Metabolic adaptation to the chronic loss of Ca2+ signaling induced by KO of IP3 receptors or the mitochondrial Ca2+ uniporter

Calcium signaling is essential for regulating many biological processes. Endoplasmic reticulum inositol trisphosphate receptors (IP₃Rs) and the mitochondrial Ca²⁺ uniporter (MCU) are key proteins that regulate intracellular Ca²⁺ concentration. Mitochondrial Ca²⁺ accumulation activates Ca²⁺-sensitive dehydrogenases of the tricarboxylic acid (TCA) cycle that maintain the biosynthetic and bioenergetic needs of both normal and cancer cells. However, the interplay between calcium signaling and metabolism is not well understood. In this study, we used human cancer cell lines (HEK293 and HeLa) with stable KOs of all three IP₃R isoforms (triple KO [TKO]) or MCU to examine metabolic and bioenergetic responses to the chronic loss of cytosolic and/or mitochondrial Ca²⁺ signaling. Our results show that TKO cells (exhibiting total loss of Ca²⁺ signaling) are viable, displaying a lower proliferation and oxygen consumption rate, with no significant changes in ATP levels, even when made to rely solely on the TCA cycle for energy production. MCU KO cells also maintained normal ATP levels but showed increased proliferation, oxygen consumption, and metabolism of both glucose and glutamine. However, MCU KO cells were unable to maintain ATP levels and died when relying solely on the TCA cycle for energy. We conclude that constitutive Ca²⁺ signaling is dispensable for the bioenergetic needs of both IP₃R TKO and MCU KO human cancer cells, likely because of adequate basal glycolytic and TCA cycle flux. However, in MCU KO cells, the higher energy expenditure associated with increased proliferation and oxygen consumption makes these cells more prone to bioenergetic failure under conditions of metabolic stress.     

Regulation of Ca2+ fluxes in rat liver mitochondria by Ca2+. Effects on Ca2+ distribution

Rat liver mitochondria are able to temporarily lower the steady-state concentration of external Ca²⁺ after having accumulated a pulse of added Ca²⁺. This has been attributed to inhibition of a putative -modulated efflux pathway [Bernardi, P. (1984)Biochim. Biophys. Acta 766, 277–282]. On the other hand, the rebounding could be due to stimulation of the uniporter by Ca²⁺ [Kröner, H. (1987)Biol. Chem. Hoppe-Seyler 369, 149–155]. By measuring unidirectional Ca²⁺ fluxes, it was found that the uniporter was stimulated during the rebounding peak both under Bernardi's and Kröner's conditions, while no effects on the efflux could be demonstrated. The rate of unidirectional efflux of Ca²⁺ was not affected by inhibition of the uniporter. It appears likely that the rebounding is due to stimulation of the uniporter rather than inhibition of efflux.     

Tissue-Specific Mitochondrial Decoding of Cytoplasmic Ca2+ Signals Is Controlled by the Stoichiometry of MICU1/2 and MCU

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Essential Role of Mitochondrial Ca2+ Uniporter in the Generation of Mitochondrial pH Gradient and Metabolism-Secretion Coupling in Insulin-releasing Cells

In pancreatic β-cells, ATP acts as a signaling molecule initiating plasma membrane electrical activity linked to Ca²⁺ influx, which triggers insulin exocytosis. The mitochondrial Ca²⁺ uniporter (MCU) mediates Ca²⁺ uptake into the organelle, where energy metabolism is further stimulated for sustained second phase insulin secretion. Here, we have studied the contribution of the MCU to the regulation of oxidative phosphorylation and metabolism-secretion coupling in intact and permeabilized clonal β-cells as well as rat pancreatic islets. Knockdown of MCU with siRNA transfection blunted matrix Ca²⁺ rises, decreased nutrient-stimulated ATP production as well as insulin secretion. Furthermore, MCU knockdown lowered the expression of respiratory chain complexes, mitochondrial metabolic activity, and oxygen consumption. The pH gradient formed across the inner mitochondrial membrane following nutrient stimulation was markedly lowered in MCU-silenced cells. In contrast, nutrient-induced hyperpolarization of the electrical gradient was not altered. In permeabilized cells, knockdown of MCU ablated matrix acidification in response to extramitochondrial Ca²⁺. Suppression of the putative Ca²⁺/H⁺ antiporter leucine zipper-EF hand-containing transmembrane protein 1 (LETM1) also abolished Ca²⁺-induced matrix acidification. These results demonstrate that MCU-mediated Ca²⁺ uptake is essential to establish a nutrient-induced mitochondrial pH gradient which is critical for sustained ATP synthesis and metabolism-secretion coupling in insulin-releasing cells.     

Advances in the Purification of the Mitochondrial Ca2+ Uniporter Using the Labeled Inhibitor 103Ru360

For many years the calcium uniporter has eluded attempts of purification, partly because of the difficulties inherent in the purification of low-abundance hydrophobic proteins (Reed and Bygrave, 1974). Liquid-phase preparative isoelectric focusing improved the fractionation of mitochondrial membrane proteins. A single 6-h run resulted in a 90-fold increase in specific activity of pooled active fractions over a semipurified fraction, allowing for enrichment of the calcium transport function in cytochrome oxidase vesicles. An additional powerful tool in the isolation of the uniporter was the use of the labeled inhibitor ¹⁰³Ru₃₆₀ as an affinity ligand; by following this procedure a protein of 18 kDa was purified in nondenatured, but rather inactive, form. The labeled protein corresponds to the protein that showed Ca²⁺ transport activity.     

Characterization of Na+-Dependent Phosphate Uptake in Cultured Fetal Rat Cortical Neurons

Abstract: Our laboratory has recently cloned and expressed a brain- and neuron-specific Na⁺-dependent inorganic phosphate (P_i) cotransporter that is constitutively expressed in neurons of the rat cerebral cortex, hippocampus, and cerebellum. We have now characterized Na⁺-dependent ³²P_i cotransport in cultured fetal rat cortical neurons, where >90% of saturable P_i uptake is Na⁺-dependent. Saturable, Na⁺-dependent ³²P_i uptake was first observed in primary cultures of cortical neurons at 7 days in vitro (DIV) and was maximal at 12 DIV. Na⁺-dependent P_i transport was optimal at physiological temperature (37°C) and pH (7.0–7.5), with apparent K_m values for P_i and Na⁺ of 54 ± 12.7 µM and 35 ± 4.2 mM, respectively. A reduction in extracellular Ca²⁺ markedly reduced (>60%) Na⁺-dependent P_i uptake, with a threshold for maximal P_i import of 1–2.5 mM CaCl₂. Primary cultures of fetal cortical neurons incubated in medium where equimolar concentrations of choline were substituted for Na⁺ had lower levels of ATP and ADP and higher levels of AMP than did those incubated in the presence of Na⁺. Furthermore, a substantial fraction of the ³²P_i cotransported with Na⁺ was concentrated in the adenine nucleotides. Inhibitors of oxidative metabolism, such as rotenone, oligomycin, or dinitrophenol, dramatically decreased Na⁺-dependent P_i import rates. These data establish the presence of a Na⁺-dependent P_i cotransport system in neurons of the CNS, demonstrate the Ca²⁺-dependent nature of ³²P_i uptake, and suggest that the neuronal Na⁺-dependent P_i cotransporter may import P_i required for the production of high-energy compounds vital to neuronal metabolism.     

Role of mitochondrial calcium uniporter‐mediated Ca2+ and iron accumulation in traumatic brain injury

Previous studies have suggested that the cellular Ca²⁺ and iron homeostasis, which can be regulated by mitochondrial calcium uniporter (MCU), is associated with oxidative stress, apoptosis and many neurological diseases. However, little is known about the role of MCU‐mediated Ca²⁺ and iron accumulation in traumatic brain injury (TBI). Under physiological conditions, MCU can be inhibited by ruthenium red (RR) and activated by spermine (Sper). In the present study, we used RR and Sper to reveal the role of MCU in mouse and neuron TBI models. Our results suggested that the Ca²⁺ and iron concentrations were obviously increased after TBI. In addition, TBI models showed a significant generation of reactive oxygen species (ROS), decrease in adenosine triphosphate (ATP), deformation of mitochondria, up‐regulation of deoxyribonucleic acid (DNA) damage and increase in apoptosis. Blockage of MCU by RR prevented Ca²⁺ and iron accumulation, abated the level of oxidative stress, improved the energy supply, stabilized mitochondria, reduced DNA damage and decreased apoptosis both in vivo and in vitro. Interestingly, Sper did not increase cellular Ca²⁺ and iron concentrations, but suppressed the Ca²⁺ and iron accumulation to benefit the mice in vivo. However, Sper had no significant impact on TBI in vitro. Taken together, our data demonstrated for the first time that blockage of MCU‐mediated Ca²⁺ and iron accumulation was essential for TBI. These findings indicated that MCU could be a novel therapeutic target for treating TBI.     

The role of Ca2+ and hemodynamics in the action of diltiazem on hepatic energy metabolism

Diltiazem causes vasoconstriction in the liver when present at high concentrations, an action that is strictly Ca²⁺-dependent. Diltiazem is also active on energy metabolism. This toxic action could be partly a consequence of hemodynamic effects. In the absence of Ca²⁺, the hemodynamic effects are no longer present and, consequently, Ca²⁺-free experiments are useful for distinguishing between hemodynamics-dependent and hemodynamics-independent effects. The experimental system used was the hemoglobin-free perfused rat liver from fed and fasted rats. Diltiazem was infused at various concentrations in the presence and absence of Ca²⁺. Several metabolic parameters were measured: lactate and pyruvate production (glycolysis), glycogenolysis, oxygen uptake, gluconeogenesis, and the cellular levels of lactate, pyruvate, glucose, AMP, ADP, and ATP. The effects of diltiazem can be divided into three groups: (1) Effects that are strictly dependent on the Ca²⁺-mediated hemodynamic action. This group comprises inhibition of oxygen uptake at all concentrations (50–500 mol/L) inhibition of lactate, pyruvate, and glucose release at high concentrations; the decrease in cellular ATP; the increase in cellular AMP; and the cellular accumulation of glucose and lactate. (2) Effects that are independent of the hemodynamic action. The most relevant effect of this type is inhibition of gluconeogenesis. (3) Effects that are influenced by Ca²⁺ but are independent of the hemodynamic effects. This is the typical case of lactate and glucose release from endogenous glycogen, whose stimulation by low diltiazem concentrations is more pronounced in the presence of Ca²⁺ than in its absence.     

Mitochondrial permeability transition in neuronal damage promoted by Ca2+ and respiratory chain complex II inhibition

Changes in mitochondrial integrity, reactive oxygen species release and Ca2+ handling are proposed to be involved in the pathogenesis of many neurological disorders including methylmalonic acidaemia and Huntington's disease, which exhibit partial mitochondrial respiratory inhibition. In this report, we studied the mechanisms by which the respiratory chain complex II inhibitors malonate, methylmalonate and 3-nitropropionate affect rat brain mitochondrial function and neuronal survival. All three compounds, at concentrations which inhibit respiration by 50%, induced mitochondrial inner membrane permeabilization when in the presence of micromolar Ca2+ concentrations. ADP, cyclosporin A and catalase prevented or delayed this effect, indicating it is mediated by reactive oxygen species and mitochondrial permeability transition (PT). PT induced by malonate was also present in mitochondria isolated from liver and kidney, but required more significant respiratory inhibition. In brain, PT promoted by complex II inhibition was stimulated by increasing Ca2+ cycling and absent when mitochondria were pre-loaded with Ca2+ or when Ca2+ uptake was prevented. In addition to isolated mitochondria, we determined the effect of methylmalonate on cultured PC12 cells and freshly prepared rat brain slices. Methylmalonate promoted cell death in striatal slices and PC12 cells, in a manner attenuated by cyclosporin A and bongkrekate, and unrelated to impairment of energy metabolism. We propose that under conditions in which mitochondrial complex II is partially inhibited in the CNS, neuronal cell death involves the induction of PT.     

Mitochondrial Ca2+ transport and permeability transition pore opening and mitochondrial energetic status

The relationship between mitochondrial Ca2+ transport and permeability transition pore (PTP) opening as well as the effects of mitochondrial energetic status on mitochondrial Ca2+ transport and PTP opening were studied. The results showed that the calcium-induced calcium release from mitochondria (mCICR) induced PTP opening. Inhibitors for electron transport of respiratory chain inhibited mCICR and PTP opening. Partial recovery of electron transport in respiratory chain resulted in partial recovery of mCICR and PTP opening. mCICR and PTP opening were also inhibited by CCCP which eliminated transmembrane proton gradient. The results indicated that mitochondrial Ca2+ transport and PTP opening are largely dependent on electron transport and energy coupling.     

Defective Mitochondrial Pyruvate Flux Affects Cell Bioenergetics in Alzheimer’s Disease-Related Models

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Mitochondrial Ca2+ transport and permeability transition pore opening and mitochondrial energetic status

The relationship between mitochondrial Ca2 transport and permeability transition pore (PTP) opening as well as the effects of mitochondrial energetic status on mitochondrial Ca2 transport and PTP opening were studied. The results showed that the calcium-induced calcium release from mitochondria (mClCR) induced PTP opening. Inhibitors for electron transport of respiratory chain inhibited mClCR and PTP opening. Partial recovery of electron transport in respiratory chain resulted in partial recovery of mClCR and PTP opening. mClCR and PTP opening were also inhibited by CCCP which eliminated transmembrane proton gradient. The results indicated that mitochondrial Ca2 transport and PTP opening are largely dependent on electron transport and energy coupling.     

The mechanism of lead-induced mitochondrial Ca2+ efflux

Addition of Pb²⁺ to rat kidney mitochondria is followed by induction of several reactions: inhibition of Ca²⁺ uptake, collapse of the transmembrane potential, oxidation of pyridine nucleotides, and a fast release of accumulated Ca²⁺. When the incubation media are supplemented with ruthenium red, the effect of Pb²⁺ on NAD(P)H oxidation, membrane , and Ca²⁺ release are not prevented if malate-glutamate are the oxidizing substrates; however, the latter two lead-induced reactions are prevented by ruthenium red if succinate is the electron donor. It is proposed that in mitochondria oxidizing NAD-dependent substrates, Pb²⁺ induces Ca²⁺ release by promoting NAD(P)H oxidation and a parallel drop in due to its binding to thiol groups, located in the cytosol side of the inner membrane. In addition, it is proposed that with succinate as substrate, the Ca²⁺-releasing effect of lead is due to the collapse of the transmembrane potential as a consequence of the uptake of Pb²⁺ through the calcium uniporter, since such effect is ruthenium red sensitive.     

Redox regulation of ER and mitochondrial Ca2+ signaling in cell survival and death

Physiological signaling by reactive oxygen species (ROS) and their pathophysiological role in cell death are well recognized. This review focuses on two ROS targets that are key to local Ca²⁺ signaling at the ER/mitochondrial interface – notably, inositol trisphosphate receptors (IP₃Rs) and the mitochondrial calcium uniporter (MCU). Both transport systems are central to molecular mechanisms in cell survival and death. Methods for the measurement of the redox state of these proteins and for the detection of ROS nanodomains are described. Recent results on the redox regulation of these proteins are reviewed.     

Passive Ca2+ Transport and Ca2+-Dependent K+ Transport in Plasmodium falciparum-Infected Red Cells

Previous reports have indicated that Plasmodium falciparum-infected red cells (pRBC) have an increased Ca²⁺ permeability. The magnitude of the increase is greater than that normally required to activate the Ca²⁺-dependent K⁺ channel (K _Ca channel) of the red cell membrane. However, there is evidence that this channel remains inactive in pRBC. To clarify this discrepancy, we have reassessed both the functional status of the K _Ca channel and the Ca²⁺ permeability properties of pRBC. For pRBC suspended in media containing Ca²⁺, K _Ca channel activation was elicited by treatment with the Ca²⁺ ionophore A23187. In the absence of ionophore the channel remained inactive. In contrast to previous claims, the unidirectional influx of Ca²⁺ into pRBC in which the Ca²⁺ pump was inhibited by vanadate was found to be within the normal range (30–55 μmol (10¹³ cells · hr)⁻¹), provided the cells were suspended in glucose-containing media. However, for pRBC in glucose-free media the Ca²⁺ influx increased to over 1 mmol (10¹³ cells · hr)⁻¹, almost an order of magnitude higher than that seen in uninfected erythrocytes under equivalent conditions. The pathway responsible for the enhanced influx of Ca²⁺ into glucose-deprived pRBC was expressed at approximately 30 hr post-invasion, and was inhibited by Ni²⁺. Possible roles for this pathway in pRBC are considered. Received: 12 May 1999/Revised: 8 July 1999     

Revealing Key Interactions in a Metabolic Network: Model of Primary Phosphate Transport in Bacteria

One of the main problems of metabolic engineering is to determine the genetically controlled limiting links of a metabolic network. We have built a model of the primary transport of inorganic phosphates (P _i ), analyzed the P _i metabolic network in Gram-negative bacteria, and determined the factors controlling the phosphate exchange. The model explains why the P _i primary transport is not observed at the release stage. The nonlinearity of primary transport and the differences in its parameters in the membrane and within the cell give rise to transport asymmetry, i.e., the P _i release rate is low as compared with the uptake rate, and is small at the background of secondary transport. Discussed is a general scheme of coordination between primary and secondary transport, which are interconnected through the substrate–product relation.     

Mitochondrial Ca2+ laMety directs fibrosis

During development, disease or in response to changes in local environmental and/or nutrient supply, cellular metabolism is substantially remodeled. Reduced mitochondrial Ca²⁺ uptake was recently reported to induce metabolic remodeling, which through stimulating alterations in the epigenome causes changes in gene expression associated with fibroblast to myofibroblast differentiation.     

Model of Sarcomeric Ca2+ Movements,Including ATP Ca2+ Binding and Diffusion,during Activation of Frog Skeletal Muscle

Cannell and Allen (1984. Biophys. J. 45:913–925) introduced the use of a multi-compartment model to estimate the time course of spread of calcium ions (Ca²⁺) within a half sarcomere of a frog skeletal muscle fiber activated by an action potential. Under the assumption that the sites of sarcoplasmic reticulum (SR) Ca²⁺ release are located radially around each myofibril at the Z line, their model calculated the spread of released Ca²⁺ both along and into the half sarcomere. During diffusion, Ca²⁺ was assumed to react with metal-binding sites on parvalbumin (a diffusible Ca²⁺- and Mg²⁺-binding protein) as well as with fixed sites on troponin. We have developed a similar model, but with several modifications that reflect current knowledge of the myoplasmic environment and SR Ca²⁺ release. We use a myoplasmic diffusion constant for free Ca²⁺ that is twofold smaller and an SR Ca²⁺ release function in response to an action potential that is threefold briefer than used previously. Additionally, our model includes the effects of Ca²⁺ and Mg²⁺ binding by adenosine 5′-triphosphate (ATP) and the diffusion of Ca²⁺-bound ATP (CaATP). Under the assumption that the total myoplasmic concentration of ATP is 8 mM and that the amplitude of SR Ca²⁺ release is sufficient to drive the peak change in free [Ca²⁺] (Δ[Ca²⁺]) to 18 μM (the approximate spatially averaged value that is observed experimentally), our model calculates that (a) the spatially averaged peak increase in [CaATP] is 64 μM; (b) the peak saturation of troponin with Ca²⁺ is high along the entire thin filament; and (c) the half-width of Δ[Ca²⁺] is consistent with that observed experimentally. Without ATP, the calculated half-width of spatially averaged Δ[Ca²⁺] is abnormally brief, and troponin saturation away from the release sites is markedly reduced. We conclude that Ca²⁺ binding by ATP and diffusion of CaATP make important contributions to the determination of the amplitude and the time course of Δ[Ca²⁺].     

Mitochondrial Efficiency-Dependent Viability of Saccharomyces cerevisiae Mutants Carrying Individual Electron Transport Chain Component Deletions

Mitochondria play a crucial role in eukaryotic cells; the mitochondrial electron transport chain (ETC) generates adenosine triphosphate (ATP), which serves as an energy source for numerous critical cellular activities. However, the ETC also generates deleterious reactive oxygen species (ROS) as a natural byproduct of oxidative phosphorylation. ROS are considered the major cause of aging because they damage proteins, lipids, and DNA by oxidation. We analyzed the chronological life span, growth phenotype, mitochondrial membrane potential (MMP), and intracellular ATP and mitochondrial superoxide levels of 33 single ETC component-deleted strains during the chronological aging process. Among the ETC mutant strains, 14 (sdh1Δ, sdh2Δ, sdh4Δ, cor1Δ, cyt1Δ, qcr7Δ, qcr8Δ, rip1Δ, cox6Δ, cox7Δ, cox9Δ, atp4Δ, atp7Δ, and atp17Δ) showed a significantly shorter life span. The deleted genes encode important elements of the ETC components succinate dehydrogenase (complex II) and cytochrome c oxidase (complex IV), and some of the deletions lead to structural instability of the membrane-F₁F₀-ATP synthase due to mutations in the stator stalk (complex V). These short-lived strains generated higher superoxide levels and produced lower ATP levels without alteration of MMP. In summary, ETC mutations decreased the life span of yeast due to impaired mitochondrial efficiency.     

Dimerization of MICU Proteins Controls Ca2+ Influx through the Mitochondrial Ca2+ Uniporter

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