Bile Acid-Induced Ca<Superscript>2+</Superscript> Efflux from Liver Mitochondria as a Factor Preventing the Formation of Mitochondrial Pores

The effect of bile acids as inducers of Ca²⁺ efflux from the matrix was studied on isolated rat liver mitochondria. Mitochondria in the presence of cyclosporin A (CsA) were energized by succinate, then loaded with Ca²⁺ and after the addition of the calcium uniporter inhibitor ruthenium red were de-energized by malonate. It was shown that under these conditions hydrophobic bile acids lithocholic and chenodeoxycholic at concentrations of 10 and 30 μM respectively and hydrophilic bile acids ursodeoxycholic and cholic at a concentration of 400 μM induce Ca²⁺ efflux from the mitochondrial matrix. It is noted that the efflux of these ions is not associated with damage of the inner mitochondrial membrane by bile acids, since it is accompanied by the generation of Δψ, i.e., the formation of the diffusion potential. It is assumed that along with induction of calcium efflux from the matrix, bile acids are also capable of transporting hydrogen and potassium ions in the opposite direction, i.e., perform H⁺/Ca²⁺ and K⁺/Ca²⁺ exchange. It was found that ruthenium red added to Ca²⁺-loaded energized mitochondria prevents the return of these ions to the matrix and weakens the effect of chenodeoxycholic acid as an inducer of the CsA-sensitive mitochondrial pore and the effect of ursodeoxycholic acid as an inducer of CsA-insensitive permeability of the inner mitochondrial membrane. We conclude that in the conditions of the calcium uniporter activity decrease, Ca²⁺ efflux from the matrix induced by bile acids can be considered as one of the mechanisms reducing their effectiveness as inducers of the Ca²⁺-dependent CsA-sensitive pore and CsA-insensitive permeability transition in mitochondria.     

A historical review of cellular calcium handling,with emphasis on mitochondria

Calcium ions are of central importance in cellular physiology, as they carry the signal activating cells to perform their programmed function. Ca²⁺ is particularly suitable for this role because of its chemical properties and because its free concentration gradient between the extra cellular and the cytosolic concentrations is very high, about four orders of magnitude. The cytosolic concentration of Ca²⁺ is regulated by binding and chelation by various substances and by transport across plasma and intracellular membranes. Various channels, transport ATPases, uniporters, and antiporters in the plasma mem brane, endoplasmic and sarcoplasmic reticulum, and mitochondria are responsible for the transport of Ca²⁺ .The regulation of these transport systems is the subject of an increasing number of studies. In this short review, we focus on the mitochondrial transporters, i.e. the calcium uniporter used for Ca²⁺ uptake, and the antiporters used for the efflux, i.e. the Ca²⁺/Na⁺ antiporter in mitochondria and the plasma membrane of excitable cells,and the Ca²⁺/nH⁺ antiporter in liver and some other mitochondrial types. Mitochondria are of special interest in that Ca²⁺ stimulates respiration and oxidative phosphorylation to meet the energy needs of activated cells. The studies on Ca²⁺ and mitochondria began in the fifties, but interest in mito chondrial Ca²⁺ handling faded in the late seventies since it had become apparent that mitochondria in resting cells contain very low Ca²⁺. Interest increased again in the nineties also because it was discovered that mitochondria and Ca²⁺ had a central role in apoptosis and necrosis. This is of special interest in calcium overload and oxidative stress conditions, when the opening of the mitochondrial permeability transition pore is stimulated.Translated from Biokhimiya,Vol. 70, No. 2, 2005, pp. 231–239.Original Russian Text Copyright © 2005 by Saris, Carafoli.This revised version was published online in April 2005 with corrections to the post codes.     

The EF-Hand Ca2+ Binding Protein MICU Choreographs Mitochondrial Ca2+ Dynamics in Arabidopsis

Plant organelle function must constantly adjust to environmental conditions, which requires dynamic coordination. Ca²⁺ signaling may play a central role in this process. Free Ca²⁺ dynamics are tightly regulated and differ markedly between the cytosol, plastid stroma, and mitochondrial matrix. The mechanistic basis of compartment-specific Ca²⁺ dynamics is poorly understood. Here, we studied the function of At-MICU, an EF-hand protein of Arabidopsis thaliana with homology to constituents of the mitochondrial Ca²⁺ uniporter machinery in mammals. MICU binds Ca²⁺ and localizes to the mitochondria in Arabidopsis. In vivo imaging of roots expressing a genetically encoded Ca²⁺ sensor in the mitochondrial matrix revealed that lack of MICU increased resting concentrations of free Ca²⁺ in the matrix. Furthermore, Ca²⁺ elevations triggered by auxin and extracellular ATP occurred more rapidly and reached higher maximal concentrations in the mitochondria of micu mutants, whereas cytosolic Ca²⁺ signatures remained unchanged. These findings support the idea that a conserved uniporter system, with composition and regulation distinct from the mammalian machinery, mediates mitochondrial Ca²⁺ uptake in plants under in vivo conditions. They further suggest that MICU acts as a throttle that controls Ca²⁺ uptake by moderating influx, thereby shaping Ca²⁺ signatures in the matrix and preserving mitochondrial homeostasis. Our results open the door to genetic dissection of mitochondrial Ca²⁺ signaling in plants.     

Mechanisms of Rapid Reactive Oxygen Species Generation in Response to Cytosolic Ca2+ or Zn2+ Loads in Cortical Neurons

Excessive “excitotoxic” accumulation of Ca²⁺ and Zn²⁺ within neurons contributes to neurodegeneration in pathological conditions including ischemia. Putative early targets of these ions, both of which are linked to increased reactive oxygen species (ROS) generation, are mitochondria and the cytosolic enzyme, NADPH oxidase (NOX). The present study uses primary cortical neuronal cultures to examine respective contributions of mitochondria and NOX to ROS generation in response to Ca²⁺ or Zn²⁺ loading. Induction of rapid cytosolic accumulation of either Ca²⁺ (via NMDA exposure) or Zn²⁺ (via Zn²⁺/Pyrithione exposure in 0 Ca²⁺) caused sharp cytosolic rises in these ions, as well as a strong and rapid increase in ROS generation. Inhibition of NOX activation significantly reduced the Ca²⁺-induced ROS production with little effect on the Zn²⁺- triggered ROS generation. Conversely, dissipation of the mitochondrial electrochemical gradient increased the cytosolic Ca²⁺ or Zn²⁺ rises caused by these exposures, consistent with inhibition of mitochondrial uptake of these ions. However, such disruption of mitochondrial function markedly suppressed the Zn²⁺-triggered ROS, while partially attenuating the Ca²⁺-triggered ROS. Furthermore, block of the mitochondrial Ca²⁺ uniporter (MCU), through which Zn²⁺ as well as Ca²⁺ can enter the mitochondrial matrix, substantially diminished Zn²⁺ triggered ROS production, suggesting that the ROS generation occurs specifically in response to Zn²⁺ entry into mitochondria. Finally, in the presence of the sulfhydryl-oxidizing agent 2,2''-dithiodipyridine, which impairs Zn²⁺ binding to cytosolic metalloproteins, far lower Zn²⁺ exposures were able to induce mitochondrial Zn²⁺ uptake and consequent ROS generation. Thus, whereas rapid acute accumulation of Zn²⁺ and Ca²⁺ each can trigger injurious ROS generation, Zn²⁺ entry into mitochondria via the MCU may do so with particular potency. This may be of particular relevance to conditions like ischemia in which cytosolic Zn²⁺ buffering is impaired due to acidosis and oxidative stress.     

Regulation of neuronal energy metabolism by calcium: Role of MCU and Aralar/malate-aspartate shuttle

Calcium is a major regulator of cellular metabolism. Calcium controls mitochondrial respiration, and calcium signaling is used to meet cellular energetic demands through energy production in the organelle. Although it has been widely assumed that Ca²⁺-actions require its uptake by mitochondrial calcium uniporter (MCU), alternative pathways modulated by cytosolic Ca²⁺ have been recently proposed. Recent findings have indicated a role for cytosolic Ca²⁺ signals acting on mitochondrial NADH shuttles in the control of cellular metabolism in neurons using glucose as fuel. It has been demonstrated that AGC1/Aralar, the component of the malate/aspartate shuttle (MAS) regulated by cytosolic Ca²⁺, participates in the maintenance of basal respiration exerted through Ca²⁺-fluxes between ER and mitochondria, whereas mitochondrial Ca²⁺-uptake by MCU does not contribute. Aralar/MAS pathway, activated by small cytosolic Ca²⁺ signals, provides in fact substrates, redox equivalents and pyruvate, fueling respiration. Upon activation and increases in workload, neurons upregulate OxPhos, cytosolic pyruvate production and glycolysis, together with glucose uptake, in a Ca²⁺-dependent way, and part of this upregulation is via Ca²⁺ signaling. Both MCU and Aralar/MAS contribute to OxPhos upregulation, Aralar/MAS playing a major role, especially at small and submaximal workloads. Ca²⁺ activation of Aralar/MAS, by increasing cytosolic NAD⁺/NADH provides Ca²⁺-dependent increases in glycolysis and cytosolic pyruvate production priming respiration as a feed-forward mechanism in response to workload. Thus, except for glucose uptake, these processes are dependent on Aralar/MAS, whereas MCU is the relevant target for Ca²⁺ signaling when MAS is bypassed, by using pyruvate or β-hydroxybutyrate as substrates.     

Effects of adenine nucleotide translocase inhibitors on dinitrophenol-induced Ca2+ efflux from pig heart mitochondria

Bongkrekic acid and atractyloside, inhibitors of adenine nucleotide translocase, do not inhibit Ca²⁺ uptake and H⁺ production by pig heart mitochondria. However, bongkrekic acid, but not atractyloside, inhibits dinitrophenol-induced Ca²⁺ efflux and H⁺ uptake. Conversely, ruthenium red blocks Ca²⁺ uptake and H⁺ production but does not prevent dinitrophenol-induced Ca²⁺ efflux and H⁺ uptake by mitochondria. These results suggest that mitochondrial Ca²⁺ uptake and release exist as two independent pathways. The efflux of Ca²⁺ from mitochondria is mediated by a bongkrekic acid sensitive component which is apparently not identical to the ruthenium red sensitive Ca²⁺ uptake carrier.     

A Biophysically Based Mathematical Model for the Kinetics of Mitochondrial Calcium Uniporter

Ca²⁺ transport through mitochondrial Ca²⁺ uniporter is the primary Ca²⁺ uptake mechanism in respiring mitochondria. Thus, the uniporter plays a key role in regulating mitochondrial Ca²⁺. Despite the importance of mitochondrial Ca²⁺ to metabolic regulation and mitochondrial function, and to cell physiology and pathophysiology, the structure and composition of the uniporter functional unit and kinetic mechanisms associated with Ca²⁺ transport into mitochondria are still not well understood. In this study, based on available experimental data on the kinetics of Ca²⁺ transport via the uniporter, a mechanistic kinetic model of the uniporter is introduced. The model is thermodynamically balanced and satisfactorily describes a large number of independent data sets in the literature on initial or pseudo-steady-state influx rates of Ca²⁺ via the uniporter measured under a wide range of experimental conditions. The model is derived assuming a multi-state catalytic binding and Eyring's free-energy barrier theory-based transformation mechanisms associated with the carrier-mediated facilitated transport and electrodiffusion. The model is a great improvement over the previous theoretical models of mitochondrial Ca²⁺ uniporter in the literature in that it is thermodynamically balanced and matches a large number of independently published data sets on mitochondrial Ca²⁺ uptake. This theoretical model will be critical in developing mechanistic, integrated models of mitochondrial Ca²⁺ handling and bioenergetics which can be helpful in understanding the mechanisms by which Ca²⁺ plays a role in mediating signaling pathways and modulating mitochondrial energy metabolism.     

Mitochondrial Ca influx and efflux rates in guinea pig cardiac mitochondria:Low and high affinity effects of cyclosporine A

Ca²⁺ plays a central role in energy supply and demand matching in cardiomyocytes by transmitting changes in excitation-contraction coupling to mitochondrial oxidative phosphorylation. Matrix Ca²⁺ is controlled primarily by the mitochondrial Ca²⁺ uniporter and the mitochondrial Na⁺/Ca²⁺ exchanger, influencing NADH production through Ca²⁺-sensitive dehydrogenases in the Krebs cycle. In addition to the well-accepted role of the Ca²⁺-triggered mitochondrial permeability transition pore in cell death, it has been proposed that the permeability transition pore might also contribute to physiological mitochondrial Ca²⁺ release. Here we selectively measure Ca²⁺ influx rate through the mitochondrial Ca²⁺ uniporter and Ca²⁺ efflux rates through Na⁺-dependent and Na⁺-independent pathways in isolated guinea pig heart mitochondria in the presence or absence of inhibitors of mitochondrial Na⁺/Ca²⁺ exchanger (CGP 37157) or the permeability transition pore (cyclosporine A). cyclosporine A suppressed the negative bioenergetic consequences (ΔΨ_m loss, Ca²⁺ release, NADH oxidation, swelling) of high extramitochondrial Ca²⁺ additions, allowing mitochondria to tolerate total mitochondrial Ca²⁺ loads of > 400 nmol/mg protein. For Ca²⁺ pulses up to 15 μM, Na⁺-independent Ca²⁺ efflux through the permeability transition pore accounted for ~ 5% of the total Ca²⁺ efflux rate compared to that mediated by the mitochondrial Na⁺/Ca²⁺ exchanger (in 5 mM Na⁺). Unexpectedly, we also observed that cyclosporine A inhibited mitochondrial Na⁺/Ca²⁺ exchanger-mediated Ca²⁺ efflux at higher concentrations (IC₅₀ = 2 μM) than those required to inhibit the permeability transition pore, with a maximal inhibition of ~ 40% at 10 μM cyclosporine A, while having no effect on the mitochondrial Ca²⁺ uniporter. The results suggest a possible alternative mechanism by which cyclosporine A could affect mitochondrial Ca²⁺ load in cardiomyocytes, potentially explaining the paradoxical toxic effects of cyclosporine A at high concentrations. This article is part of a Special Issue entitled: Mitochondria and Cardioprotection.     

Molecularly Distinct Routes of Mitochondrial Ca2+ Uptake Are Activated Depending on the Activity of the Sarco/Endoplasmic Reticulum Ca2+ ATPase (SERCA)

The transfer of Ca²⁺ across the inner mitochondrial membrane is an important physiological process linked to the regulation of metabolism, signal transduction, and cell death. While the definite molecular composition of mitochondrial Ca²⁺ uptake sites remains unknown, several proteins of the inner mitochondrial membrane, that are likely to accomplish mitochondrial Ca²⁺ fluxes, have been described: the novel uncoupling proteins 2 and 3, the leucine zipper-EF-hand containing transmembrane protein 1 and the mitochondrial calcium uniporter. It is unclear whether these proteins contribute to one unique mitochondrial Ca²⁺ uptake pathway or establish distinct routes for mitochondrial Ca²⁺ sequestration. In this study, we show that a modulation of Ca²⁺ release from the endoplasmic reticulum by inhibition of the sarco/endoplasmatic reticulum ATPase modifies cytosolic Ca²⁺ signals and consequently switches mitochondrial Ca²⁺ uptake from an uncoupling protein 3- and mitochondrial calcium uniporter-dependent, but leucine zipper-EF-hand containing transmembrane protein 1-independent to a leucine zipper-EF-hand containing transmembrane protein 1- and mitochondrial calcium uniporter-mediated, but uncoupling protein 3-independent pathway. Thus, the activity of sarco/endoplasmatic reticulum ATPase is significant for the mode of mitochondrial Ca²⁺ sequestration and determines which mitochondrial proteins might actually accomplish the transfer of Ca²⁺ across the inner mitochondrial membrane. Moreover, our findings herein support the existence of distinct mitochondrial Ca²⁺ uptake routes that might be essential to ensure an efficient ion transfer into mitochondria despite heterogeneous cytosolic Ca²⁺ rises.     

Permeability transition pore of the inner mitochondrial membrane can operate in two open states with different selectivities

Prooxidants induce release of Ca²⁺ from mitochondria through the giant solute pore in the mitochondrial inner membrane. However, under appropriate conditions prooxidants can induce Ca²⁺ release without inducing a nonspecific permeability change. Prooxidant-induced release of Ca²⁺ isselective. Presumably, this is the result of the operation of a permeability pathway for H⁺ coupled to the reversal of the Ca²⁺ uniporter, the latter generating the selectivity. The solute pore and prooxidant-induced Ca²⁺-specific pathways exhibit common sensitivities to a set of inhibitors and activators. It is proposed that the pore can operate in two open states: (1) permeable to H⁺ only and (2) permeable to solutes of M_r<1500. Under some conditions, prooxidants induce the H⁺-selective state which, in turn, collapses the inner membrane potential and permits selective loss of Ca²⁺ via the Ca²⁺ uniporter.     

Diacylglycerols Activate Mitochondrial Cationic Channel(s) and Release Sequestered Ca<Superscript>2+</Superscript>

Mitochondria contribute to cytosolic Ca²⁺ homeostasis through several uptake and release pathways. Here we report that 1,2-sn-diacylglycerols (DAGs) induce Ca²⁺ release from Ca²⁺-loaded mammalian mitochondria. Release is not mediated by the uniporter or the Na⁺/Ca²⁺ exchanger, nor is it attributed to putative catabolites. DAGs-induced Ca²⁺ efflux is biphasic. Initial release is rapid and transient, insensitive to permeability transition inhibitors, and not accompanied by mitochondrial swelling. Following initial rapid release of Ca²⁺ and relatively slow reuptake, a secondary progressive release of Ca²⁺ occurs, associated with swelling, and mitigated by permeability transition inhibitors. The initial peak of DAGs-induced Ca²⁺ efflux is abolished by La³⁺ (1 mM) and potentiated by protein kinase C inhibitors. Phorbol esters, 1,3-diacylglycerols and 1-monoacylglycerols do not induce mitochondrial Ca²⁺ efflux. Ca²⁺-loaded mitoplasts devoid of outer mitochondrial membrane also exhibit DAGs-induced Ca²⁺ release, indicating that this mechanism resides at the inner mitochondrial membrane. Patch clamping brain mitoplasts reveal DAGs-induced slightly cation-selective channel activity that is insensitive to bongkrekic acid and abolished by La³⁺. The presence of a second messenger-sensitive Ca²⁺ release mechanism in mitochondria could have an important impact on intracellular Ca²⁺ homeostasis.     

Mitochondrial Ca2+ Uptake 1 (MICU1) and Mitochondrial Ca2+ Uniporter (MCU) Contribute to Metabolism-Secretion Coupling in Clonal Pancreatic ��-Cells

In pancreatic β-cells, uptake of Ca²⁺ into mitochondria facilitates metabolism-secretion coupling by activation of various matrix enzymes, thus facilitating ATP generation by oxidative phosphorylation and, in turn, augmenting insulin release. We employed an siRNA-based approach to evaluate the individual contribution of four proteins that were recently described to be engaged in mitochondrial Ca²⁺ sequestration in clonal INS-1 832/13 pancreatic β-cells: the mitochondrial Ca²⁺ uptake 1 (MICU1), mitochondrial Ca²⁺ uniporter (MCU), uncoupling protein 2 (UCP2), and leucine zipper EF-hand-containing transmembrane protein 1 (LETM1). Using a FRET-based genetically encoded Ca²⁺ sensor targeted to mitochondria, we show that a transient knockdown of MICU1 or MCU diminished mitochondrial Ca²⁺ uptake upon both intracellular Ca²⁺ release and Ca²⁺ entry via L-type channels. In contrast, knockdown of UCP2 and LETM1 exclusively reduced mitochondrial Ca²⁺ uptake in response to either intracellular Ca²⁺ release or Ca²⁺ entry, respectively. Therefore, we further investigated the role of MICU1 and MCU in metabolism-secretion coupling. Diminution of MICU1 or MCU reduced mitochondrial Ca²⁺ uptake in response to d-glucose, whereas d-glucose-triggered cytosolic Ca²⁺ oscillations remained unaffected. Moreover, d-glucose-evoked increases in cytosolic ATP and d-glucose-stimulated insulin secretion were diminished in MICU1- or MCU-silenced cells. Our data highlight the crucial role of MICU1 and MCU in mitochondrial Ca²⁺ uptake in pancreatic β-cells and their involvement in the positive feedback required for sustained insulin secretion.     

The gatekeepers of mitochondrial calcium influx: MICU1 and MICU2

The receptor‐evoked Ca²⁺ signal is sensed and translated by mitochondria. Physiological cytoplasmic Ca²⁺ ([Ca²⁺]_c) oscillations result in mitochondrial Ca²⁺ ([Ca²⁺]_m) oscillations, while large and sustained [Ca²⁺]_c increase results in a pathologic increase in basal [Ca²⁺]_m and in Ca²⁺ accumulation. The physiological [Ca²⁺]_m signal regulates [Ca²⁺]_c and stimulates oxidative metabolism, while excess Ca²⁺ accumulation causes cell stress leading to cell death. [Ca²⁺]_m is determined by Ca²⁺ uptake mediated by the mitochondria Ca²⁺ uniporter (MCU) channel and by Na⁺‐ and H⁺‐coupled Ca²⁺ extrusion 1 .     

Modulation of calcium signalling by mitochondria

In this review we will attempt to summarise the complex and sometimes contradictory effects that mitochondria have on different forms of calcium signalling. Mitochondria can influence Ca²⁺ signalling indirectly by changing the concentration of ATP, NAD(P)H, pyruvate and reactive oxygen species — which in turn modulate components of the Ca²⁺ signalling machinery i.e. buffering, release from internal stores, influx from the extracellular solution, uptake into cellular organelles and extrusion by plasma membrane Ca²⁺ pumps. Mitochondria can directly influence the calcium concentration in the cytosol of the cell by importing Ca²⁺ via the mitochondrial Ca²⁺ uniporter or transporting Ca²⁺ from the interior of the organelle into the cytosol by means of Na⁺/Ca²⁺ or H⁺/Ca²⁺ exchangers. Considerable progress in understanding the relationship between Ca²⁺ signalling cascades and mitochondrial physiology has been accumulated over the last few years due to the development of more advanced optical techniques and electrophysiological approaches.     

Outstanding questions regarding the permeation,selectivity, and regulation of the mitochondrial calcium uniporter

The recent discovery of genes encoding the mitochondrial calcium (Ca²⁺) uniporter has revealed new opportunities for studying how abnormal Ca²⁺ signals cause disease. Ca²⁺ transport across the mitochondrial inner membrane is highly regulated, and the uniporter is the channel that acts as a major portal for Ca²⁺ influx. Low amounts of mitochondrial Ca²⁺ can boost ATP synthesis, but excess amounts, such as following cytoplasmic Ca²⁺ overload in heart failure, triggers mitochondrial failure and cell death. In fact, precisely because mitochondrial Ca²⁺ transport is so tightly regulated, a fundamental understanding of how the uniporter functions is necessary. Two key uniporter features allow Ca²⁺ influx without mitochondrial damage during normal physiology. First, the channel is significantly more selective than other known Ca²⁺ channels. This prevents the permeation of other ions and uncoupling of the electrochemical gradient. Second, the uniporter becomes active at only high Ca²⁺ concentrations, preventing a resting leak of cytoplasmic Ca²⁺ itself. Now possessing the identities of the various proteins forming the uniporter, we can proceed with efforts to define the molecular determinants of permeation, selectivity and Ca²⁺-regulation.     

Further studies on the effect of phosphoenolpyruvate on respiration-dependent calcium transport by rat heart mitochondria

Phosphoenolpyruvate was found to depress extra oxygen consumption associated with Ca²⁺-induced respiratory jump by rat heart mitochondria. Addition of phosphoenolpyruvate to mitochondria which have accumulated Ca²⁺ in the presence of glutamate and inorganic phosphate causes the release of Ca²⁺ from mitochondria. The phosphoenolpyruvate-stimulated Ca²⁺ efflux can be observed with mitochondria loaded with low initial Ca²⁺ concentration (0.12 mM) in the incubation medium. Measurements of mitochondrial H⁺ translocation produced by addition of Ca²⁺ to respiring mitochondria show that phosphoenolpyruvate depresses H⁺ ejection and enhances H⁺ uptake by mitochondria. The Ca²⁺-releasing effect of phosphoenolpyruvate was found to be significantly stronger than that produced by rotenone when added to mitochondria loaded with Ca²⁺ in the presence of glutamate and inorganic phosphate. Dithiothreitol cannot overcome the effect of phosphoenolpyruvate on mitochondrial Ca²⁺ transport.     

Inhibition of mitochondrial calcium uptake rather than efflux impedes calcium release by inositol-1,4,5-trisphosphate-sensitive receptors

Mitochondria modulate cellular Ca²⁺ signals by accumulating the ion via a uniporter and releasing it via Na⁺- or H⁺-exchange. In smooth muscle, inhibition of mitochondrial Ca²⁺ uptake inhibits Ca²⁺ release from the sarcoplasmic reticulum (SR) via inositol-1,4,5-trisphosphate-sensitive receptors (IP₃R). At least two mechanisms may explain this effect. First, localised uptake of Ca²⁺ by mitochondria may prevent negative feedback by cytosolic Ca²⁺ on IP₃R activity, or secondly localised provision of Ca²⁺ by mitochondrial efflux may maintain IP₃R function or SR Ca²⁺ content. To distinguish between these possibilities the role of mitochondrial Ca²⁺ efflux on IP₃R function was examined. IP₃ was liberated in freshly isolated single colonic smooth muscle cells and mitochondrial Na⁺–Ca²⁺ exchanger inhibited with CGP-37157 (10 μM). Mitochondria accumulated Ca²⁺ during IP₃-evoked [Ca²⁺]_c rises and released the ion back to the cytosol (within 15 s) when mitochondrial Ca²⁺ efflux was active. When mitochondrial Ca²⁺ efflux was inhibited by CGP-37157, an extensive and sustained loading of mitochondria with Ca²⁺ occurred after IP₃-evoked Ca²⁺ release. IP₃-evoked [Ca²⁺]_c rises were initially unaffected, then only slowly inhibited by CGP-37157. IP₃R activity was required for inhibition to occur; incubation with CGP-37157 for the same duration without IP₃ release did not inhibit IP₃R. CGP-37157 directly inhibited voltage-gated Ca²⁺ channel activity, however SR Ca²⁺ content was unaltered by the drug. Thus, the gradual decline of IP₃R function that followed mitochondrial Na⁺–Ca²⁺ exchanger inhibition resulted from a gradual overload of mitochondria with Ca²⁺, leading to a reduced capacity for Ca²⁺ uptake. Localised uptake of Ca²⁺ by mitochondria, rather than mitochondrial Ca²⁺ efflux, appears critical for maintaining IP₃R activity.     

An Integrated Mitochondrial ROS Production and Scavenging Model: Implications for Heart Failure

It has been observed experimentally that cells from failing hearts exhibit elevated levels of reactive oxygen species (ROS) upon increases in energetic workload. One proposed mechanism for this behavior is mitochondrial Ca²⁺ mismanagement that leads to depletion of ROS scavengers. Here, we present a computational model to test this hypothesis. Previously published models of ROS production and scavenging were combined and reparameterized to describe ROS regulation in the cellular environment. Extramitochondrial Ca²⁺ pulses were applied to simulate frequency-dependent changes in cytosolic Ca²⁺. Model results show that decreased mitochondrial Ca²⁺uptake due to mitochondrial Ca²⁺ uniporter inhibition (simulating Ru360) or elevated cytosolic Na⁺, as in heart failure, leads to a decreased supply of NADH and NADPH upon increasing cellular workload. Oxidation of NADPH leads to oxidation of glutathione (GSH) and increased mitochondrial ROS levels, validating the Ca²⁺ mismanagement hypothesis. The model goes on to predict that the ratio of steady-state [H₂O₂]_m during 3Hz pacing to [H₂O₂]_m at rest is highly sensitive to the size of the GSH pool. The largest relative increase in [H₂O₂]_m in response to pacing is shown to occur when the total GSH and GSSG is close to 1 mM, whereas pool sizes below 0.9 mM result in high resting H₂O₂ levels, a quantitative prediction only possible with a computational model.     

Ca2+ translocation in Ehrlich ascites tumor cells

Summary Ca²⁺ uptake into Ehrlich ascites tumor cells was studied at 0°C in the presence of mitochondrial inhibitors, conditions that minimized complications caused by sequestration of Ca²⁺ into organelles or by excretion. Under these conditions Ruthenium Red inhibited Ca²⁺ uptake, but other previously implicated ions, such as P_i or Mg²⁺, had no effect. Valinomycin either inhibited or slightly stimulated Ca²⁺ uptake depending on the presence of excess K⁺ on the outside or inside of the cell, respectively. Nigericin inhibited Ca²⁺ transport. Based on these data we propose an electrogenic uptake of Ca²⁺, possibly via a Ca²⁺/H⁺ antiport mechanism.The observation that glucose inhibited Ca²⁺ uptake suggested that in Ehrlich ascites tumor cells an energy-driven Ca²⁺ expulsion mechanism is operative, similar to that in erythrocytes. Plasma membrane preparations of ascites tumor cells were found to contain a Ca²⁺-dependent ATPase. These preparations, when incorporated into liposomes in an inside-out orientation, catalyzed an ATP-dependent uptake of Ca²⁺.     

NCLX Protein,but Not LETM1, Mediates Mitochondrial Ca2+ Extrusion,Thereby Limiting Ca2+-induced NAD(P)H Production and Modulating Matrix Redox State

Mitochondria capture and subsequently release Ca²⁺ ions, thereby sensing and shaping cellular Ca²⁺ signals. The Ca²⁺ uniporter MCU mediates Ca²⁺ uptake, whereas NCLX (mitochondrial Na/Ca exchanger) and LETM1 (leucine zipper-EF-hand-containing transmembrane protein 1) were proposed to exchange Ca²⁺ against Na⁺ or H⁺, respectively. Here we study the role of these ion exchangers in mitochondrial Ca²⁺ extrusion and in Ca²⁺-metabolic coupling. Both NCLX and LETM1 proteins were expressed in HeLa cells mitochondria. The rate of mitochondrial Ca²⁺ efflux, measured with a genetically encoded indicator during agonist stimulations, increased with the amplitude of mitochondrial Ca²⁺ ([Ca²⁺]_mt) elevations. NCLX overexpression enhanced the rates of Ca²⁺ efflux, whereas increasing LETM1 levels had no impact on Ca²⁺ extrusion. The fluorescence of the redox-sensitive probe roGFP increased during [Ca²⁺]_mt elevations, indicating a net reduction of the matrix. This redox response was abolished by NCLX overexpression and restored by the Na⁺/Ca²⁺ exchanger inhibitor {"type":"entrez-protein","attrs":{"text":"CGP37157","term_id":"875406365","term_text":"CGP37157"}}CGP37157. The [Ca²⁺]_mt elevations were associated with increases in the autofluorescence of NAD(P)H, whose amplitude was strongly reduced by NCLX overexpression, an effect reverted by Na⁺/Ca²⁺ exchange inhibition. We conclude that NCLX, but not LETM1, mediates Ca²⁺ extrusion from mitochondria. By controlling the duration of matrix Ca²⁺ elevations, NCLX contributes to the regulation of NAD(P)H production and to the conversion of Ca²⁺ signals into redox changes.