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Mitochondrial pharmacology turns its sights on the Ca2+ uniporter

Mitochondrial pharmacology turns its sights on the Ca2+ uniporter DS16570511 Citation: Cell Death Discovery (2017) 3, 17064; doi:10.1038/cddiscovery.2017.64 OPEN Official journal of the Cell Death Differentiation Association www.nature.com/cddiscovery EDITORIAL 2+ Mitochondrial pharmacology turns its sights on the Ca uniporter Cell Death Discovery (2017) 3, 17064; doi:10.1038/cddiscovery.2017.64; Calcium uptake into mitochondria was first discovered in the 1960s by measuring radiolabelled calcium influx into isolated published online 25 September 2017 3,4 mitochondria from rat kidney homogenates. The biochemical research that followed took advantage of ruthenium red and lanthanum compounds which are potent inhibitors of calcium flux To use a well-known phrase, mitochondria are the ‘power house of through calcium selective pores. The problem with these the cell’ and produce ATP, the energetic currency of life. compounds is that they are not cell permeable and therefore Although this is a vital role, mitochondria also have many other useful only in studying isolated mitochondria or mitoplasts crucial roles including cell death pathways such as collapse of the (mitochondria without outer membranes). This has limited the mitochondrial membrane potential by opening the mitochondrial interpretation of the results to the level of the mitochondria rather permeability transition pore (mPTP). These roles are profoundly than the context of the cell or the whole organ or indeed the affected by the entry of calcium into the mitochondria. organism. Mitochondrial calcium increases the likelihood of mPTP opening, Exploration of the more cellular aspects of mitochondrial regulation of metabolic rate by activation of metabolic enzymes calcium uptake has been possible through use of aequorin, a (dehydrogenases), reactive oxygen species production, signalling calcium-sensitive luminescent protein from jellyfish that detects (Ca-calmodulin) and influencing the pattern of global calcium changes in calcium concentrations in bioluminescence assays. oscillations in response to receptor stimulation. Mitochondrially tagged aequorin has been exploited to report Cytoplasm Outer mitochondrial membrane Site of action of cell / mitochondrial Mitochondrial membrane permeable small molecule calcium uptake Intermembrane space drugs such as, DS16570511 protein 1 and 2 (MICU 1, 2) Mitochondrial calcium uniporter complex Inner mitochondrial membrane Mitochondrial matrix Mitochondrial calcium uniporter (MCU) Essential MCU regulator (EMRE) Mitochondrial calcium MCUb uniporter regulator (MCUR1) 2+ Ca Figure 1. The site of action of small molecule drug DS16570511. The cell and mitochondrial membrane permeable small molecule drug DS16570511 acts at the mitochondrial calcium uniporter complex. This is a schematic representation of the mitochondrial calcium uniporter complex because the cross talk and interaction sites for all the regulatory components of the complex are not fully understood. MICU1 was the first member of the complex to be found, it has calcium sensitive domain and is a regulatory subunit. Next the pore forming subunit, the mitochondrial calcium uniporter was found. Other members of the MCU complex are reviewed elsewhere. Editorial changes in intramitochondrial calcium concentrations and over block calcium entry rather than being a compound that collapses the years has been of enormous value in understanding the driving force for calcium entry. The advantage of using cell mitochondrial calcium uptake. In terms of overall calcium permeable drugs is that the fast-acting effects avoid the concentration, it is worth noting that mitochondria accumulate compensatory mechanisms that can often cloud interpretation orders of magnitude higher levels than free cytosolic calcium of knockout studies. Most exciting of all is the ability to examine concentrations and the driving force for this staggering calcium the effects of the rapidly acting drugs in cells and indeed in ex vivo accumulation is the mitochondrial membrane potential (−180 mV) organs and potentially in vivo. In the ex vivo Langendorff model, generated by the respiratory chain proton gradient. It has DS16570511 prevented calcium overload, which is usually emerged that the mitochondrial calcium uptake mechanism observed when hearts are perfused with a high calcium appeared to have a relatively low affinity for calcium which concentration solution. supports the idea that microdomains must reach much higher One caveat is that newly discovered drugs rarely remain truly local calcium concentrations for calcium uptake to occur. What specific for long as other effects at different sites gradually has also proved interesting is that at rest mitochondria do not become apparent. Ruthenium derivatives such as ruthenium red accumulate calcium and that uptake occurs after physiological and Ru360 have been potent and powerful tools but cell receptor stimulation, such as leukotriene receptor activation in permeable small molecules are a much better proposition for immune cells. research and potential therapeutics. Unfortunately, DS16570511 Almost 50 years after discovering that mitochondria accumulate has a relatively low potency compared with ruthenium red but calcium, a significant change in our understanding occurred from hopefully the emergence of small molecule drugs with a an ingenious combination of both proteomics and genomics potency that matches the ruthenium compounds is just around which revealed the identity of a mitochondrial calcium uniporter the corner. It is also unclear whether DS16570511 binds to MCU (MCU). This uniporter was found to be responsible for the or one of the multiple auxiliary subunits within the complex 7,8 radiolabelled calcium uptake observed half a century earlier. (Figure 1). In addition to not knowing the binding site of Finding the uniporter was achieved using the Mitocarta database. DS16570511, there is also no current indication of the molecular The database is a compilation of mouse genes that were found by mechanisms of its action. mass spectroscopy analysis of isolated mitochondria and Despite the unknowns there are opportunities here to learn from other proteins known to have a mitochondrial target more and it will be fascinating to see how this drug will affect sequence. This database was mined for candidate ruthenium red mPTP opening and cell death in heart ischaemia reperfusion injury sensitive mitochondrial calcium uptake proteins which appear in models. Depending where the inhibitor binds, it may also reveal the genome phylogenetically after yeast (which do not have the nature of cross-talk between MCU complex members and a MCU). molecular mechanisms of calcium uptake regulation. Perhaps, we This was an exciting time as it marked a shift from the can even look forward to understanding the nature of how the biochemical approaches into molecular approaches such as mircodomains are organised and cross-talk with mitochondrial silencing RNA, overexpression, dominant negatives and knockout associated membranes and other organelles such as the mice. There has indeed been an explosion of deeper under- endoplasmic reticulum. standing of MCU, including the emergence of many auxiliary regulatory proteins to form what is now known as the ‘mitochondrial uniporter complex’. This is perhaps no surprise to COMPETING INTERESTS those studying mitochondrial calcium uptake as the first protein The authors declare no conflict of interest. identified in the search was an integral member of the MCU complex, mitochondrial calcium uptake protein 1. It has calcium sensitive domain to detect calcium concentration changes, making it the gatekeeper of mitochondrial calcium 1 2 uptake but without any other characteristics of a uniporter or Nina M Storey and David G Lambert channel it was clearly not the protein responsible for calcium Department of Molecular and Cell Biology, University of Leicester, influx (Figure 1). Leicester LE1 9HN, UK and A number of drugs targeted to the mitochondria have been Department of Cardiovascular Sciences, University of Leicester, developed such as Mito Q an antioxidant acting to ‘mop up’ Division of Anaesthesia, Critical Care and Pain Management, reactive oxygen species and prevent damage. This may prove to Leicester Royal Infirmary, Leicester LE2 7LX, UK be highly beneficial in cardiovascular diseases. The paper Correspondence: NM Storey (ns140@le.ac.uk) described here may mark the dawn of the next era of research into mitochondrial calcium because it describes a novel, cell PUBLISHER’S NOTE permeable and specific mitochondrial calcium uptake inhibitor. Perhaps this will provide the next step change in our under- Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. standing of the function and role of MCU and its complex. Kon et al. have used high-throughput screening to identify a cell permeable drug DS16570511, which inhibits mitochondrial REFERENCES calcium entry not only in isolated mitochondria but also in cells 1 Szabadkai G, Duchen MR. Mitochondria: the hub of cellular Ca2+ signaling. Phy- and in the whole heart. Now the rapid effects of a cell permeable, siology (Bethesda) 2008; 23:84–94. fast-acting mitochondrial calcium uptake inhibitor can be 2 Kamer KJ, Mootha VK. The molecular era of the mitochondrial calcium uniporter. observed in cells and tissues with the potential to provide Nat Rev Mol Cell Biol 2015; 16: 545–553. invaluable insight into the roles and functioning of the MCU 3 Deluca HF, Engstrom GW. Calcium uptake by rat kidney mitochondria. Proc Natl complex. Acad Sci USA 1961; 47: 1744–1750. Kon et al. have screened a library to pull out compounds that 4 Vasington FD, Murphy JV. Ca ion uptake by rat kidney mitochondria and inhibit mitochondrial targeted aequorin luminescence but did not its dependence on respiration and phosphorylation. JBiol Chem 1962; 237: inhibit a cytosolic aequorin luminescence. This compound 2670–2677. inhibited mitochondrial calcium uptake while having no effect 5 Brand MD, Chen CH, Lehninger AL. Stoichiometry of H+ ejection during on mitochondrial membrane potential tested by the indicator respiration-dependent accumulation of Ca2+ by rat liver mitochondria. J Biol JC-10. This is good evidence that DS16570511 is more likely to Chem 1976; 251: 968–974. Cell Death Discovery (2017) 17064 Official journal of the Cell Death Differentiation Association Editorial 6 Samanta K, Douglas S, Parekh AB. Mitochondrial calcium uniporter MCU 11 Kon N, Murakoshi M, Isobe A, Kagechika K, Miyoshi N, Nagayama T. DS16570511 is a small-molecule inhibitor of the mitochondrial calcium uniporter. Cell Death supports cytoplasmic Ca2+ oscillations, store-operated Ca2+ entry and Ca2 Discov 2017; 3: 17045. +-dependent gene expression in response to receptor stimulation. PLoS One 2014; 9: e101188. 7 Baughman JM, Perocchi F, Girgis HS, Plovanich M, Belcher-Timme CA, Sancak Y This work is licensed under a Creative Commons Attribution 4.0 et al. Integrative genomics identifies MCU as an essential component of the International License. The images or other third party material in this mitochondrial calcium uniporter. Nature 2011; 476:341–345. article are included in the article’s Creative Commons license, unless indicated 8 De Stefani D, Raffaello A, Teardo E, Szabo I, Rizzuto R. A forty-kilodalton protein of otherwise in the credit line; if the material is not included under the Creative Commons the inner membrane is the mitochondrial calcium uniporter. Nature 2011; 476: license, users will need to obtain permission from the license holder to reproduce the 336–340. material. To view a copy of this license, visit http://creativecommons.org/licenses/ 9 Logan A, Murphy MP. Using chemical biology to assess and modulate mito- by/4.0/ chondria: progress and challenges. Interface Focus 2017; 7: 20160151. 10 Silva FS, Simoes RF, Couto R, Oliveira PJ. Targeting mitochondria in cardiovascular diseases. Curr Pharm Des 2016; 22:5698–5717. © The Author(s) 2017 Official journal of the Cell Death Differentiation Association Cell Death Discovery (2017) 17064 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Cell Death Discovery Springer Journals

Mitochondrial pharmacology turns its sights on the Ca2+ uniporter

Cell Death Discovery , Volume 3 (1) – Sep 25, 2017

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References (14)

Publisher
Springer Journals
Copyright
Copyright © 2017 by The Author(s)
Subject
Life Sciences; Life Sciences, general; Biochemistry, general; Cell Biology; Stem Cells; Apoptosis; Cell Cycle Analysis
eISSN
2058-7716
DOI
10.1038/cddiscovery.2017.64
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Abstract

DS16570511 Citation: Cell Death Discovery (2017) 3, 17064; doi:10.1038/cddiscovery.2017.64 OPEN Official journal of the Cell Death Differentiation Association www.nature.com/cddiscovery EDITORIAL 2+ Mitochondrial pharmacology turns its sights on the Ca uniporter Cell Death Discovery (2017) 3, 17064; doi:10.1038/cddiscovery.2017.64; Calcium uptake into mitochondria was first discovered in the 1960s by measuring radiolabelled calcium influx into isolated published online 25 September 2017 3,4 mitochondria from rat kidney homogenates. The biochemical research that followed took advantage of ruthenium red and lanthanum compounds which are potent inhibitors of calcium flux To use a well-known phrase, mitochondria are the ‘power house of through calcium selective pores. The problem with these the cell’ and produce ATP, the energetic currency of life. compounds is that they are not cell permeable and therefore Although this is a vital role, mitochondria also have many other useful only in studying isolated mitochondria or mitoplasts crucial roles including cell death pathways such as collapse of the (mitochondria without outer membranes). This has limited the mitochondrial membrane potential by opening the mitochondrial interpretation of the results to the level of the mitochondria rather permeability transition pore (mPTP). These roles are profoundly than the context of the cell or the whole organ or indeed the affected by the entry of calcium into the mitochondria. organism. Mitochondrial calcium increases the likelihood of mPTP opening, Exploration of the more cellular aspects of mitochondrial regulation of metabolic rate by activation of metabolic enzymes calcium uptake has been possible through use of aequorin, a (dehydrogenases), reactive oxygen species production, signalling calcium-sensitive luminescent protein from jellyfish that detects (Ca-calmodulin) and influencing the pattern of global calcium changes in calcium concentrations in bioluminescence assays. oscillations in response to receptor stimulation. Mitochondrially tagged aequorin has been exploited to report Cytoplasm Outer mitochondrial membrane Site of action of cell / mitochondrial Mitochondrial membrane permeable small molecule calcium uptake Intermembrane space drugs such as, DS16570511 protein 1 and 2 (MICU 1, 2) Mitochondrial calcium uniporter complex Inner mitochondrial membrane Mitochondrial matrix Mitochondrial calcium uniporter (MCU) Essential MCU regulator (EMRE) Mitochondrial calcium MCUb uniporter regulator (MCUR1) 2+ Ca Figure 1. The site of action of small molecule drug DS16570511. The cell and mitochondrial membrane permeable small molecule drug DS16570511 acts at the mitochondrial calcium uniporter complex. This is a schematic representation of the mitochondrial calcium uniporter complex because the cross talk and interaction sites for all the regulatory components of the complex are not fully understood. MICU1 was the first member of the complex to be found, it has calcium sensitive domain and is a regulatory subunit. Next the pore forming subunit, the mitochondrial calcium uniporter was found. Other members of the MCU complex are reviewed elsewhere. Editorial changes in intramitochondrial calcium concentrations and over block calcium entry rather than being a compound that collapses the years has been of enormous value in understanding the driving force for calcium entry. The advantage of using cell mitochondrial calcium uptake. In terms of overall calcium permeable drugs is that the fast-acting effects avoid the concentration, it is worth noting that mitochondria accumulate compensatory mechanisms that can often cloud interpretation orders of magnitude higher levels than free cytosolic calcium of knockout studies. Most exciting of all is the ability to examine concentrations and the driving force for this staggering calcium the effects of the rapidly acting drugs in cells and indeed in ex vivo accumulation is the mitochondrial membrane potential (−180 mV) organs and potentially in vivo. In the ex vivo Langendorff model, generated by the respiratory chain proton gradient. It has DS16570511 prevented calcium overload, which is usually emerged that the mitochondrial calcium uptake mechanism observed when hearts are perfused with a high calcium appeared to have a relatively low affinity for calcium which concentration solution. supports the idea that microdomains must reach much higher One caveat is that newly discovered drugs rarely remain truly local calcium concentrations for calcium uptake to occur. What specific for long as other effects at different sites gradually has also proved interesting is that at rest mitochondria do not become apparent. Ruthenium derivatives such as ruthenium red accumulate calcium and that uptake occurs after physiological and Ru360 have been potent and powerful tools but cell receptor stimulation, such as leukotriene receptor activation in permeable small molecules are a much better proposition for immune cells. research and potential therapeutics. Unfortunately, DS16570511 Almost 50 years after discovering that mitochondria accumulate has a relatively low potency compared with ruthenium red but calcium, a significant change in our understanding occurred from hopefully the emergence of small molecule drugs with a an ingenious combination of both proteomics and genomics potency that matches the ruthenium compounds is just around which revealed the identity of a mitochondrial calcium uniporter the corner. It is also unclear whether DS16570511 binds to MCU (MCU). This uniporter was found to be responsible for the or one of the multiple auxiliary subunits within the complex 7,8 radiolabelled calcium uptake observed half a century earlier. (Figure 1). In addition to not knowing the binding site of Finding the uniporter was achieved using the Mitocarta database. DS16570511, there is also no current indication of the molecular The database is a compilation of mouse genes that were found by mechanisms of its action. mass spectroscopy analysis of isolated mitochondria and Despite the unknowns there are opportunities here to learn from other proteins known to have a mitochondrial target more and it will be fascinating to see how this drug will affect sequence. This database was mined for candidate ruthenium red mPTP opening and cell death in heart ischaemia reperfusion injury sensitive mitochondrial calcium uptake proteins which appear in models. Depending where the inhibitor binds, it may also reveal the genome phylogenetically after yeast (which do not have the nature of cross-talk between MCU complex members and a MCU). molecular mechanisms of calcium uptake regulation. Perhaps, we This was an exciting time as it marked a shift from the can even look forward to understanding the nature of how the biochemical approaches into molecular approaches such as mircodomains are organised and cross-talk with mitochondrial silencing RNA, overexpression, dominant negatives and knockout associated membranes and other organelles such as the mice. There has indeed been an explosion of deeper under- endoplasmic reticulum. standing of MCU, including the emergence of many auxiliary regulatory proteins to form what is now known as the ‘mitochondrial uniporter complex’. This is perhaps no surprise to COMPETING INTERESTS those studying mitochondrial calcium uptake as the first protein The authors declare no conflict of interest. identified in the search was an integral member of the MCU complex, mitochondrial calcium uptake protein 1. It has calcium sensitive domain to detect calcium concentration changes, making it the gatekeeper of mitochondrial calcium 1 2 uptake but without any other characteristics of a uniporter or Nina M Storey and David G Lambert channel it was clearly not the protein responsible for calcium Department of Molecular and Cell Biology, University of Leicester, influx (Figure 1). Leicester LE1 9HN, UK and A number of drugs targeted to the mitochondria have been Department of Cardiovascular Sciences, University of Leicester, developed such as Mito Q an antioxidant acting to ‘mop up’ Division of Anaesthesia, Critical Care and Pain Management, reactive oxygen species and prevent damage. This may prove to Leicester Royal Infirmary, Leicester LE2 7LX, UK be highly beneficial in cardiovascular diseases. The paper Correspondence: NM Storey (ns140@le.ac.uk) described here may mark the dawn of the next era of research into mitochondrial calcium because it describes a novel, cell PUBLISHER’S NOTE permeable and specific mitochondrial calcium uptake inhibitor. Perhaps this will provide the next step change in our under- Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. standing of the function and role of MCU and its complex. Kon et al. have used high-throughput screening to identify a cell permeable drug DS16570511, which inhibits mitochondrial REFERENCES calcium entry not only in isolated mitochondria but also in cells 1 Szabadkai G, Duchen MR. Mitochondria: the hub of cellular Ca2+ signaling. Phy- and in the whole heart. Now the rapid effects of a cell permeable, siology (Bethesda) 2008; 23:84–94. fast-acting mitochondrial calcium uptake inhibitor can be 2 Kamer KJ, Mootha VK. The molecular era of the mitochondrial calcium uniporter. observed in cells and tissues with the potential to provide Nat Rev Mol Cell Biol 2015; 16: 545–553. invaluable insight into the roles and functioning of the MCU 3 Deluca HF, Engstrom GW. Calcium uptake by rat kidney mitochondria. Proc Natl complex. Acad Sci USA 1961; 47: 1744–1750. Kon et al. have screened a library to pull out compounds that 4 Vasington FD, Murphy JV. Ca ion uptake by rat kidney mitochondria and inhibit mitochondrial targeted aequorin luminescence but did not its dependence on respiration and phosphorylation. JBiol Chem 1962; 237: inhibit a cytosolic aequorin luminescence. This compound 2670–2677. inhibited mitochondrial calcium uptake while having no effect 5 Brand MD, Chen CH, Lehninger AL. Stoichiometry of H+ ejection during on mitochondrial membrane potential tested by the indicator respiration-dependent accumulation of Ca2+ by rat liver mitochondria. J Biol JC-10. This is good evidence that DS16570511 is more likely to Chem 1976; 251: 968–974. Cell Death Discovery (2017) 17064 Official journal of the Cell Death Differentiation Association Editorial 6 Samanta K, Douglas S, Parekh AB. Mitochondrial calcium uniporter MCU 11 Kon N, Murakoshi M, Isobe A, Kagechika K, Miyoshi N, Nagayama T. DS16570511 is a small-molecule inhibitor of the mitochondrial calcium uniporter. Cell Death supports cytoplasmic Ca2+ oscillations, store-operated Ca2+ entry and Ca2 Discov 2017; 3: 17045. +-dependent gene expression in response to receptor stimulation. PLoS One 2014; 9: e101188. 7 Baughman JM, Perocchi F, Girgis HS, Plovanich M, Belcher-Timme CA, Sancak Y This work is licensed under a Creative Commons Attribution 4.0 et al. Integrative genomics identifies MCU as an essential component of the International License. The images or other third party material in this mitochondrial calcium uniporter. Nature 2011; 476:341–345. article are included in the article’s Creative Commons license, unless indicated 8 De Stefani D, Raffaello A, Teardo E, Szabo I, Rizzuto R. A forty-kilodalton protein of otherwise in the credit line; if the material is not included under the Creative Commons the inner membrane is the mitochondrial calcium uniporter. Nature 2011; 476: license, users will need to obtain permission from the license holder to reproduce the 336–340. material. To view a copy of this license, visit http://creativecommons.org/licenses/ 9 Logan A, Murphy MP. Using chemical biology to assess and modulate mito- by/4.0/ chondria: progress and challenges. Interface Focus 2017; 7: 20160151. 10 Silva FS, Simoes RF, Couto R, Oliveira PJ. Targeting mitochondria in cardiovascular diseases. Curr Pharm Des 2016; 22:5698–5717. © The Author(s) 2017 Official journal of the Cell Death Differentiation Association Cell Death Discovery (2017) 17064

Journal

Cell Death DiscoverySpringer Journals

Published: Sep 25, 2017

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