Mitochondrial calcium homeostasis in endothelial cells

For a long time, mitochondria were thought to serve as passive Ca2+ sinks that accumulate Ca2+ along the organelle`s negative membrane potential. This paradigm has changed radically. Nowadays, mitochondria are known to specifically respond to environmental Ca2+ and to contribute actively to the sophisticated regulation of the spatial and temporal patterns of intracellular Ca2+ signaling. Accordingly, our recent work describes mitochondria to be essential for the maintenance of capacitative Ca2+ entry, the accomplishment of Ca2+ refilling of the endoplasmic reticulum and Ca2+-dependent protein folding. Although these findings foster our understanding of the physiological role of mitochondria in Ca2+ signaling, the actual proteins involved in mitochondrial Ca2+ homeostasis, the molecular mechanisms of its regulation and the interdependency with other ions are largely unknown. In our previous grant, we demonstrated that two isoforms of the uncoupling protein family, UCP2 and UCP3, are essential for mitochondrial Ca2+ uniport (Trenker et al. Nat. Cell Biol. in press). However, each protein alone failed to accomplish Ca2+ fluxes in a heterologous environment, thus, pointing to additional proteins that might be essential to assemble the mitochondrial Ca2+ uniporter. Accordingly, in work package A, we will explore UCP2 and UCP3 as fundamental components of the mitochondrial Ca2+ uniporter. In particular, utilizing UCP2/UCP3 as bait the protein composition of the assumed signalplex that forms the mitochondrial Ca2+ uniporter, the functional interaction of UCP2- and UCP3-containing signalplexes with intracellular sites of Ca2+ release/uptake and Ca2+ entry and the importance of existing phosphorylation sites for UCP2/UCP3 function and activity will be explored. Our recent findings on the contribution of UCP2/UCP3 on mitochondrial Ca2+ uptake apparently stands, against the common hypothesis on the physiological uncoupling function of these proteins. Consequently, work package C is designed to provide a critical in-depth evaluation whether the fundamental contribution of UCP2/UCP3 to MCU can represent the molecular mechanism behind already reported phenomena by correlating their impact on mitochondrial Ca2+ uptake with mitochondrial oxidative phosphorylation and ROS generation. Another important aspect of mitochondrial Ca2+ signaling is its interrelation with the cellular Na+ homeostasis. Mitochondrial permeability for these ions is strongly associated with mitochondrial Ca2+ homeostasis and the initiation of endothelial Ca2+ signaling is accompanied with activation of Na+ -permeable ion channels or antiporters in the plasma membrane. Moreover, Na+ dependent Ca2+ efflux from mitochondria strictly depends on SERCA activity. Consequently, work package C is designated to explore the importance of Na+ -permeable plasma membrane ion channels and NCXpm and SERCA activity for mitochondrial Ca2+ homeostasis in endothelial cells. The project outcome will reveal molecular insights of mitochondrial Ca2+ homeostasis and principles of its regulation. Furthermore the recently discovered "Ca 2+ function" of UCP2 and UCP3 will be critical evaluated in comparison to already existing data regarding these UCP isoforms. Despite this grant focuses on mitochondrial Ca2+ homoestasis in endothelial cells as well defined test models, the research outcome will have important implications for Ca2+ handling in other cell types as well. This aspect deserves to be explored subsequently to the molecular assessment implemented in this project.

This project aimed to investigate the molecular mechanisms of mitochondrial Ca2+ uptake in intact cells. The rational of this project builds on our previous findings that the novel uncoupling proteins (UCP) 2 and 3 are fundamentally involved in mitochondrial Ca2+ sequestration in endothelial cells. Notably, these finding was the first report regarding the molecular identification of a contributor to mitochondrial Ca2+ uptake, a mechanism that is of utmost importance for regulating most mitochondrial functions. The project consisted of three work packages that were elaborated according to the proposal. In work package A, the molecular composition of UCP2/3-dependent mitochondrial Ca2+ uptake machinery was studied. For this purpose a high-end proteomics approach was successfully utilized that revealed ten novel interaction partners for mitochondrial UCP2 in human cells. Subsequently the identification of these proteins by mass spectrometry, functional evaluations on their contribution to mitochondrial Ca2+ sequestration were launched and are still under investigation. Notably, already one candidate protein was found to be essential for UCP2/3-dependent mitochondrial Ca2+ uptake. This work includes siRNA-mediated knock-down, rescue experiments and the use of protein-specific knock-out cells. Furthermore, we could characterize small molecular inhibitors of the UCP2/3- dependent mitochondrial Ca2+ uptake. These compounds are currently tested regarding their specificity and may serve as leading compounds for the design of a new class of drugs against mitochondria-related disease. In work package B, the structure - function relationship of UCP3 was investigated by applying tools of bioinformatics for identification of essential domains for the protein`s Ca2+ function. Notably, two distinct domains in the intermembrane loop 2 of UCP3 were found to be important for the Ca2+ sensitivity to facilitate Ca2+ uptake into the mitochondria. Hence, these two Ca2+ sensitivities correlated with the properties of the UCP2/3-dependent mitochondrial uptake of high (i.e. intracellularly released) and low (i.e. via the store-operated pathway entering) Ca2+. In course of this work, two additional contributors to mitochondrial Ca2+ uptake, Letm1 and MICU1 were investigated. Our data indicate that these proteins are essential for distinct mitochondrial Ca2+ uptake routes that may coexist or be established specifically depending on the cell type. In work package C, the impact of Na+ on mitochondrial homeostasis was investigated and revealed a crucial importance of mitochondrial Na+ /Ca 2+ exchanger under conditions of excessive Ca2+ entry. Overall, this project achieved significant progress in the molecular and functional understanding of mitochondrial Ca2+ uptake. These data may serve as basis for further investigations and development of a new class of drugs that are targeted to mitochondrial Ca2+ uptake, a strategy that might be suitable to treat mitochondria-related diseases, such as neurodegenerative diseases, (cardio-)myopathies or vascular dysfunction.