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.     

Characterization of Mg2+ inhibition of mitochondrial Ca2+ uptake by a mechanistic model of mitochondrial Ca2+ uniporter

Ca(2+) is an important regulatory ion and alteration of mitochondrial Ca(2+) homeostasis can lead to cellular dysfunction and apoptosis. Ca(2+) is transported into respiring mitochondria via the Ca(2+) uniporter, which is known to be inhibited by Mg(2+). This uniporter-mediated mitochondrial Ca(2+) transport is also shown to be influenced by inorganic phosphate (Pi). Despite a large number of experimental studies, the kinetic mechanisms associated with the Mg(2+) inhibition and Pi regulation of the uniporter function are not well established. To gain a quantitative understanding of the effects of Mg(2+) and Pi on the uniporter function, we developed here a mathematical model based on known kinetic properties of the uniporter and presumed Mg(2+) inhibition and Pi regulation mechanisms. The model is extended from our previous model of the uniporter that is based on a multistate catalytic binding and interconversion mechanism and Eyring's free energy barrier theory for interconversion. The model satisfactorily describes a wide variety of experimental data sets on the kinetics of mitochondrial Ca(2+) uptake. The model also appropriately depicts the inhibitory effect of Mg(2+) on the uniporter function, in which Ca(2+) uptake is hyperbolic in the absence of Mg(2+) and sigmoid in the presence of Mg(2+). The model suggests a mixed-type inhibition mechanism for Mg(2+) inhibition of the uniporter function. This model is critical for building mechanistic models of mitochondrial bioenergetics and Ca(2+) handling to understand the mechanisms by which Ca(2+) mediates signaling pathways and modulates energy metabolism.     

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.     

Mitochondrial calcium channels

Mitochondrial Ca²⁺ handling plays an important role in energy production and various cellular signaling processes. Mitochondrial Ca²⁺ uptake is regulated by the mitochondrial Ca²⁺ uniporter (MCU), at least one non-MCU Ca²⁺ channel and possibly a mitochondrial ryanodine receptor. Two distinct mechanisms mediate Ca²⁺ outward transport, the Na⁺-dependent (mNCX) and the Na⁺-independent Ca²⁺ efflux. In recent years we gained more insight into the regulation and function of these different Ca²⁺ transport mechanisms. However, the precise physiological role and the molecular structure of all mitochondrial Ca²⁺ transporters and channels still has to be determined.     

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.     

A Biophysically Based Mathematical Model for the Kinetics of Mitochondrial Na-Ca Antiporter

Sodium-calcium antiporter is the primary efflux pathway for Ca²⁺ in respiring mitochondria, and hence plays an important role in mitochondrial Ca²⁺ homeostasis. Although experimental data on the kinetics of Na⁺-Ca²⁺ antiporter are available, the structure and composition of its functional unit and kinetic mechanisms associated with the Na⁺-Ca²⁺ exchange (including the stoichiometry) remains unclear. To gain a quantitative understanding of mitochondrial Ca²⁺ homeostasis, a biophysical model of Na⁺-Ca²⁺ antiporter is introduced that is thermodynamically balanced and satisfactorily describes a number of independent data sets under a variety of experimental conditions. The model is based on a multistate catalytic binding mechanism for carrier-mediated facilitated transport and Eyring's free energy barrier theory for interconversion and electrodiffusion. The model predicts the activating effect of membrane potential on the antiporter function for a 3Na⁺:1Ca²⁺ electrogenic exchange as well as the inhibitory effects of both high and low pH seen experimentally. The model is useful for further development of mechanistic integrated models of mitochondrial Ca²⁺ handling and bioenergetics to understand the mechanisms by which Ca²⁺ plays a role in mitochondrial signaling pathways and energy metabolism.     

Mitochondrial Ca uptake with and without the formation of high-Ca microdomains

The mitochondrial Ca²⁺ uniporter has low affinity for Ca²⁺, therefore it has been assumed that submicromolar Ca²⁺ signals cannot induce mitochondrial Ca²⁺ uptake. The close apposition of the plasma membrane or the endoplamic reticulum (ER) to the mitochondria and the limited Ca²⁺ diffusion in the cytoplasm result in the formation of perimitochondrial high-Ca²⁺ microdomains (HCMDs) capable of activating mitochondrial Ca²⁺ uptake. The possibility of mitochondrial Ca²⁺ uptake at low submicromolar [Ca²⁺]_c has not yet been generally accepted.Earlier we found in permeabilized glomerulosa, luteal and pancreatic β cells that [Ca²⁺]_m increased when [Ca²⁺]_c was raised from 60 nM to less than 200 nM. Here we report data obtained from H295R (adrenocortical) cells transfected with ER-targeted GFP. Cytoplasmic Ca²⁺ response to angiotensin II was different in mitochondrion-rich and mitochondrion-free domains. The mitochondrial Ca²⁺ response to angiotensin II correlated with GFP fluorescence indicating the vicinity of ER. When the cells were exposed to K⁺ (inducing Ca²⁺ influx), no correlation was found between the mitochondrial Ca²⁺ signal and the vicinity of the plasma membrane or the ER. The results presented here provide evidence that mitochondrial Ca²⁺ uptake may occur both with and without the formation of HCMDs within the same cell.     

Mitochondrial Ca channels: Great unknowns with important functions

Mitochondria process local and global Ca²⁺ signals. Thereby the spatiotemporal patterns of mitochondrial Ca²⁺ signals determine whether the metabolism of these organelles is adjusted or cell death is executed. Mitochondrial Ca²⁺ channels of the inner mitochondrial membrane (IMM) actually implement mitochondrial uptake from cytosolic Ca²⁺ rises. Despite great efforts in the past, the identity of mitochondrial Ca²⁺ channels is still elusive. Numerous studies aimed to characterize mitochondrial Ca²⁺ uniport channels and provided a detailed profile of these great unknowns with important functions. This mini-review revisits previous research on the mechanisms of mitochondrial Ca²⁺ uptake and aligns them with most recent findings.     

Mitochondrial calcium signalling and cell death: Approaches for assessing the role of mitochondrial Ca uptake in apoptosis

Local Ca²⁺ transfer between adjoining domains of the sarcoendoplasmic reticulum (ER/SR) and mitochondria allows ER/SR Ca²⁺ release to activate mitochondrial Ca²⁺ uptake and to evoke a matrix [Ca²⁺] ([Ca²⁺]_m) rise. [Ca²⁺]_m exerts control on several steps of energy metabolism to synchronize ATP generation with cell function. However, calcium signal propagation to the mitochondria may also ignite a cell death program through opening of the permeability transition pore (PTP). This occurs when the Ca²⁺ release from the ER/SR is enhanced or is coincident with sensitization of the PTP. Recent studies have shown that several pro-apoptotic factors, including members of the Bcl-2 family proteins and reactive oxygen species (ROS) regulate the Ca²⁺ sensitivity of both the Ca²⁺ release channels in the ER and the PTP in the mitochondria. To test the relevance of the mitochondrial Ca²⁺ accumulation in various apoptotic paradigms, methods are available for buffering of [Ca²⁺], for dissipation of the driving force of the mitochondrial Ca²⁺ uptake and for inhibition of the mitochondrial Ca²⁺ transport mechanisms. However, in intact cells, the efficacy and the specificity of these approaches have to be established. Here we discuss mechanisms that recruit the mitochondrial calcium signal to a pro-apoptotic cascade and the approaches available for assessment of the relevance of the mitochondrial Ca²⁺ handling in apoptosis. We also present a systematic evaluation of the effect of ruthenium red and Ru360, two inhibitors of mitochondrial Ca²⁺ uptake on cytosolic [Ca²⁺] and [Ca²⁺]_m in intact cultured cells.     

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.     

Mitochondrial Ca2+ uptake with and without the formation of high-Ca2+ microdomains

The mitochondrial Ca²⁺ uniporter has low affinity for Ca²⁺, therefore it has been assumed that submicromolar Ca²⁺ signals cannot induce mitochondrial Ca²⁺ uptake. The close apposition of the plasma membrane or the endoplamic reticulum (ER) to the mitochondria and the limited Ca²⁺ diffusion in the cytoplasm result in the formation of perimitochondrial high-Ca²⁺ microdomains (HCMDs) capable of activating mitochondrial Ca²⁺ uptake. The possibility of mitochondrial Ca²⁺ uptake at low submicromolar [Ca²⁺]_c has not yet been generally accepted.Earlier we found in permeabilized glomerulosa, luteal and pancreatic β cells that [Ca²⁺]_m increased when [Ca²⁺]_c was raised from 60 nM to less than 200 nM. Here we report data obtained from H295R (adrenocortical) cells transfected with ER-targeted GFP. Cytoplasmic Ca²⁺ response to angiotensin II was different in mitochondrion-rich and mitochondrion-free domains. The mitochondrial Ca²⁺ response to angiotensin II correlated with GFP fluorescence indicating the vicinity of ER. When the cells were exposed to K⁺ (inducing Ca²⁺ influx), no correlation was found between the mitochondrial Ca²⁺ signal and the vicinity of the plasma membrane or the ER. The results presented here provide evidence that mitochondrial Ca²⁺ uptake may occur both with and without the formation of HCMDs within the same cell.     

Mitochondrial Free [Ca] Increases during ATP/ADP Antiport and ADP Phosphorylation: Exploration of Mechanisms

ADP influx and ADP phosphorylation may alter mitochondrial free [Ca²⁺] ([Ca²⁺]_m) and consequently mitochondrial bioenergetics by several postulated mechanisms. We tested how [Ca²⁺]_m is affected by H₂PO₄⁻ (P_i), Mg²⁺, calcium uniporter activity, matrix volume changes, and the bioenergetic state. We measured [Ca²⁺]_m, membrane potential, redox state, matrix volume, pH_m, and O₂ consumption in guinea pig heart mitochondria with or without ruthenium red, carboxyatractyloside, or oligomycin, and at several levels of Mg²⁺ and P_i. Energized mitochondria showed a dose-dependent increase in [Ca²⁺]_m after adding CaCl₂ equivalent to 20, 114, and 485 nM extramatrix free [Ca²⁺] ([Ca²⁺]_e); this uptake was attenuated at higher buffer Mg²⁺. Adding ADP transiently increased [Ca²⁺]_m up to twofold. The ADP effect on increasing [Ca²⁺]_m could be partially attributed to matrix contraction, but was little affected by ruthenium red or changes in Mg²⁺ or P_i. Oligomycin largely reduced the increase in [Ca²⁺]_m by ADP compared to control, and [Ca²⁺]_m did not return to baseline. Carboxyatractyloside prevented the ADP-induced [Ca²⁺]_m increase. Adding CaCl₂ had no effect on bioenergetics, except for a small increase in state 2 and state 4 respiration at 485 nM [Ca²⁺]_e. These data suggest that matrix ADP influx and subsequent phosphorylation increase [Ca²⁺]_m largely due to the interaction of matrix Ca²⁺ with ATP, ADP, P_i, and cation buffering proteins in the matrix.     

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.     

Mitochondrial Ca<Superscript>2+</Superscript> uptake pathways

Calcium (Ca²⁺) plays diverse roles in all living organisms ranging from bacteria to humans. It is a structural element for bones, an essential mediator of excitation-contraction coupling, and a universal second messenger in the regulation of ion channel, enzyme and gene expression activities. In mitochondria, Ca²⁺ is crucial for the control of energy production and cellular responses to metabolic stress. Ca²⁺ uptake by the mitochondria occurs by the uniporter mechanism. The Mitochondrial Ca2+ Uniporter (MCU) protein has recently been identified as a core component responsible for mitochondrial Ca²⁺ uptake. MCU knockout (MCU KO) studies have identified a number of important roles played by this high capacity uptake pathway. Interestingly, this work has also shown that MCU-mediated Ca²⁺ uptake is not essential for vital cell functions such as muscle contraction, energy metabolism and neurotransmission. Although mitochondrial Ca²⁺ uptake was markedly reduced, MCU KO mitochondria still contained low but detectable levels of Ca²⁺. In view of the fundamental importance of Ca²⁺ for basic cell signalling, this finding suggests the existence of other currently unrecognized pathways for Ca²⁺ entry. We review the experimental evidence for the existence of alternative Ca²⁺ influx mechanisms and propose how these mechanisms may play an integral role in mitochondrial Ca²⁺ signalling.     

Mitochondrial Calcium Uniporter MCU Supports Cytoplasmic Ca2+ Oscillations,Store-Operated Ca2+ Entry and Ca2+-Dependent Gene Expression in Response to Receptor Stimulation

Ca²⁺ flux into mitochondria is an important regulator of cytoplasmic Ca²⁺ signals, energy production and cell death pathways. Ca²⁺ uptake can occur through the recently discovered mitochondrial uniporter channel (MCU) but whether the MCU is involved in shaping Ca²⁺ signals and downstream responses to physiological levels of receptor stimulation is unknown. Here, we show that modest stimulation of leukotriene receptors with the pro-inflammatory signal LTC₄ evokes a series of cytoplasmic Ca²⁺ oscillations that are rapidly and faithfully propagated into mitochondrial matrix. Knockdown of MCU or mitochondrial depolarisation, to reduce the driving force for Ca²⁺ entry into the matrix, prevents the mitochondrial Ca²⁺ rise and accelerates run down of the oscillations. The loss of cytoplasmic Ca²⁺ oscillations appeared to be a consequence of enhanced Ca²⁺-dependent inactivation of InsP₃ receptors, which arose from the loss of mitochondrial Ca²⁺ buffering. Ca²⁺ dependent gene expression in response to leukotriene receptor activation was suppressed following knockdown of the MCU. In addition to buffering Ca²⁺ release, mitochondria also sequestrated Ca²⁺ entry through store-operated Ca²⁺ channels and this too was prevented following loss of MCU. MCU is therefore an important regulator of physiological pulses of cytoplasmic Ca²⁺.     

Mitochondrial Ca transport and permeability transition in zebrafish (Danio rerio)

We have studied mitochondrial Ca²⁺ transport and the permeability transition (PT) in the teleost zebrafish (Danio rerio), a key model system for human diseases. Permeabilized zebrafish embryo cells displayed a mitochondrial energy-dependent Ca²⁺ uptake system that, like the Ca²⁺ uniporter of mammals, was inhibited by ruthenium red. Zebrafish mitochondria underwent a Ca²⁺-dependent PT that displayed Pi-dependent desensitization by cyclosporin A, and responded appropriately to key modulators of the mammalian PT pore (voltage, pH, ubiquinone 0, dithiol oxidants and cross linkers, ligands of the adenine nucleotide translocator, arachidonic acid). Opening of the pore was documented in intact cells, where it led to death that could largely be prevented by cyclosporin A. Our results represent a necessary step toward the use of zebrafish for the screening and validation of PTP inhibitors of potential use in human diseases, as recently shown for collagen VI muscular dystrophy [Telfer et al., 2010].     

Modulation of Intracellular Calcium Waves and Triggered Activities by Mitochondrial Ca Flux in Mouse Cardiomyocytes

Recent studies have suggested that mitochondria may play important roles in the Ca²⁺ homeostasis of cardiac myocytes. However, it is still unclear if mitochondrial Ca²⁺ flux can regulate the generation of Ca²⁺ waves (CaWs) and triggered activities in cardiac myocytes. In the present study, intracellular/cytosolic Ca²⁺ (Ca_i ²⁺) was imaged in Fluo-4-AM loaded mouse ventricular myocytes. Spontaneous sarcoplasmic reticulum (SR) Ca²⁺ release and CaWs were induced in the presence of high (4 mM) external Ca²⁺ (Ca_o ²⁺). The protonophore carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (FCCP) reversibly raised basal Ca_i ²⁺ levels even after depletion of SR Ca²⁺ in the absence of Ca_o ²⁺ , suggesting Ca²⁺ release from mitochondria. FCCP at 0.01 - 0.1 µM partially depolarized the mitochondrial membrane potential (Δψ _m) and increased the frequency and amplitude of CaWs in a dose-dependent manner. Simultaneous recording of cell membrane potentials showed the augmentation of delayed afterdepolarization amplitudes and frequencies, and induction of triggered action potentials. The effect of FCCP on CaWs was mimicked by antimycin A (an electron transport chain inhibitor disrupting Δψ _m) or Ru360 (a mitochondrial Ca²⁺ uniporter inhibitor), but not by oligomycin (an ATP synthase inhibitor) or iodoacetic acid (a glycolytic inhibitor), excluding the contribution of intracellular ATP levels. The effects of FCCP on CaWs were counteracted by the mitochondrial permeability transition pore blocker cyclosporine A, or the mitochondrial Ca²⁺ uniporter activator kaempferol. Our results suggest that mitochondrial Ca²⁺ release and uptake exquisitely control the local Ca²⁺ level in the micro-domain near SR ryanodine receptors and play an important role in regulation of intracellular CaWs and arrhythmogenesis.     

SLC25A23 augments mitochondrial Ca2+ uptake,interacts with MCU,and induces oxidative stress–mediated cell death

Emerging findings suggest that two lineages of mitochondrial Ca²⁺ uptake participate during active and resting states: 1) the major eukaryotic membrane potential–dependent mitochondrial Ca²⁺ uniporter and 2) the evolutionarily conserved exchangers and solute carriers, which are also involved in ion transport. Although the influx of Ca²⁺ across the inner mitochondrial membrane maintains metabolic functions and cell death signal transduction, the mechanisms that regulate mitochondrial Ca²⁺ accumulation are unclear. Solute carriers—solute carrier 25A23 (SLC25A23), SLC25A24, and SLC25A25—represent a family of EF-hand–containing mitochondrial proteins that transport Mg-ATP/Pi across the inner membrane. RNA interference–mediated knockdown of SLC25A23 but not SLC25A24 and SLC25A25 decreases mitochondrial Ca²⁺ uptake and reduces cytosolic Ca²⁺ clearance after histamine stimulation. Ectopic expression of SLC25A23 EF-hand–domain mutants exhibits a dominant-negative phenotype of reduced mitochondrial Ca²⁺ uptake. In addition, SLC25A23 interacts with mitochondrial Ca²⁺ uniporter (MCU; CCDC109A) and MICU1 (CBARA1) while also increasing I_MCU. In addition, SLC25A23 knockdown lowers basal mROS accumulation, attenuates oxidant-induced ATP decline, and reduces cell death. Further, reconstitution with short hairpin RNA–insensitive SLC25A23 cDNA restores mitochondrial Ca²⁺ uptake and superoxide production. These findings indicate that SLC25A23 plays an important role in mitochondrial matrix Ca²⁺ influx.     

Mitochondrial inhibitors activate influx of external Ca in sea urchin sperm

Sea urchin sperm have a single mitochondrion which, aside from its main ATP generating function, may regulate motility, intracellular Ca²⁺ concentration ([Ca²⁺]_i) and possibly the acrosome reaction (AR). We have found that acute application of agents that inhibit mitochondrial function via differing mechanisms (CCCP, a proton gradient uncoupler, antimycin, a respiratory chain inhibitor, oligomycin, a mitochondrial ATPase inhibitor and CGP37157, a Na⁺/Ca²⁺ exchange inhibitor) increases [Ca²⁺]_i with at least two differing profiles. These increases depend on the presence of extracellular Ca²⁺, which indicates they involve Ca²⁺ uptake and not only mitochondrial Ca²⁺ release. The plasma membrane permeation pathways activated by the mitochondrial inhibitors are permeable to Mn²⁺. Store-operated Ca²⁺ channel (SOC) blockers (Ni²⁺, SKF96365 and Gd²⁺) and internal-store ATPase inhibitors (thapsigargin and bisphenol) antagonize Ca²⁺ influx induced by the mitochondrial inhibitors. The results indicate that the functional status of the sea urchin sperm mitochondrion regulates Ca²⁺ entry through SOCs. As neither CCCP nor dicycloexyl carbodiimide (DCCD), another mitochondrial ATPase inhibitor, eliminate the oligomycin induced increase in [Ca²⁺]_i, apparently oligomycin also has an extra mitochondrial target.