Structural insights into the Ca2+-dependent gating of the human mitochondrial calcium uniporter.
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ABSTRACT:
SUBMITTER: Wang Y
PROVIDER: S-EPMC7442490 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Mitochondrial calcium uptake is crucial to the regulation of eukaryotic Ca2+ homeostasis and is mediated by the mitochondrial calcium uniporter (MCU). While MCU alone can transport Ca2+ in primitive eukaryotes, metazoans require an essential single membrane-spanning auxiliary component called EMRE to form functional channels; however, the molecular mechanism of EMRE regulation remains elusive. Here, we present the cryo-EM structure of the human MCU-EMRE complex, which defines the interactions between MCU and EMRE as well as pinpoints the juxtamembrane loop of MCU and extended linker of EMRE as the crucial elements in the EMRE-dependent gating mechanism among metazoan MCUs. The structure also features the dimerization of two MCU-EMRE complexes along an interface at the N-terminal domain (NTD) of human MCU that is a hotspot for post-translational modifications. Thus, the human MCU-EMRE complex, which constitutes the minimal channel components among metazoans, provides a framework for future mechanistic studies on MCU.
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Project description:Calcium (Ca(2+)) flux into the matrix is tightly controlled by the mitochondrial Ca(2+) uniporter (MCU) due to vital roles in cell death and bioenergetics. However, the precise atomic mechanisms of MCU regulation remain unclear. Here, we solved the crystal structure of the N-terminal matrix domain of human MCU, revealing a β-grasp-like fold with a cluster of negatively charged residues that interacts with divalent cations. Binding of Ca(2+) or Mg(2+) destabilizes and shifts the self-association equilibrium of the domain toward monomer. Mutational disruption of the acidic face weakens oligomerization of the isolated matrix domain and full-length human protein similar to cation binding and markedly decreases MCU activity. Moreover, mitochondrial Mg(2+) loading or blockade of mitochondrial Ca(2+) extrusion suppresses MCU Ca(2+)-uptake rates. Collectively, our data reveal that the β-grasp-like matrix region harbors an MCU-regulating acidic patch that inhibits human MCU activity in response to Mg(2+) and Ca(2+) binding.
| S-EPMC5035232 | biostudies-literature
Project description:AimsMitochondrial Ca2+ homeostasis is crucial for balancing cell survival and death. The recent discovery of the molecular identity of the mitochondrial Ca2+ uniporter pore (MCU) opens new possibilities for applying genetic approaches to study mitochondrial Ca2+ regulation in various cell types, including cardiac myocytes. Basal tyrosine phosphorylation of MCU was reported from mass spectroscopy of human and mouse tissues, but the signaling pathways that regulate mitochondrial Ca2+ entry through posttranslational modifications of MCU are completely unknown. Therefore, we investigated α1-adrenergic-mediated signal transduction of MCU posttranslational modification and function in cardiac cells.Resultsα1-adrenoceptor (α1-AR) signaling translocated activated proline-rich tyrosine kinase 2 (Pyk2) from the cytosol to mitochondrial matrix and accelerates mitochondrial Ca2+ uptake via Pyk2-dependent MCU phosphorylation and tetrametric MCU channel pore formation. Moreover, we found that α1-AR stimulation increases reactive oxygen species production at mitochondria, mitochondrial permeability transition pore activity, and initiates apoptotic signaling via Pyk2-dependent MCU activation and mitochondrial Ca2+ overload.InnovationOur data indicate that inhibition of α1-AR-Pyk2-MCU signaling represents a potential novel therapeutic target to limit or prevent mitochondrial Ca2+ overload, oxidative stress, mitochondrial injury, and myocardial death during pathophysiological conditions, where chronic adrenergic stimulation is present.ConclusionThe α1-AR-Pyk2-dependent tyrosine phosphorylation of the MCU regulates mitochondrial Ca2+ entry and apoptosis in cardiac cells.
| S-EPMC4116095 | biostudies-literature
Project description:Cardiomyocyte Ca2+ is buffered by mitochondria via the mitochondrial calcium uniporter (MCU) complex. The MCU complex consists of pore-forming proteins including the mitochondrial calcium uniporter (MCU), and regulatory proteins such as mitochondrial calcium uptake proteins 1 and 2 (MICU1/2). The stoichiometry of these proteins influences the sensitivity to Ca2+ and activity of the complex. However, the factors that regulate their gene expression remain incompletely understood. Long non-coding RNAs (lncRNAs) regulate gene expression through various mechanisms, and we recently found that the lncRNA Tug1 affected the expression of MCU-associated genes. To further explore this, we knocked down Tug1 (Tug1 KD) in H9c2 rat cardiomyocytes using antisense LNA oligo. This led to increased MCU protein expression yet this did not enhance a marker of mitochondrial Ca2+ uptake. RNA-seq revealed that Tug1 KD increased Mcu and led to differential expression of genes and pathways related to Ca2+ regulation in the heart. To understand the effect of this on Ca2+ signalling, we measured phosphorylation of Ca2+/calmodulin-dependent protein kinase II (CaMKII) and its downstream target cAMP Response Element-Binding protein (CREB), a transcription factor known to promote Mcu gene expression. Tug1 KD attenuated the increase in CAMKII and CREB phosphorylation in response to ionomycin, a Ca2+ ionophore. Inhibition of CaMKII, but not CREB, partially prevented the Tug1 KD mediated increase in Mcu. Together, these data suggest that Tug1 modulates MCU expression via a mechanism that may involve CAMKII and CREB. The Tug1 mediated regulation of MCU on mitochondrial Ca2+ uptake, may have functional consequences for cellular Ca2+ handling which could have implications for cardiac disease.
2023-07-22 | GSE237787 | GEO
Project description:The mitochondrial calcium uniporter (MCU) plays essential roles in mitochondrial calcium homeostasis and regulates cellular functions, such as energy synthesis, cell growth, and development. Thus, MCU activity is tightly controlled by its regulators as well as post-translational modification, including phosphorylation by protein kinases such as proline-rich tyrosine kinase 2 (Pyk2) and AMP-activated protein kinase (AMPK). In our in vitro kinase assay, the MCU N-terminal domain (NTD) was phosphorylated by protein kinase C isoforms (PKCβII, PKCδ, and PKCε) localized in the mitochondrial matrix. In addition, we found the conserved S92 was phosphorylated by the PKC isoforms. To reveal the structural effect of MCU S92 phosphorylation (S92p), we determined crystal structures of the MCU NTD of S92E and D119A mutants and analysed the molecular dynamics simulation of WT and S92p. We observed conformational changes of the conserved loop2-loop4 (L2-L4 loops) in MCU NTDS92E, NTDD119A, and NTDS92p due to the breakage of the S92-D119 hydrogen bond. The results suggest that the phosphorylation of S92 induces conformational changes as well as enhancements of the negative charges at the L2-L4 loops, which may affect the dimerization of two MCU-EMRE tetramers.
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Project description:Rods and cones use intracellular Ca2+ to regulate many functions, including phototransduction and neurotransmission. The Mitochondrial Calcium Uniporter (MCU) complex is thought to be the primary pathway for Ca2+ entry into mitochondria in eukaryotes. We investigate the hypothesis that mitochondrial Ca2+ uptake via MCU influences phototransduction and energy metabolism in photoreceptors using a mcu-/- zebrafish and a rod photoreceptor-specific Mcu-/- mouse. Using genetically encoded Ca2+ sensors to directly examine Ca2+ uptake in zebrafish cone mitochondria, we found that loss of MCU reduces but does not eliminate mitochondrial Ca2+ uptake. Loss of MCU does not lead to photoreceptor degeneration, mildly affects mitochondrial metabolism, and does not alter physiological responses to light, even in the absence of the Na+/Ca2+, K+ exchanger. Our results reveal that MCU is dispensable for vertebrate photoreceptor function, consistent with its low expression and the presence of an alternative pathway for Ca2+ uptake into photoreceptor mitochondria.
| S-EPMC7525533 | biostudies-literature
Project description:Ca2+ entry into mitochondria is through the mitochondrial calcium uniporter complex (MCUcx), a Ca2+-selective channel composed of five subunit types. Two MCUcx subunits (MCU and EMRE) span the inner mitochondrial membrane, while three Ca2+-regulatory subunits (MICU1, MICU2, and MICU3) reside in the intermembrane space. Here, we provide rigorous analysis of Ca2+ and Na+ fluxes via MCUcx in intact isolated mitochondria to understand the function of MICU subunits. We also perform direct patch clamp recordings of macroscopic and single MCUcx currents to gain further mechanistic insights. This comprehensive analysis shows that the MCUcx pore, composed of the EMRE and MCU subunits, is not occluded nor plugged by MICUs during the absence or presence of extramitochondrial Ca2+ as has been widely reported. Instead, MICUs potentiate activity of MCUcx as extramitochondrial Ca2+ is elevated. MICUs achieve this by modifying the gating properties of MCUcx allowing it to spend more time in the open state.
| S-EPMC8437439 | biostudies-literature
Project description:The mitochondrial calcium uniporter is a multi-subunit Ca2+-activated Ca2+ channel, made up of the pore-forming MCU protein, a metazoan-specific EMRE subunit, and MICU1/MICU2, which mediate Ca2+ activation. It has been established that metazoan MCU requires EMRE binding to conduct Ca2+, but how EMRE promotes MCU opening remains unclear. Here, we demonstrate that EMRE controls MCU activity via its transmembrane helix, while using an N-terminal PKP motif to strengthen binding with MCU. Opening of MCU requires hydrophobic interactions mediated by MCU residues near the pore's luminal end. Enhancing these interactions by single mutation allows human MCU to transport Ca2+ without EMRE. We further show that EMRE may facilitate MCU opening by stabilizing the open state in a conserved MCU gating mechanism, present also in non-metazoan MCU homologs. These results provide insights into the evolution of the uniporter machinery and elucidate the mechanism underlying the physiologically crucial EMRE-dependent MCU activation process.
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Project description:Mitochondria from many eukaryotic clades take up large amounts of calcium (Ca(2+)) via an inner membrane transporter called the uniporter. Transport by the uniporter is membrane potential dependent and sensitive to ruthenium red or its derivative Ru360 (ref. 1). Electrophysiological studies have shown that the uniporter is an ion channel with remarkably high conductance and selectivity. Ca(2+) entry into mitochondria is also known to activate the tricarboxylic acid cycle and seems to be crucial for matching the production of ATP in mitochondria with its cytosolic demand. Mitochondrial calcium uniporter (MCU) is the pore-forming and Ca(2+)-conducting subunit of the uniporter holocomplex, but its primary sequence does not resemble any calcium channel studied to date. Here we report the structure of the pore domain of MCU from Caenorhabditis elegans, determined using nuclear magnetic resonance (NMR) and electron microscopy (EM). MCU is a homo-oligomer in which the second transmembrane helix forms a hydrophilic pore across the membrane. The channel assembly represents a new solution of ion channel architecture, and is stabilized by a coiled-coil motif protruding into the mitochondrial matrix. The critical DXXE motif forms the pore entrance, which features two carboxylate rings; based on the ring dimensions and functional mutagenesis, these rings appear to form the selectivity filter. To our knowledge, this is one of the largest membrane protein structures characterized by NMR, and provides a structural blueprint for understanding the function of this channel.
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Project description:Mitochondria play an essential role in maintaining intraneuronal calcium homeostasis. Mitochondrial calcium uniporter (MCU) is a determined major brain mitochondrial calcium entry pathway. Activated MCU-mediated mitochondrial calcium overloading has been linked with brain mitochondrial pathology in disease conditions. Cyclophilin D (CypD)-mediated mitochondrial permeability transition (mPT) favors mitochondrial calcium efflux. The physiological function of CypD-mediated mPT has received increasing recognition. However, the regulatory role of CypD-mediated mPT in brain mitochondrial calcium dynamics in response to mitochondrial calcium accumulation via MCU has not been comprehensively studied. Here, by adopting purified brain mitochondria, we have determined an effect of CypD and CypD-mediated mPT against mitochondrial calcium overloading. In addition, blockade of CypD pharmaceutically or genetically blunts brain mitochondrial MCU's sensitivity to its inhibitor. Therefore, our findings suggest that CypD-mediated mPT is a mitochondrial compensatory response to MCU-mediated excess mitochondrial calcium accumulation. Moreover, CypD may potentially modulate MCU function in calcium-stressed mitochondria.
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