Regulatory mechanisms of mitochondrial calcium uptake by the calcium uniporter complex

Abstract

Mitochondria play an important role in energy conversion as well as in intracellular calcium (Ca²⁺) storage. Ca²⁺ uptake from the cytosol to the mitochondria is mediated by the calcium uniporter, which functions as a Ca²⁺ ion channel. However, the molecular composition of this uniporter has remained unclear until recently. The Ca²⁺ ion channel consists of seven subunits. The yeast reconstitution technique revealed that the mitochondrial calcium uniporter (MCU) and essential MCU regulatory element (EMRE) are the core subunits of the complex. Furthermore, detailed structure-function analyses of the core subunits (MCU and EMRE) were performed. In this review, the regulatory mechanism of mitochondrial Ca²⁺ uptake is discussed.

Significance

The calcium uniporter complex, which is involved in calcium uptake in mitochondria, is a novel calcium ion channel whose component molecules have recently been identified. The regulatory mechanisms of calcium uptake are unique, and several subunits play important roles in the regulation of this system. In particular, the functions of the two core subunits, MCU and EMRE, which function as pores for calcium uptake, have been studied intensively. In this review, we focus on the identification of the core subunits from multiple subunits and their structure-function analysis, which has revealed the mechanism of calcium uptake regulation.

Introduction

The calcium ion (Ca²⁺) deemed the “ion of life and death,” plays a vital role in a variety of biological processes, from muscle contraction and fertilization to cell death. Hence, the intracellular concentration of Ca²⁺ is strictly regulated. The mitochondria are important regulators of Ca²⁺ in eukaryotic cells and abnormalities in mitochondrial Ca²⁺ dynamics have been associated with a variety of conditions, including ischemia-reperfusion injury, Ulrich muscular dystrophy, and Alzheimer’s and Huntington’s disease. For this reason, intracellular Ca²⁺ has attracted much attention as a target for drug discovery. However, until recently, the molecular mechanisms involved in the regulation of mitochondrial Ca²⁺ concentration were largely unknown. Over the past few years, a number of molecules comprising the calcium uniporters have been identified, which include a pore-forming subunit bound to several regulatory subunits. The identification of relevant molecules has allowed for deeper questioning of how this complex takes up Ca²⁺ and why this uptake mechanism is important for the functioning of cells and many organs. In this article, the latest developments in the molecular mechanism of Ca²⁺ uptake by calcium uniporters are introduced and the prospects for drug discovery targeting calcium uniporters are discussed in consideration with previous findings and the confusion surrounding the three-dimensional (3D) structure over the past two years.

This review article is an extended version of the mitochondrial calcium ion channel, calcium uniporter, published in SEIBUTSU-BUTSURI Vol.61, p.157–161 (2021) [1].

Mitochondrial Calcium Uniporter (MCU)

Research on mitochondrial Ca²⁺ uptake dates back to the 1960s, when several research groups reported that mitochondria purified from animal tissues had the ability to take up and retain Ca²⁺ [2]. Ca²⁺ uptake has been observed in the mitochondria of a wide range of organisms, with the exception of some fungi, highlighting the importance of this ion throughout the evolutionary process. Ca²⁺ uptake is driven by membrane potential across the mitochondrial inner membrane via the calcium uniporter (Figure 1A) and does not require exchange with other anions or cations [3]. The calcium uniporter is unique in that it actively uptakes Ca²⁺ only when the extramitochondrial Ca²⁺ concentration is relatively high (>10 μM). A 2004 electrophysiological study by Kirichok et al. [4] revealed that the Ca²⁺-selective conductance of the mitochondrial membranes is high, indicating that the calcium uniporter is a channel-like transporter. However, the molecules involved in mitochondrial Ca²⁺ uptake remained unknown.

Figure 1 

Mitochondrial calcium uptake. (A) Mitochondria and mitochondrial calcium uniporter. (B) The subunits of the MCU complex.

In 1980, Denton and McCormack [5] reported that Ca²⁺ uptake into the mitochondrial matrix via the calcium uniporter activated three types of dehydrogenases in the matrix and was involved in metabolic regulation. Subsequently, Jouaville et al. [6] reported that MCUs regulated the cytoplasmic Ca²⁺ wave to increase the intracellular Ca²⁺ concentration. Thereafter, Duchen [7] found that excessive Ca²⁺ uptake into the mitochondria via the calcium uniporter increased the permeability of the mitochondrial inner membrane (permeability transition), which triggered cell death. This permeability transition is believed to be the cause of ischemia-reperfusion injury, Ulrich muscular dystrophy, and Alzheimer’s and Huntington’s disease. Thus, the calcium uniporter has recently attracted attention as a therapeutic target for various diseases.

Molecular Mechanism of Ca²⁺ Uptake by the Calcium Uniporter

While research on the physiological role of the calcium uniporter has continued to progress, understanding of the molecular mechanism underlying Ca²⁺ uptake by the calcium uniporters has been stagnant for more than 50 years. In 2008, Mootha et al. at Harvard University created the MitoCarta database of mitochondrial proteins and in 2010, the mitochondrial calcium uptake 1 (MICU1) was identified by comparing the mitochondrial proteins between yeast without mitochondrial Ca²⁺ uptake and those of trypanosomes with Ca²⁺ uptake [8]. The identification of MICU1 led to the identification of the MCU [9,10] and the paralogs MICU2 [11], MICU3 [11], MCUb [12], and mitochondrial calcium uniporter regulator 1 (MCUR1) [13] in 2012 and the essential MCU regulatory element (EMRE) in 2013 [14]. Most of these subunits are distributed throughout the body, while MICU3 is brain-specific. Currently, calcium uniporters are thought to function as complex channels composed of six to seven subunits (Figure 1B). It has been suggested that calcium uniporters oligomerize to form channel pores that are opened and closed by other subunits, although the functions of these individual subunits remain unclear. Previously reported Ca²⁺ channels form multi-pass transmembrane domains, whereas no multi-pass transmembrane domains have been reported in calcium uniporters. Because of this structural dissimilarity, calcium uniporters are categorized as novel class in Ca²⁺ channels; thus, their structure-function relationships are of particular interest. In addition, familial cardiomyopathy in Europeans with point mutations in the MICU1 has been also reported. Therefore, research on the calcium uniporter would help to clarify the relationship between mitochondrial Ca²⁺ uptake and diseases at the molecular level [15].

Determination of the Minimum Subunit Essential for Mitochondrial Ca²⁺ Uptake

Although seven subunits that are involved in the mitochondrial Ca²⁺ uptake have been identified, the essential subunit for Ca²⁺ uptake remains unclear. Until now, studies on the molecular mechanism of calcium uniporters have been conducted using animal cells lacking specific subunits of Ca²⁺ ion channels. However, this approach is insufficient to determine whether the MCU alone has the capability to uptake Ca²⁺ because it would require the deletion of multiple genes, but the possibility that the function of the deleted gene might be replaced by another subunit cannot be ruled out. To avoid this issue, we focused on yeast. The function of Ca²⁺ uptake into the mitochondria is widely conserved between humans and fungi, but interestingly, it is defective in the yeast Saccharomyces cerevisiae and no orthologs of the MCU subunit group are found in the yeast genome. Taking advantage of this property, it is possible to express specific mammalian subunits of the calcium uniporter in yeast. This method allows for analysis of the function of individual subunits without secondary effects derived from endogenous subunits. Thus, subunits of the mouse calcium uniporter were expressed in yeast to determine whether Ca²⁺ uptake activity was observed in the mitochondria. The results showed that when expressed individually, none of the subunits, including the MCU, had the capability to actively uptake Ca²⁺, suggesting that our concept might be wrong. However, later co-expression experiments with all combinations of the discovered subunits found that co-expression of only MCU with EMRE resulted in significant Ca²⁺ uptake activity (Figure 2). This finding confirmed that among the seven known subunits, the MCU and EMRE are the minimum subunits required for Ca²⁺ uptake [16,17], which paved the way for detailed structure-function analysis of these two subunits.

Figure 2 

Relative Ca²⁺ uptake of the yeast mitochondria with mammalian MCU subunit(s). The activities of Ca²⁺ uptake in the mitochondria isolated from yeast with mammalian MCU subunit(s) were compared.

Role of EMRE in Mitochondrial Ca²⁺ Uptake

Previous findings suggest that MCUs form a channel pore. However, the specific role of EMRE remains unclear, as its Ca²⁺ uptake function was only found to be lost when it was knocked out. Therefore, the structure and function of EMRE were investigated using a yeast expression system.

Structure-function Analysis of EMRE

First, the potential contribution of EMRE to the stability of the MCU was investigated. We prepared yeast mitochondria expressing MCU, EMRE, or both and examined their protein levels. As a result, the amount of MCUs present in mitochondria did not change when expressed alone or co-expressed with EMRE. Therefore, EMRE was not considered to be a contributing factor to the stability of the MCU [16,17]. In general, several acidic amino acids present in ion channels contribute to the accumulation and binding of substrate cations. Therefore, mutants of the acidic amino acids of EMRE conserved among species were created to examine the effects of each on Ca²⁺ uptake. As a result, mitochondria expressing any of the mutants exhibited Ca²⁺ uptake activity comparable to that of the wild-type EMRE. The results suggest that EMRE does not directly bind Ca²⁺ or contribute to Ca²⁺ uptake. On the other hand, various mutants other than acidic amino acids were constructed and analyzed, which revealed no Ca²⁺ uptake by the Pro60 mutant (P60A) or the mutant lacking Ser85–Asn90 (Δ85–90) (Figure 3A).

Figure 3 

Functional analysis of EMRE using a yeast expression system. (A) Identification of amino-acid regions of the mouse EMRE critical for Ca²⁺ uptake. (B) The amino-acid residue(s) with important roles in the functions of EMRE. Functional model of the roles of EMRE in mitochondrial Ca²⁺ uptake.

Importance of the Interaction between EMRE and the MCU

Since EMRE was found to interact with the MCU, immunoprecipitation analysis was performed to determine the effects of the interaction between the loss-of-function EMRE mutants and MCU. The results showed that neither P60A nor Δ85–90 interacted with the MCU, indicating that the interaction between EMRE and the MCU is essential for Ca²⁺ uptake into the mitochondria and that the interaction involves Pro60 on the matrix side and Ser85–Asn90 on the cytoplasmic side of the EMRE (Figure 3A). This finding suggests that EMRE and the MCU interact on both sides through the inner membrane. Based on the close interaction between EMRE and the MCU, we conclude that EMRE functions as a structural factor that holds the channel pore formed by the MCU in an open state (Figure 3B). Recently, the 3D structure of the human MCU/EMRE complex has been elucidated by cryo-electron microscopy [18]. In this structure, EMRE was found to fix the channel pore formed by MCU in the open state, as predicted by the authors based on biochemical analysis.

Regulatory Mechanism of Ca²⁺ Uptake by the MCU Revealed by Structure-function Analysis

The MCU, a subunit of the calcium uniporter that forms the channel pore, has been the most intensively studied. The DXXE motif consists of two transmembrane regions (TM1 and TM2) separated by Asp and Glu residues, and two coiled-coil domains (CC1 and CC2) flanking two transmembrane regions (Figure 4A) [19]. Previous studies have shown that the DXXE motif functions as a Ca²⁺-selective filter, which is an essential structure for ion channels [20–24]. However, the function of the domains, other than the DXXE motif, and how Ca²⁺ enters the matrix through the domains located in the inner membrane or matrix after being selected by the cytoplasmic DXXE motif remained unclear. Therefore, the yeast reconstitution system established earlier was employed for structure-function analysis of various domains of the MCU. First, mutants of the mouse MCU lacking various amino-acid regions were expressed together with EMRE in yeast to examine the mitochondrial Ca²⁺ uptake activity. The results showed that the deletion of two coiled-coil domains (CC1 and CC2) abolished the Ca²⁺ uptake function (Figure 4B). Next, point mutations were introduced to inhibit the formation of the coiled-coil structures of CC1 or CC2 to determine whether either is required for Ca²⁺ uptake. The results revealed that both mutations abolished the Ca²⁺ uptake function, suggesting that Ca²⁺ uptake requires the coiled-coil structures of both CC1 and CC2 of the MCU. We then investigated why the coiled-coil structure of MCU is required for Ca²⁺ uptake. Previous studies reported that the MCU interacts with the regulatory subunits MCUR1 and EMRE [13,14]. However, our analysis showed that MCUR1 and EMRE are irrelevant to the functional importance of either domain, indicating that CC1 and CC2 of the MCU do not form a coiled-coil structure with the regulatory subunits, but rather between CC1 and CC2, which is important for channel pore formation in the MCU [25,26].

Figure 4 

Identification of amino-acid regions of the mouse MCU critical for Ca²⁺ uptake. (A) Domains/motifs and topology of the mitochondrial inner membrane (IM) of the mouse MCU. NTD, N-terminal domain; CC, coiled-coil domain; TM, transmembrane region. (B) The activities of Ca²⁺ uptake in the mitochondria isolated from yeast with each MCU deletion mutant were compared.

Functional Role of the Coiled-coil Structure in the 3D Structure of MCU

Over the past two years, several groups have successively elucidated the 3D structure of the MCU. Oxenoid et al. [20] analyzed the structure of the C. elegans MCU lacking the N-terminal domain by nuclear magnetic resonance (NMR) spectroscopy and found that it is a pentamer and forms a channel pore. We consider that the C. elegans MCU lacking the N-terminal domain were analyzed due to the technical limitation in the analyses of protein structure using NMR. However, in this structural model, the coiled-coil domain consisted of five CC2s forming a pentameric coiled-coil with a single superhelix and CC1 existing around the pentameric coiled-coil of CC2 without forming a coiled-coil structure. Our biochemical studies showed that CC1 formed a pentameric coiled-coil structure. Although both CC1 and CC2 had a coiled-coil structure, the pentamer model did not reflect our results. This confusion continued until 2 years later, when four independent research groups reported the 3D structure of fungal MCUs containing an N-terminal domain [21–24]. In these four reports, all MCUs constituted the channel pore as a tetramer. In these structures, the CC1 and CC2 domains were oriented antiparallel to each other to form a coiled-coil structure. The structural features of this MCU tetramer were consistent with our biochemical findings. Furthermore, we constructed a model of the mouse MCU structure based on this fungal MCU structure and compared it with the results of our previous biochemical analyses, which confirmed that the six amino-acid residues with lost activity by point mutation analysis were all located at the junction of two α-helices that form a coiled-coil structure (Figure 5). This suggests that the tetrameric model reflects the physiological structure of the MCU on the mitochondrial inner membrane, at least in the case of Ca²⁺ uptake. Although it is unknown why the C. elegans MCU was observed as a pentamer, it is possible that the MCU underwent a conformational change or the loss of the N-terminal domain had some unknown structural effect.

Figure 5 

Coiled-coil structure of the mouse MCU model. (Right) Close-up views of the coiled-coil structure. The hydrophobic amino-acid residues located at the position where CC1 and CC2 were the closest to one another are shown in orange.

The structural mouse model included a cavity in the middle of the region of the MCU tetramer exposed to the mitochondrial interior. This suggests that Ca²⁺ entering the channel from outside of the mitochondria through the ion-selective filter diffuses from the channel lumen into the mitochondrial lumen through this cavity, possibly due to the formation of the coiled-coil structure by the interaction of CC1 and CC2. This structure probably acts as a stopper that prevents the formation of the framework of an open umbrella to expand the Ca²⁺-diffusion space at the channel exit (Figure 6). The coiled-coil structure of the other ion channels was found to have a function in expanding the Ca²⁺-diffusion space at the channel exit. Based on the findings of the role of the coiled-coil structure in the function of other ion channels, it is possible that the coiled-coil structure of the MCU functions as a switch that regulates the opening and closing of the channel pore. In this case, the coiled-coil domain may be a promising target site for drug discovery to regulate Ca²⁺ uptake into the mitochondria.

Figure 6 

Functional model of coiled-coil domains of the MCU. There is ample space for Ca²⁺ to diffuse between the transmembrane region and coiled-coil domain; thus, Ca²⁺ which has passed the Ca²⁺ selective filter is considered to enter the matrix mainly through this space.

Conclusion

Here, we reviewed the regulatory function of Ca²⁺ channel pore opening and closing in the MCU and the role of EMRE in regulating this function using a unique method of reconstituting mammalian proteins into yeast mitochondria. This yeast reconstitution technique has already been used for large-scale screening of MCU inhibitors [27] and is expected to be applied in future for drug development. With recent technological innovations in 3D structural analysis, including cryo-electron microscopy, we are accumulating findings on the major questions of how the MCU selectively channels Ca²⁺ into mitochondria and the role of EMRE in Ca²⁺ permeation. However, further studies are required to determine the actual number of EMRE molecules bound to the MCU on the mitochondrial membrane (stoichiometric ratio) and the functional roles of the accessory subunits. In addition, interesting issues, such as the mechanism by which mitochondrial Ca²⁺ uptake activity differs among tissues, remain to be addressed. Clarification of the molecular mechanisms that regulate Ca²⁺ transport by calcium uniporters is expected to lead to the development of new therapeutic agents for various diseases that originate from mitochondrial Ca²⁺ uptake.

Conflict of Interest

The authors declare that there are no conflicts of interest.

Author Contributions

A.Y wrote the manuscript with A.W under the supervision of T.Y

Data Availability

The evidence data generated and/or analyzed during the current study are available from the corresponding author on reasonable request.

Acknowledgments

We thank Professor Yasuo Shinohara from the Institute for Advanced Enzyme Research, University of Tokushima. We would also like to express our gratitude to the late Dr. Hiroshi Terada, President of Niigata University of Pharmacy and Applied Life Sciences and pray for the repose of his soul. We would like to express our gratitude to the Ministry of Education, Culture, Sports, Science, and Technology (MEXT) for financial support via a Grant-in-Aid for Scientific Research C, the Kowa Foundation for Life Sciences, and the Takeda Science Foundation.

References

• [1]   Yamamoto,  T.,  Yamada,  A.,  Shinohara,  Y.,  Watanabe,  A. The molecular mechanisms of mitochondrial calcium ion channel, calcium uniporter. SEIBUTSU BUTSURI. 61, 157–161 (2021). https://doi.org/10.2142/biophys.61.157
• [2]   DeLuca,  H. F.,  Engstrom,  G. W. Calcium uptake by rat kidney mitochondria. Proc. Natl. Acad. Sci. U.S.A. 47, 1744–1750 (1961). https://doi.org/10.1073/pnas.47.11.1744
• [3]   Gunter,  T. E.,  Pfeiffer,  D. R. Mechanisms by which mitochondria transport calcium. Am. J. Physiol. 258, C755–C786 (1990). https://doi.org/10.1152/ajpcell.1990.258.5.C755
• [4]   Kirichok,  Y.,  Krapivinsky,  G.,  Clapham,  D. E. The mitochondrial calcium uniporter is a highly selective ion channel. Nature 427, 360–364 (2004). https://doi.org/10.1038/nature02246
• [5]   Denton,  R. M.,  McCormack,  J. G. The role of calcium in the regulation of mitochondrial metabolism. Biochem. Soc. Trans. 8, 266–268 (1980). https://doi.org/10.1042/bst0080266
• [6]   Jouaville,  L. S.,  Ichas,  F.,  Holmuhamedov,  E. L.,  Camacho,  P.,  Lechleiter,  J. D. Synchronization of calcium waves by mitochondrial substrates in Xenopus laevis oocytes. Nature 377, 438–441 (1995). https://doi.org/10.1038/377438a0
• [7]   Duchen,  M. R. Mitochondria and calcium: From cell signalling to cell death. J. Physiol. 529, 57–68 (2000). https://doi.org/10.1111/j.1469-7793.2000.00057.x
• [8]   Perocchi,  F.,  Gohil,  V. M.,  Girgis,  H. S.,  Bao,  X. R.,  McCombs,  J. E.,  Palmer,  A. E., et al. MICU1 encodes a mitochondrial EF hand protein required for Ca²⁺ uptake. Nature 467, 291–296 (2010). https://doi.org/10.1038/nature09358
• [9]   Baughman,  J. M.,  Perocchi,  F.,  Girgis,  H. S.,  Plovanich,  M.,  Belcher-Timme,  C. A.,  Sancak,  Y., et al. Integrative genomics identifies MCU as an essential component of the mitochondrial calcium uniporter. Nature 476, 341–345 (2011). https://doi.org/10.1038/nature10234
• [10]   De Stefani,  D.,  Raffaello,  A.,  Teardo,  E.,  Szabó,  I.,  Rizzuto,  R. A forty-kilodalton protein of the inner membrane is the mitochondrial calcium uniporter. Nature 476, 336–340 (2011). https://doi.org/10.1038/nature10230
• [11]   Plovanich,  M.,  Bogorad,  R. L.,  Sancak,  Y.,  Kamer,  K. J.,  Strittmatter,  L.,  Li,  A. A., et al. MICU2, a paralog of MICU1, resides within the mitochondrial uniporter complex to regulate calcium handling. PLoS One 8, e55785 (2013). https://doi.org/10.1371/journal.pone.0055785
• [12]   Raffaello,  A.,  De Stefani,  D.,  Sabbadin,  D.,  Teardo,  E.,  Merli,  G.,  Picard,  A., et al. The mitochondrial calcium uniporter is a multimer that can include a dominant-negative pore-forming subunit. EMBO J. 32, 2362–2376 (2013). https://doi.org/10.1038/emboj.2013.157
• [13]   Mallilankaraman,  K.,  Cárdenas,  C.,  Doonan,  P. J.,  Chandramoorthy,  H. C.,  Irrinki,  K. M.,  Golenár,  T., et al. MCUR1 is an essential component of mitochondrial Ca²⁺ uptake that regulates cellular metabolism. Nat. Cell Biol. 14, 1336–1343 (2012). https://doi.org/10.1038/ncb2622
• [14]   Sancak,  Y.,  Markhard,  A. L.,  Kitami,  T.,  Kovács-Bogdán,  E.,  Kamer,  K. J.,  Udeshi,  N. D., et al. EMRE is an essential component of the mitochondrial calcium uniporter complex. Science 342, 1379–1382 (2013). https://doi.org/10.1126/science.1242993
• [15]   Logan,  C. V.,  Szabadkai,  G.,  Sharpe,  J. A.,  Parry,  D. A.,  Torelli,  S.,  Childs,  A. M., et al. Loss-of-function mutations in MICU1 cause a brain and muscle disorder linked to primary alterations in mitochondrial calcium signaling. Nat. Genet. 46, 188–193 (2014). https://doi.org/10.1038/ng.2851
• [16]   Yamamoto,  T.,  Yamagoshi,  R.,  Harada,  K.,  Kawano,  M.,  Minami,  N.,  Ido,  Y., et al. Analysis of the structure and function of EMRE in a yeast expression system. Biochim. Biophys. Acta Bioenerg. 1857, 831–839 (2016). https://doi.org/10.1016/j.bbabio.2016.03.019
• [17]   Yamamoto,  T.,  Ozono,  M.,  Watanabe,  A.,  Yamada,  A. The function of EMRE in mitochondrial calcium uptake system. Journal of Japanese Biochemical Society 89, 453–457 (2017). https://doi.org/10.14952/SEIKAGAKU.2017.890453
• [18]   Wang,  Y.,  Nguyen,  N. X.,  She,  J.,  Zeng,  W.,  Yang,  Y.,  Bai,  X., et al. Structural mechanism of EMRE-dependent gating of the human mitochondrial calcium uniporter. Cell 177, 1252–1261 (2019). https://doi.org/10.1016/j.cell.2019.03.050
• [19]   Bick,  A. G.,  Calvo,  S. E.,  Mootha,  V. K. Evolutionary diversity of the mitochondrial calcium uniporter. Science 336, 886 (2012). https://doi.org/10.1126/science.1214977
• [20]   Oxenoid,  K.,  Dong,  Y.,  Cao,  C.,  Cui,  T.,  Sancak,  Y.,  Markhard,  A. L., et al. Architecture of the mitochondrial calcium uniporter. Nature 533, 269–273 (2016). https://doi.org/10.1038/nature17656
• [21]   Yoo,  J.,  Wu,  M.,  Yin,  Y.,  Herzik,  M. A.,  Lander Jr.,  G. C.,  Lee,  S. Y. Cryo-EM structure of a mitochondrial calcium uniporter. Science 361, 506–511 (2018). https://doi.org/10.1126/science.aar4056
• [22]   Nguyen,  N. X.,  Armache,  J. P.,  Lee,  C.,  Yang,  Y.,  Zeng,  W.,  Mootha,  V. K., et al. Cryo-EM structure of a fungal mitochondrial calcium uniporter. Nature 559, 570–574 (2018). https://doi.org/10.1038/s41586-018-0333-6
• [23]   Fan,  C.,  Fan,  M.,  Orlando,  B. J.,  Fastman,  N. M.,  Zhang,  J.,  Xu,  Y., et al. X-ray and cryo-EM structures of the mitochondrial calcium uniporter. Nature 559, 575–579 (2018). https://doi.org/10.1038/s41586-018-0330-9
• [24]   Baradaran,  R.,  Wang,  C.,  Siliciano,  A. F.,  Long,  S. B. Cryo-EM structures of fungal and metazoan mitochondrial calcium uniporters. Nature 559, 580–584 (2018). https://doi.org/10.1038/s41586-018-0331-8
• [25]   Yamamoto,  T.,  Ozono,  M.,  Watanabe,  A.,  Maeda,  K.,  Nara,  A.,  Hashida,  M., et al. Functional analysis of coiled-coil domains of MCU in mitochondrial calcium uptake. Biochim. Biophys. Acta Bioenerg. 1860, 148061 (2019). https://doi.org/10.1016/j.bbabio.2019.148061
• [26]   Yamamoto,  T. The Molecular mechanisms of mitochondrial calcium uptake by calcium uniporter. YAKUGAKU ZASSHI 141, 491–499 (2021). https://doi.org/10.1248/yakushi.20-00204-1
• [27]   Arduino,  D.,  Wettmarshausen,  J.,  Vais,  H.,  Navas-Navarro,  P.,  Cheng,  Y.,  Leimpek,  A., et al. Systematic identification of MCU modulators by orthogonal interspecies chemical screening. Mol. Cell 67, 711–723 (2017). https://doi.org/10.1016/j.molcel.2017.07.019