In yeast, Ca2+ and octylguanidine interact with porin (VDAC) preventing the mitochondrial permeability transition

In yeast, Ca(2+) and long chain alkylguanidines interact with mitochondria modulating the opening of the yeast mitochondrial unspecific channel. Mammalians possess a similar structure, the mitochondrial permeability transition pore. The composition of these pores is under debate. Among other components, the voltage-dependent anion channel has been proposed as a component of either pore. In yeast from an industrial strain, octylguanidine and calcium closed the yeast mitochondrial unspecific channel. Here, the effects of the cations Ca(2+) or octylguanidine and the voltage-dependent anion channel effector decavanadate were evaluated in yeast mitochondria from either a wild type or a voltage-dependent anion channel deletion laboratory strain. It was observed that in the absence of voltage-dependent anion channel, the yeast mitochondrial unspecific channel was desensitized to Ca(2+), octylguanidine or decavanadate but remained sensitive to phosphate. It is thus suggested that in yeast mitochondria, the voltage-dependent anion channel has a cation binding site where Ca(2+) and octylguanidine interact, conferring cation sensitivity to the yeast mitochondrial unspecific channel.     

The permeability transition pore as a mitochondrial calcium release channel: A critical appraisal

Mitochondria from a variety of sources possess an inner membrane channel, the permeability transition pore. The pore is a voltage-dependent channel, activated by matrix Ca²⁺ and inhibited by matrix H⁺, which can be blocked by cyclosporin A, presumably after binding to mitochondrial cyclophilin. The physiological function of the permeability transition pore remains unknown. Here we evaluate its potential role as a fast Ca²⁺ release channel involved in mitochondrial and cellular Ca²⁺ homeostasis. We (i) discuss the theoretical and experimental reasons why mitochondria need a fast, inducible Ca²⁺ release channel; (ii) analyze the striking analogies between the mitochondrial permeability transition pore and the sarcoplasmic reticulum ryanodine receptor-Ca²⁺ release channel; (iii) argue that the permeability transition pore can act as a selective release channel for Ca²⁺ despite its apparent lack of selectivity for the transported speciesin vitro; and (iv) discuss the importance of mitochondria in cellular Ca²⁺ homeostasis, and how disruption of this function could impinge upon cell viability, particularly under conditions of oxidative stress.     

Closure of the yeast mitochondria unspecific channel (YMUC) unmasks a Mg2+ and quinine sensitive K+ uptake pathway in Saccharomyces cerevisiae

The K⁺ uptake pathways in yeast mitochondria are still undefined. Nonetheless, the K⁺-mediated mitochondrial swelling observed in the absence of phosphate (PO₄) and in the presence of a respiratory substrate has led to propose that large K⁺ movements occur in yeast mitochondria. Thus, the uptake of K⁺ by isolated yeast mitochondria was evaluated. Two parallel experiments were conducted to evaluate K⁺ transport; these were mitochondrial swelling and the uptake of the radioactive K⁺ analog ⁸⁶Rb⁺. The opening of the yeast mitochondrial unspecific channel (YMUC) was regulated by different PO₄ concentrations. The high protein concentrations used to measure ⁸⁶Rb⁺ uptake resulted in a slight stabilization of the transmembrane potential at 0.4 mM PO₄ but not at 0 or 4 mM PO₄. At 4 mM PO₄ swelling was inhibited while, in contrast, ⁸⁶Rb⁺ uptake was still observed. The results suggest that an energy-dependent K⁺ uptake mechanism was unmasked when the YMUC was closed. To further analyze the properties of this K⁺ uptake system, the Mg²⁺ and quinine sensitivity of both swelling and ⁸⁶Rb⁺ uptake were evaluated. Under the conditions where the unspecific pore was closed, K⁺ transport sensitivity to Mg²⁺ and quinine increased. In addition, when Zn²⁺ was added as an antiport inhibitor, uptake of ⁸⁶Rb⁺ increased. It is suggested that in yeast mitochondria, the K⁺ concentration is highly regulated by the equilibrium of uptake and exit of this cation through two specific transporters.     

In Saccharomyces cerevisiae,cations control the fate of the energy derived from oxidative metabolism through the opening and closing of the yeast mitochondrial unselective channel

The yeast mitochondrial unspecific channel (YMUC) sensitivity to inorganic (Ca²⁺ or Mg²⁺) or organic (hexyl or octyl-guanidine) cations was measured. The rate of oxygen consumption in State 3 and State 4, the transmembrane potential (), mitochondrial swelling, and the polyethylene-glycol mediated recontraction were used to follow opening of the YMUC. Addition of 0.4 mM PO₄ did not close the YMUC, although it did enhance the sensitivity to Ca²⁺ (I₅₀ decreased from 50 to 0.3 mM) and Mg²⁺ (I₅₀ decreased from 5 to 0.83 mM Mg²⁺). The Ca²⁺ concentration needed to close the YMUC was higher than the concentrations usually observed in the cell. Nonetheless, Mg²⁺, Ca²⁺, and PO₄ exhibited additive effects. These cations did not inhibit contraction of preswollen mitochondria, suggesting that the YMUC/cation interaction was labile. Octyl-guanidine (OG-I₅₀ 7.5 M) was the only cation which inhibited mitochondrial recontraction, probably as a result of membrane binding stabilization through its hydrophobic tail. The PO₄-dependent, Ca²⁺/Mg²⁺-mediated closure of the YMUC may be a means to control the proportion of oxidative energy producing ATP or being lost as heat.     

In vitro characterization of scaffold-free three-dimensional mesenchymal stem cell aggregates

Cerebral ischemia is a key pathophysiological feature of various brain insults. Inadequate oxygen supply can manifest regionally in stroke or as a result of traumatic brain injury or globally following cardiac arrest, all leading to irreversible brain damage. Mitochondrial function is essential for neuronal survival, since neurons critically depend on ATP synthesis generated by mitochondrial oxidative phosphorylation. Mitochondrial activity depends on Ca²⁺ and is fueled either by Ca²⁺ from the extracellular space when triggered by neuronal activity or by Ca²⁺ released from the endoplasmic reticulum (ER) and taken up through specialized contact sites between the ER and mitochondria known as mitochondrial-associated ER membranes. The coordination of these Ca²⁺ pools is required to synchronize mitochondrial respiration rates and ATP synthesis to physiological demands. In this review, we discuss the role of the proteins involved in mitochondrial Ca²⁺ homeostasis in models of ischemia. The proteins include those important for the Ca²⁺-dependent motility of mitochondria and for Ca²⁺ transfer from the ER to mitochondria, the tethering proteins that bring the two organelles together, inositol 1,4,5-triphosphate receptors that enable Ca²⁺ release from the ER, voltage-dependent anion channels that allow Ca²⁺ entry through the highly permeable outer mitochondrial membrane and the mitochondrial Ca²⁺ uniporter together with its regulatory proteins that permit Ca²⁺ entry into the mitochondrial matrix. Finally, we address those proteins important for the extrusion of Ca²⁺ from the mitochondria such as the mitochondrial Na⁺/Ca²⁺ exchanger or, if the mitochondrial Ca²⁺ concentration exceeds a certain threshold, the mitochondrial permeability transition pore.     

The effect of taurine on the ion transport system in mitochondria

The effect of taurine on the ATP-dependent mitochondrial swelling that characterizes the activity of mitochondrial ATP-dependent K⁺ channel and the formation of Ca²⁺-dependent pores, different in sensitivity to cyclosporin A, has been studied in rat liver mitochondria. It has been shown that taurine in micromolar concentrations (0.5–125 μM) stimulates the energy-dependent swelling of mitochondria. Taurine in physiological concentrations (0.5–20 mM) has no effect on the ATP-dependent swelling and the formation of cyclosporin A-insensitive Pal/Ca²⁺-activated pore in mitochondria. Taurine in these concentrations increased the rate of cyclosporin A-sensitive swelling of mitochondria induced by Ca²⁺ and P_i and reduced the Ca²⁺ capacity of mitochondria. The different effects of physiological taurine concentrations on the ATP-dependent transport of K⁺ and Ca²⁺ ions in mitochondrial membranes as compared with cell membranes are discussed.     

The Voltage-dependent Anion Channel in Endoplasmic/Sarcoplasmic Reticulum: Characterization,Modulation and Possible Function

In recent years, it has been recognized that there is a metabolic coupling between the cytosol, ER/SR and mitochondria. In this cross-talk, mitochondrial Ca²⁺ homeostasis and ATP production and supply play a major role. The primary transporter of adenine nucleotides, Ca²⁺and other metabolites into and out of mitochondria is the voltage-dependent anion channel (VDAC) located at the outer mitochondrial membrane, at a crucial position in the cell. VDAC has been established as a key player in mitochondrial metabolite and ion signaling and it has also been proposed that VDAC is present in extramitochondrial membranes. Thus, regulation of VDAC, as the main interface between mitochondrial and cellular metabolism, by other molecules is of utmost importance. This article reviews localization and function of VDAC, and focuses on VDAC as a skeletal muscle sarcoplasmic reticulum channel. The regulation of VDAC activity by associated proteins and by inhibitors is also presented. Several aspects of the physiological relevance of VDAC to Ca²⁺ homeostasis and mitochondria-mediated apoptosis will be discussed.     

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 Ca<Superscript>2+</Superscript> cycle mediated by the palmitate-activated cyclosporin a-insensitive pore

Earlier we found that in isolated rat liver mitochondria the reversible opening of the mitochondrial cyclosporin A-insensitive pore induced by low concentrations of palmitic acid (Pal) plus Ca²⁺ results in the brief loss of Δψ [Mironova et al., J Bioenerg Biomembr (2004), 36:171–178]. Now we report that Pal and Ca²⁺, increased to 30 and 70 nmol/mg protein respectively, induce a stable and prolonged (10 min) partial depolarization of the mitochondrial membrane, the release of Ca²⁺ and the swelling of mitochondria. Inhibitors of the Ca²⁺ uniporter, ruthenium red and La³⁺, as well as EGTA added in 10 min after the Pal/Ca²⁺-activated pore opening, prevent the release of Ca²⁺ and repolarize the membrane to initial level. Similar effects can be observed in the absence of exogeneous Pal, upon mitochondria accumulating high [Sr²⁺], which leads to the activation of phospholipase A₂ and appearance of endogenous fatty acids. The paper proposes a new model of the mitochondrial Ca²⁺ cycle, in which Ca²⁺ uptake is mediated by the Ca²⁺ uniporter and Ca²⁺ efflux occurs via a short-living Pal/Ca²⁺-activated pore.     

Functional analysis of coiled-coil domains of MCU in mitochondrial calcium uptake

The mitochondrial calcium uniporter (MCU) complex is a highly-selective calcium channel. This complex consists of MCU, mitochondrial calcium uptake proteins (MICUs), MCU regulator 1 (MCUR1), essential MCU regulator element (EMRE), etc. MCU, which is the pore-forming subunit, has 2 highly conserved coiled-coil domains (CC1 and CC2); however, their functional roles are unknown. The yeast expression system of mammalian MCU and EMRE enables precise reconstitution of the properties of the mammalian MCU complex in yeast mitochondria. Using the yeast expression system, we here showed that, when MCU mutant lacking CC1 or CC2 was expressed together with EMRE in yeast, their mitochondrial Ca²⁺-uptake function was lost. Additionally, point mutations in CC1 or CC2, which were expected to prevent the formation of the coiled coil, also disrupted the Ca²⁺-uptake function. Thus, it is essential for the Ca²⁺ uptake function of MCU that the coiled-coil structure be formed in CC1 and CC2. The loss of function of those mutated MCUs was also observed in the mitochondria of a yeast strain lacking the yeast MCUR1 homolog. Also, in the D. discoideum MCU, which has EMRE-independent Ca²⁺-uptake function, the deletion of either CC1 or CC2 caused the loss of function. These results indicated that the critical functions of CC1 and CC2 were independent of other regulatory subunits such as MCUR1 and EMRE, suggesting that CC1 and CC2 might be essential for pore formation by MCUs themselves. Based on the tetrameric structure of MCU, we discussed the functional roles of the coiled-coil domains of MCU.     

Properties of the permeability transition in VDAC1−/− mitochondria

Opening of the permeability transition pore (PTP), a high-conductance mitochondrial channel, causes mitochondrial dysfunction with Ca²⁺ deregulation, ATP depletion, release of pyridine nucleotides and of mitochondrial apoptogenic proteins. Despite major efforts, the molecular nature of the PTP remains elusive. A compound library screening led to the identification of a novel high affinity PTP inhibitor (Ro 68-3400), which labeled a ∼32 kDa protein that was identified as isoform 1 of the voltage-dependent anion channel (VDAC1) [A.M. Cesura, E. Pinard, R. Schubenel, V. Goetschy, A. Friedlein, H. Langen, P. Polcic, M.A. Forte, P. Bernardi, J.A. Kemp, The voltage-dependent anion channel is the target for a new class of inhibitors of the mitochondrial permeability transition pore. J. Biol. Chem. 278 (2003) 49812–49818]. In order to assess the role of VDAC1 in PTP formation and activity, we have studied the properties of mitochondria from VDAC1^−/− mice. The basic properties of the PTP in VDAC1^−/− mitochondria were indistinguishable from those of strain-matched mitochondria from wild-type CD1 mice, including inhibition by Ro 68-3400, which labeled identical proteins of 32 kDa in both wild-type and VDAC1^−/− mitochondria. The labeled protein could be separated from all VDAC isoforms. While these results do not allow to exclude that VDAC is part of the PTP, they suggest that VDAC is not the target for PTP inhibition by Ro 68-3400.     

Distinct characteristics of Ca-induced depolarization of isolated brain and liver mitochondria

Ca²⁺-induced mitochondrial depolarization was studied in single isolated rat brain and liver mitochondria. Digital imaging techniques and rhodamine 123 were used for mitochondrial membrane potential measurements. Low Ca²⁺ concentrations (about 30-100 nM) initiated oscillations of the membrane potential followed by complete depolarization in brain mitochondria. In contrast, liver mitochondria were less sensitive to Ca²⁺; 20 μM Ca²⁺ was required to depolarize liver mitochondria. Ca²⁺ did not initiate oscillatory depolarizations in liver mitochondria, where each individual mitochondrion depolarized abruptly and irreversibly. Adenine nucleotides dramatically reduced the oscillatory depolarization in brain mitochondria and delayed the onset of the depolarization in liver mitochondria. In both type of mitochondria, the stabilizing effect of adenine nucleotides completely abolished by an inhibition of adenine nucleotide translocator function with carboxyatractyloside, but was not sensitive to bongkrekic acid. Inhibitors of mitochondrial permeability transition cyclosporine A and bongkrekic acid also delayed Ca²⁺-depolarization. We hypothesize that the oscillatory depolarization in brain mitochondria is associated with the transient conformational change of the adenine nucleotide translocator from a specific transporter to a non-specific pore, whereas the non-oscillatory depolarization in liver mitochondria is caused by the irreversible opening of the pore.     

Induction of permeability of the inner membrane of yeast mitochondria

The current view on apoptosis is given, with a special emphasis placed on apoptosis in yeasts. Induction of a non-specific permeability transition pore (mPTP) in mammalian and yeast mitochondria is described, particularly in mitochon-dria from Yarrowia lipolytica and Dipodascus (Endomyces) magnusii yeasts, which are aerobes possessing the fully competent respiratory chain with all three points of energy conservation and well-structured mitochondria. They were examined for their ability to induce an elevated permeability transition of the inner mitochondrial membrane, being subjected to virtually all conditions known to induce the mPTP in animal mitochondria. Yeast mitochondria do not form Ca²⁺-dependent pores, neither the classical Ca²⁺/P_i-dependent, cyclosporin A-sensitive pore even under deenergization of mitochondria or depletion of the intramitochondrial nucleotide pools, nor a pore induced in mammalian mitochondria upon concerted action of moderate Ca²⁺ concentrations (in the presence of the Ca²⁺ ionophore ETH129) and saturated fatty acids. No pore formation was found in yeast mitochondria in the presence of elevated phosphate concentrations at acidic pH values. It is concluded that the permeability transition in yeast mitochondria is not coupled with Ca²⁺ uptake and is differently regulated compared to the mPTP of animal mitochondria.     

Induction of a non-specific permeability transition in mitochondria from Yarrowia lipolytica and Dipodascus (Endomyces) magnusii yeasts

In this study we used tightly-coupled mitochondria from Yarrowia lipolytica and Dipodascus (Endomyces) magnusii yeasts, possessing a respiratory chain with the usual three points of energy conservation. High-amplitude swelling and collapse of the membrane potential were used as parameters for demonstrating induction of the mitochondrial permeability transition due to opening of a pore (mPTP). Mitochondria from Y. lipolytica, lacking a natural mitochondrial Ca²⁺ uptake pathway, and from D. magnusii, harboring a high-capacitive, regulated mitochondrial Ca²⁺ transport system (Bazhenova et al. J Biol Chem 273:4372–4377, 1998a; Bazhenova et al. Biochim Biophys Acta 1371:96–100, 1998b; Deryabina and Zvyagilskaya Biochemistry (Moscow) 65:1352–1356, 2000; Deryabina et al. J Biol Chem 276:47801–47806, 2001) were very resistant to Ca²⁺ overload. However, exposure of yeast mitochondria to 50–100 μM Ca²⁺ in the presence of the Ca²⁺ ionophore ETH129 induced collapse of the membrane potential, possibly due to activation of the fatty acid-dependent Ca²⁺/nH⁺-antiporter, with no classical mPTP induction. The absence of response in yeast mitochondria was not simply due to structural limitations, since large-amplitude swelling occurred in the presence of alamethicin, a hydrophobic, helical peptide, forming voltage-sensitive ion channels in lipid membranes. Ca²⁺- ETH129-induced activation of the Ca²⁺/H⁺-antiport system was inhibited and prevented by bovine serum albumin, and partially by inorganic phosphate and ATP. We subjected yeast mitochondria to other conditions known to induce the permeability transition in animal mitochondria, i.e., Ca²⁺ overload (in the presence of ETH129) combined with palmitic acid (Mironova et al. J Bioenerg Biomembr 33:319–331, 2001; Sultan and Sokolove Arch Biochem Biophys 386:37–51, 2001), SH-reagents, carboxyatractyloside (an inhibitor of the ADP/ATP translocator), depletion of intramitochondrial adenine nucleotide pools, deenergization of mitochondria, and shifting to acidic pH values in the presence of high phosphate concentrations. None of the above-mentioned substances or conditions induced a mPTP-like pore. It is thus evident that the permeability transition in yeast mitochondria is not coupled with Ca²⁺ uptake and is differently regulated compared to the mPTP of animal mitochondria.     

Activity of the yeast vacuolar TRP channel TRPY1 is inhibited by Ca2+–calmodulin binding

Transient receptor potential (TRP) cation channels, which are conserved across mammals, flies, fish, sea squirts, worms, and fungi, essentially contribute to cellular Ca²⁺ signaling. The activity of the unique TRP channel in yeast, TRP yeast channel 1 (TRPY1), relies on the vacuolar and cytoplasmic Ca²⁺ concentration. However, the mechanism(s) of Ca²⁺-dependent regulation of TRPY1 and possible contribution(s) of Ca²⁺-binding proteins are yet not well understood. Our results demonstrate a Ca²⁺-dependent binding of yeast calmodulin (CaM) to TRPY1. TRPY1 activity was increased in the cmd1–6 yeast strain, carrying a non–Ca²⁺-binding CaM mutant, compared with the parent strain expressing wt CaM (Cmd1). Expression of Cmd1 in cmd1–6 yeast rescued the wt phenotype. In addition, in human embryonic kidney 293 cells, hypertonic shock-induced TRPY1-dependent Ca²⁺ influx and Ca²⁺ release were increased by the CaM antagonist ophiobolin A. We found that coexpression of mammalian CaM impeded the activity of TRPY1 by reinforcing effects of endogenous CaM. Finally, inhibition of TRPY1 by Ca²⁺–CaM required the cytoplasmic amino acid stretch E₃₃–Y₉₂. In summary, our results show that TRPY1 is under inhibitory control of Ca²⁺–CaM and that mammalian CaM can replace yeast CaM for this inhibition. These findings add TRPY1 to the innumerable cellular proteins, which include a variety of ion channels, that use CaM as a constitutive or dissociable Ca²⁺-sensing subunit, and contribute to a better understanding of the modulatory mechanisms of Ca²⁺–CaM.     

Coupling of K+-gating and permeation with Ca2+ block in the Ca2+-Activated K+ channel inChara australis

Summary The patch-clamp technique is used here to investigate the kinetics of Ca²⁺ block in single high-conductance Ca²⁺-activated K⁺ channels. These channels are detected in the membrane surounding cytoplasmic drops fromChara australis, a membrane which originates from the tonoplast of the parent cell. The amplitudes and durations of single channel events are measured over a wide range of membrane potential (–300 to 200 mV). Ca²⁺ on either side of the channel reduces its K⁺ conductance and alters its ion-gating characteristics in a voltage-dependent manner. This Ca²⁺-induced attenuation of conductance is analyzed using the theory of diffusion-limited ion flow through pores. Interaction of external Ca²⁺ with the channel's ion-gating mechanism is examined in terms of a kinetic model for ion-gating that includes two voltage-dependent gating mechanisms. The kinetics of channel block by external Ca²⁺ indicates that (i) external Ca²⁺ binds at two sites, a superficial site and a deep site, located at 8 and 40% along the trans-pore potential difference, (ii) the external vestibule cannot be occupied by more than one Ca²⁺ or K⁺, and (iii) the kinetics of Ca²⁺ binding at the deep site is coupled with that of a voltage-dependent gate on the external side of the channel. Kinetics of channel block by internal Ca²⁺ indicates that more than one Ca²⁺ is involved.     

Intracellular Ca<Superscript>2+</Superscript> Pools and Fluxes in Cardiac Muscle-Derived H9c2 Cells

Relevant Ca²⁺ pools and fluxes in H9c2 cells have been studied using fluorescent indicators and Ca²⁺-mobilizing agents. Vasopressin produced a cytoplasmic Ca²⁺ peak with half-maximal effective concentration of 6 nM, whereas thapsigargin-induced Ca²⁺ increase showed half-maximal effect at 3 nM. Depolarization of the mitochondrial inner membrane by protonophore was also associated with an increase in cytoplasmic Ca²⁺. Ionomycin induced a small and sustained depolarization, while thapsigargin had a small but transient effect. The thapsigargin-sensitive Ca²⁺ pool was also sensitive to ionomycin, whereas the protonophore-sensitive Ca²⁺ pool was not. The vasopressin-induced cytoplasmic Ca²⁺ signal, which caused a reversible discharge of the sarco-endoplasmic reticulum Ca²⁺ pool, was sensed as a mitochondrial Ca²⁺ peak but was unaffected by the permeability transition pore inhibitor cyclosporin A. The mitochondrial Ca²⁺ peak was affected by cyclosporin A when the Ca²⁺ signal was induced by irreversible discharge of the intracellular Ca²⁺ pool, i.e., adding thapsigargin. These observations indicate that the mitochondria interpret the cytoplasmic Ca²⁺ signals generated in the reticular store.     

The role of mitochondrial palmitate/Ca<Superscript>2+</Superscript>-activated pore in palmitate-induced apoptosis

The evidence of possible involvement of the mitochondrial cyclosporin A-insensitive palmitate/Ca²⁺-activated pore in palmitate-induced apoptosis is presented. It has been established that the opening of the palmitate/Ca²⁺-activated pore results in the high-amplitude swelling of mitochondria and the release of the apoptosis-inducing factor from organelles. These processes are accompanied by a short-term slight decrease of membrane potential, which recovers in 1 min. The possible role of the palmitate/Ca²⁺-activated pore in the induction of palmitate-induced apoptosis is discussed.     

Restoration of CA2+-inhibited oxidative phosphorylation in cardiac mitochondria by mitochondrial Ca2+ unloading

Mitochondria, the major source of cellular ATP, display high vulnerability to metabolic stress, in particular to excessive Ca²⁺ loading. Here, we show that Ca²⁺-inhibited mitochondrial ATP generation could be restored through stimulated Ca²⁺ discharge from mitochondrial matrix. This was demonstrated using a Ca²⁺ ionophore or through Na⁺/Ca²⁺ exchange-mediated decrease of mitochondrial Ca²⁺ load. Furthermore, diazoxide, a mitochondrial potassium channel opener, which maintained mitochondrial Ca²⁺ homeostasis, also restored Ca²⁺-inhibited ATP synthesis and preserved the structural integrity of Ca²⁺-challenged mitochondria. Thus, under conditions of excessive mitochondrial Ca²⁺ overload targeting mitochondrial Ca²⁺ transport pathways restores oxidative phosphorylation required for vital cellular processes. This study, therefore, identifies an effective strategy capable to rescue Ca²⁺-disrupted mitochondrial energetics.     

线粒体和细胞内钙自稳平衡

线粒体对胞浆钙信号调节作用的研究已经历较长时间.近年,随着研究方法和技术的不断改进,发现在绝大多数生理条件下,线粒体都能参与胞内钙通信过程.线粒体可感受其周围钙微区的存在从而摄取钙,又可以通过钠-钙交换和大分子孔道将钙释放出来,因此可以调节胞浆钙信号的时空特性,影响相关的细胞功能.但是,由于技术上的局限性,目前的研究仍然存在模糊不清和自相矛盾之处,有待于进一步研究.