
the elucidation of
interactors, as described below
The search of the other mitochondrial Ca 2+ channel components has been proceeding expansively in the last decade (see next paragraph for a detailed timeline) and it is presently still actively ongoing. The discoveries of many different groups worldwide have been indeed instrumental to provide cell biologists with new knowledge on the functional role of mitochondrial Ca 2+ and with new tools for the genetic and molecular intervention on global Ca 2+ signaling and cell energetics.
Discovery and Characterization of the MCU Complex Components
chronicle of MCU discovery starts with two pioneering studies published in 2011
which was identified one year earlier [ 40 ]. These reports clearly demonstrated that the transient downregulation of MCU inhibits the mitochondrial accumulation of Ca 2+ that follows the IP 3 -generating agonist in stimulated cells. Of note, the blunted mitochondrial Ca2+ uptake response occurs without changes in the mitochondria morphology or membrane potential of the MCU-silenced cells Sequence
topology analyses revealed that both the N
termini of MCU are located
mitochondrial matrix and that MCU is endowed with two transmembrane domains, linked by a short highly conserved acidic loop exposed
intermembrane space (IMS) which contains the so-called acidic “DIME” motif. The acidic residues present in this stretch (in particular E257, D261, E264) are critical for the Ca 2+ transport since, if substituted with uncharged residues, MCU mutants failed to rescue the mitochondrial Ca 2+ uptake in MCU-silenced cells [ 41 ] [ 42 ] . However, the definitive description of MCU 3D structure had to wait till the very last years, when cryo-EM
X-ray diffraction analyses finally allowed the resolution of full-length MCU structure [ 44 ] [ 45 ] [ 46 ] [ 47 ] [ 48 ] [ 49 ] [ 50 ] [ 51 ] . These studies coherently confirmed that purified MCU from different sources (fungi and metazoan) arranges in a tetramer, confuting previous assumptions on a putative pentameric MCU architecture [ 52 ] . Notably, the cryo-EM data also unveiled the exact position of the MCU channel selectivity filter, in which the DIME motif is fundamental for the coordination
ions and which was definitively shown to reside at the beginning of the second transmembrane α-helix [ 50 ] and not in the linker region between the two transmembrane helices, as previously suggested [ 52 ] . More recently, the structure of the human MCU together with its auxiliary component EMRE was obtained [ 53 ] . Each human MCU arranges in tetramers and each subunit complexes with one EMRE peptide. Differently from the three described for fungal MCU, human MCU appears organized in four domains, which are:
i) the N -terminal domain (NTD), (ii) the linker helix domain (LHD) —absent in fungi—, (iii) the coiled-coil domain (CCD), and iv) the transmembrane domain (TMD) ( Figure 2
The MCU holocomplex structure. Schematic representation of the MCU holocomplex (uniplex) components and their relevant domains: the pore-forming subunit MCU (light blue) with the two transmembrane (TM) and coiled-coil (CC) domains and the linker helix domain (LHD); the essential mitochondrial Ca2+ uniporter regulator EMRE (yellow); the mitochondrial Ca2+ uptake proteins MICU1 (violet) and MICU2 (purple), with the EF-hands relevant for the MICU dimer interaction highlighted. The critical residues of the MCU DIME motif forming the Ca2+ selectivity filter are indicated, together with the MICU1 residues of the K-R ring coordinating the MCU acidic region.
, in the
very last year, further insight into
modulation and function has been gained thanks to
achievement of the human MCU -MICUs holocomplex structure in both the Ca 2+- free and Ca 2+ -bound state by several independent reports.
The gating mechanism by which MICU1 regulates the uniporter activity via the conformational change triggered by Ca2+ was finally unveiled
Figure 3. The MCU complex activity at low and high intracellular Ca2+ concentration. Schematic representation of the proteins involved in the MCU complex-mediated mitochondrial Ca2+ uptake: the pore-forming subunits MCU (light blue) and MCUb (green), the essential mitochondrial Ca2+ uniporter regulator EMRE (yellow), the mitochondrial Ca2+ uptake proteins MICU1 / MICU1.1 (violet), MICU2/ MICU3 (purple), and the MCU regulator 1 MCUR1 (light violet). The EF-hand Ca2+ binding domains of MICU proteins are indicated as little circles. At low intracellular Ca2+ concentration, the cation does not permeate through the MCU channel since the heterodimers formed by MICU1/MICU1.1–MICU2/MICU3 block the channel pore, thus preventing Ca2+ flux in resting conditions. Differently, at high intracellular Ca2+ concentration, MICU proteins undergo conformational changes relieving the inhibition on MCU and positively regulating channel activity, leading to an efficient mitochondrial Ca2+ uptake. OMM, outer mitochondrial membrane; IMM, inner mitochondrial membrane.
A large body of experimental evidence on the MCU complex functional role has accumulated since the discovery of the MCU. The genetic manipulation of the MCU led to the generation of germline and tissue-specific transgenic models [ 55 ] [ 56 ] [ 57 ] [ 58 ] [ 59 ] [ 60 ] [ 61 ] [ 62 ] , which provided pivotal tools for understanding the pathophysiological implications of the mitochondrial Ca 2+ signaling in vivo that would have otherwise remained unexplored.
In 2013, three independent groups showed that the
As for mammals, the
, some years later, it has been reported that the block of MCU-dependent Ca2+ uptake affects
The genetic manipulation of MCU in C. elegans model also provided additional interesting notions on its role in organism physiology. Indeed, MCU −/− C. elegans is viable and grossly normal, mirroring what was found in the first MCU −/− mouse model described [ 55 ] , even though it presents some defects in the epidermal wound repair mechanisms. More recently, the characterization of C. elegans deficient of a functional MCU [ 68 ] suggested that uniporter activity is essential for mitochondrial Ca 2+ transfer during high-intensity stimulation of the worm pharynx muscle. However, a lot still remains to be explored on the role of MCU in this model and most of the knowledge on mitochondrial Ca 2+ regulation of muscle physiology has been achieved using the mammalian mouse model where MCU expression was genetically targeted, which we will briefly review in the following paragraphs.
In 2013, Raffaello and co-authors discovered that
MCU is not the only pore-forming subunit of the mitochondrial Ca2+ uniporter, since an alternative MCU isoform
exists, named MCUb , which crosses the IMM and associates to MCU to form the calcium channel Interestingly
displays different expression levels in the different mammalian tissues, and the MCUb:MCU proportion appears also the distinctive feature ensuring the appropriate mitochondrial Ca2+ current to each cell type [ 69 ] [ 71 ]. For instance, a high MCUb/MCU ratio (3:1) is typical of cells with low mitochondrial Ca2+ transients , such as adult cardiomyocytes (
Figure 4. MCU holocomplex composition in different tissues. Schematic representation of the tissue-specific components of the MCU holo-complex. The presence of a relatively high MCUb:MCU ratio in the heart ensures a re-duced Ca2+ load in cardiomyocyte mitochondria. On the contrary, the expression of the MICU1.1 variant and MICU3 determines an elevated Ca2+ flux in the mitochondria of skeletal muscle fibers and neurons, respectively. IMS, inter-membrane space; IMM, inner mitochondrial membrane.
These lines of evidence highlight the importance of MCU:MCUb proportion in the control of mitochondrial Ca 2+ uniporter activity and further investigation will be of fundamental relevance for the understanding of its role in the pathophysiology of different tissues.
MICU1 was actually the first component to be described as the regulator of the long-sought MCU channel and its identification even anticipated that of the pore-forming subunit MCU (
The downregulation of MICU1
was initially shown to abolish mitochondrial Ca2+ influx in intact and permeabilized HeLa cells [ 40 ] . Later studies also demonstrated that the absence of MICU1 also leads to an adaptive Ca2+ accumulation inside the mitochondria matrix, triggering excessive ROS production and the consequent higher sensitivity to apoptotic stress. In addition, the ability of MICU1 to sense cytosolic Ca 2+ levels confers to MICU1 the capacity to set the threshold for the activation of mitochondrial Ca 2+ uptake. Nevertheless, this occurs without altering the overall kinetics of the channel [ 75 ] [ 76 ] . Two in vivo studies, performed in MICU1 -/- mouse models have strengthened this concept and gave additional insight into the physiological role of this MCU regulator. In more detail, the
characterization of MICU1-/- transgenic mice reveals that, despite partial postnatal mortality, the viable animals show marked ataxia and muscle weakness
role of MICU1 in the regulation of
crucial metabolic steps of
has emerged. Indeed, MICU1 was shown to inhibit
the mitochondrial transport of pyruvate and fatty acids
and, interestingly, this MICU1 function appears independent of Ca 2+ and MCU core-complex composition [ 80 ]
This study reveals a mechanism that controls the MCU-mediated Ca 2+ flux machinery and that relies on TCA cycle substrate availability. According to this view, the MICU1 regulatory axis acts as a metabolic homeostatic circuit to protect cells from the risk of bioenergetic crisis and mitochondrial Ca 2+ overload during periods of nutrient stress. Altogether these findings [ 77 ] [ 79 ] [ 80 ] highlight once more the crucial importance of the fine-tuning of mitochondrial Ca 2+ uptake by Ca 2+ and MICUs, especially in the context of promotion of cell survival under stress conditions.
to that, new important functions of MICU1 in the control of mitochondrial cristae junctions have been revealed by the recent super-resolution structured illumination microscopy (SIM) and electron microscopy studies of Gottschalk and collaborators [ 81 ] . According to the authors
appears to act as a Ca 2+ -dependent regulator of cristae junctions’ integrity and cytochrome c localization. Another intriguing feature of MICU1 regulatory function on the determination of uniporter cation selectivity has recently emerged [ 82 ] . MICU1 was revealed as the primary
responsible for conferring and ensuring the stringent MCU selectivity for Ca2+
when present, MICU1 impedes Mn 2+ ions to cross the MCU channel pore; on the contrary, in the absence of MICU1, both Ca 2+ and Mn 2+ cations can enter the mitochondrial matrix. This additional MICU1 checkpoint
of fundamental importance
survival of cells sensitive to Mn2+
such as neurons
crucial safeguard against cellular toxicity due to manganese in neurodegenerative diseases [ 82 ] . 3.4. MICU1.1 A
variant of MICU1, named MICU1.1, has been discovered by our group as an alternative splicing product of the
first information about the existence of other genuine MCU regulators, in addition to MICU1, was provided by human genome sequencing studies a few years later [ 84 ] ( Figure 1 ). Initially known as EF-hand domain-containing family member A1 (EFHA1), the mitochondrial calcium uptake protein 2 or MICU2, was found to be a paralog of MICU1 Although the functional role of MICU2 is still controversial, recent findings clarify some key aspects of uniporter modulation by this regulator. In mammalian cells, for example, it
has been shown that MICU2 positively regulates MCU activation by controlling the cytosolic [Ca2+
] threshold for the relief of MICU1-mediated inhibition of MCU
This function allows MICU2 to restrict the spatial Ca 2+ crosstalk between inositol 1,4,5-trisphosphate receptor (InsP3R) and MCU channels [ 86 ] . Subsequently, the generation and characterization of the MICU2 −/− mouse model highlighted other important properties of this regulator [ 87 ] .
MICU2 genetic ablation produces a decrease in the threshold for mitochondrial Ca2+ uptake due to loss of the gatekeeping activity and overall loss of MCU-dependent Ca2+ influx due to the destabilization of the entire uniporter complex.
These findings lead to the conclusion that the amount of MCU and MICUs proteins is crucial to maintain the stability of the whole complex. Moreover, some phenotypic features of the MICU2 −/− mice are in common with models of cardiac pathologies, suggesting that MICU2 may act as a possible cardioprotective factor.
MICU3, previously known as EFHA2, is another MICU1 paralog
, originally identified by the same genetic sequence analysis that described MICU2 [ 84 ] ( Regarding its functional role, our group showed that
acts as a positive regulator of mitochondrial Ca2+ uptake through MICU1 [ 88 ] . Indeed, MICU3 forms heterodimers exclusively with MICU1 , but not with MICU2, and the MICU1-MICU3 interaction leads to a significant increase of mitochondrial Ca 2+ uptake, demonstrating the stimulatory action of MICU3 on uniporter activity [ 88 ]. Moreover, MICU3 downregulation blocks Ca 2+ influx elicited by synaptic activity in primary cortical neurons, suggesting a specific role of this MCU regulator on neuronal function [ 88 ] . This line of evidence lets to hypothesize that the primary role of MICU3 is to enhance MCU opening to ensure mitochondrial Ca2+ uptake in response to both small and fast cytosolic Ca2+ rises, typical of synaptic neuronal stimulation (
The Essential MCU REgulator (EMRE)
is an additional constituent of the uniporter, discovered by quantitative mass spectrometry analysis of affinity-purified MCU complex components
revealed: the m-AAA protease-mediated degradation of EMRE is an essential event to guarantee the correct MCU-MICU proteins’ assembly [ 95 ] . Indeed, the deficiency of m-AAA leads to EMRE accumulation, which prevents the MCU-MICUs association by competing for MCU binding. This generates a constitutively active MCU-EMRE channel that finally induces mitochondrial Ca2+ overload and eventually death of neuronal cells.