Control of mitochondrial membrane potential and ROS formation by reversible phosphorylation of cytochrome c oxidase

Phosphorylation of isolated cytochrome c oxidase from bovine kidney and heart, and of the reconstituted heart enzyme, with protein kinase A, cAMP and ATP turns on the allosteric ATP-inhibition at high ATP/ADP ratios. Also incubation of isolated bovine liver mitochondria only with cAMP and ATP turns on, and subsequent incubation with Ca²⁺ turns off the allosteric ATP-inhibition of cytochrome c oxidase. In the bovine heart enzyme occur only three consensus sequences for cAMP-dependent phosphorylation (in subunits I, III and Vb). The evolutionary conservation of RRYS⁴⁴¹ at the cytosolic side of subunit I, together with the above results, suggest that phosphorylation of Ser⁴⁴¹ turns on the allosteric ATP-inhibition of cytochrome c oxidase. The results support the 'molecular-physiological hypothesis' [29], which proposes a low mitochondrial membrane potential through the allosteric ATP-inhibition. A hormone- or agonist-stimulated increase of cellular [Ca²⁺]] is suggested to activate a mitochondrial protein phosphatase which dephosphorylates cytochrome c oxidase, turns off the allosteric ATP-inhibition and results in increase of mitochondrial membrane potential and ROS formation.     

The role of mitochondrial membrane potential in ischemic heart failure

The molecular events occurring during myocardial infarction and cardioprotection are described with an emphasis on the changes of the mitochondrial membrane potential (ΔΨ_m). The low ΔΨ_m values of the normal beating heart (100–140 mV) are explained by the allosteric ATP-inhibition of cytochrome c oxidase (CcO) through feedback inhibition by ATP at high [ATP]/[ADP] ratios. During ischemia the mechanism is reversibly switched off by signaling through reactive oxygen species (ROS). At reperfusion high ΔΨ_m values cause a burst of ROS production leading to apoptosis and/or necrosis. Ischemic preconditioning is suggested to cause additional phosphorylation of CcO, protecting the enzyme from immediate dephosphorylation via ROS signaling.     

Revisiting Kadenbach: Electron flux rate through cytochrome c‐oxidase determines the ATP‐inhibitory effect and subsequent production of ROS

Mitochondrial respiration is the predominant source of ATP. Excessive rates of electron transport cause a higher production of harmful reactive oxygen species (ROS). There are two regulatory mechanisms known. The first, according to Mitchel, is dependent on the mitochondrial membrane potential that drives ATP synthase for ATP production, and the second, the Kadenbach mechanism, is focussed on the binding of ATP to Cytochrome c Oxidase (CytOx) at high ATP/ADP ratios, which results in an allosteric conformational change to CytOx, causing inhibition. In times of stress, ATP‐dependent inhibition is switched off and the activity of CytOx is exclusively determined by the membrane potential, leading to an increase in ROS production. The second mechanism for respiratory control depends on the quantity of electron transfer to the Heme aa3 of CytOx. When ATP is bound to CytOx the enzyme is inhibited, and ROS formation is decreased, although the mitochondrial membrane potential is increased.     

Control of mitochondrial membrane potential and ROS formation by reversible phosphorylation of cytochrome c oxidase

Phosphorylation of isolated cytochrome c oxidase from bovine kidney and heart, and of the reconstituted heart enzyme, with protein kinase A, cAMP and ATP turns on the allosteric ATP-inhibition at high ATP/ADP ratios. Also incubation of isolated bovine liver mitochondria only with cAMP andATP turns on, and subsequent incubation with Ca2+ turns off the allosteric ATP-inhibition of cytochrome c oxidase. In the bovine heart enzyme occur only three consensus sequences for cAMP-dependent phosphorylation (in subunits I, III and Vb). The evolutionary conservation of RRYS441 at the cytosolic side of subunit I, together with the above results, suggest that phosphorylation of Ser441 turns on the allosteric ATP-inhibition of cytochrome c oxidase. The results support the 'molecular-physiological hypothesis' [29], which proposes a low mitochondrial membrane potential through the allosteric ATP-inhibition. A hormone- or agonist-stimulated increase of cellular [Ca2+] is suggested to activate a mitochondrial protein phosphatase which dephosphorylates cytochrome c oxidase, turns off the allosteric ATP-inhibition and results in increase of mitochondrial membrane potential and ROS formation.     

Multiple phosphorylations of cytochrome c oxidase and their functions

Cytochrome c oxidase (COX), the terminal enzyme of the mitochondrial electron transport chain, is regulated by isozyme expression, allosteric effectors such as the ATP/ADP ratio, and reversible phosphorylation. Of particular interest is the "allosteric ATP-inhibition," which has been hypothesized to keep the mitochondrial membrane potential at low healthy values (<140 mV), thus preventing the formation of superoxide radical anions, which have been implicated in multiple degenerative diseases. It has been proposed that the "allosteric ATP-inhibition" is switched on by the protein kinase A-dependent phosphorylation of COX. The goal of this study was to identify the phosphorylation site(s) involved in the "allosteric ATP-inhibition" of COX. We report the mass spectrometric identification of four new phosphorylation sites in bovine heart COX. The identified phosphorylation sites include Tyr-218 in subunit II, Ser-1 in subunit Va, Ser-2 in subunit Vb, and Ser-1 in subunit VIIc. With the exception of Ser-2 in subunit Vb, the identified phosphorylation sites were found in enzyme samples with and without "allosteric ATP inhibition," making Ser-2 of subunit Vb a candidate site enabling allosteric regulation. We therefore hypothesize that additional phosphorylation(s) may be required for the "allosteric ATP-inhibition," and that these sites may be easily dephosphorylated or difficult to identify by mass spectrometry.     

Mitochondrial energy metabolism is regulated via nuclear-coded subunits of cytochrome c oxidase

A new mechanism on regulation of mitochondrial energy metabolism is proposed on the basis of reversible control of respiration by the intramitochondrial ATP/ADP ratio and slip of proton pumping (decreased H+/e- stoichiometry) in cytochrome c oxidase (COX) at high proton motive force delta p. cAMP-dependent phosphorylation of COX switches on and Ca2+-dependent dephosphorylation switches off the allosteric ATP-inhibition of COX (nucleotides bind to subunit IV). Control of respiration via phosphorylated COX by the ATP/ADP ratio keeps delta p (mainly delta psi(m)) low. Hormone induced Ca2+-dependent dephosphorylation results in loss of ATP-inhibition, increase of respiration and delta p with consequent slip in proton pumping. Slip in COX increases the free energy of reaction, resulting in increased rates of respiration, thermogenesis and ATP-synthesis. Increased delta psi(m) stimulates production of reactive oxygen species (ROS), mutations of mitochondrial DNA and accelerates aging. Slip of proton pumping without dephosphorylation and increase of delta p is found permanently in the liver-type isozyme of COX (subunit VIaL) and at high intramitochondrial ATP/ADP ratios in the heart-type isozyme (subunit VIaH). High substrate pressure (sigmoidal v/s kinetics), palmitate and 3,5-diiodothyronine (binding to subunit Va) increase also delta p, ROS production and slip but without dephosphorylation of COX.     

Phosphorylation and kinetics of mammalian cytochrome c oxidase

The influence of protein phosphorylation on the kinetics of cytochrome c oxidase was investigated by applying Western blotting, mass spectrometry, and kinetic measurements with an oxygen electrode. The isolated enzyme from bovine heart exhibited serine, threonine, and/or tyrosine phosphorylation in various subunits, except subunit I, by using phosphoamino acid-specific antibodies. The kinetics revealed slight inhibition of oxygen uptake in the presence of ATP, as compared with the presence of ADP. Mass spectrometry identified the phosphorylation of Ser-34 at subunit IV and Ser-4 and Thr-35 at subunit Va. Incubation of the isolated enzyme with protein kinase A, cAMP, and ATP resulted in serine and threonine phosphorylation of subunit I, which was correlated with sigmoidal inhibition kinetics in the presence of ATP. This allosteric ATP-inhibition of cytochrome c oxidase was also found in rat heart mitochondria, which had been rapidly prepared in the presence of protein phosphatase inhibitors. The isolated rat heart enzyme, prepared from the mitochondria by blue native gel electrophoresis, showed serine, threonine, and tyrosine phosphorylation of subunit I. It is concluded that the allosteric ATP-inhibition of cytochrome c oxidase, previously suggested to keep the mitochondrial membrane potential and thus the reactive oxygen species production in cells at low levels, occurs in living cells and is based on phosphorylation of cytochrome c oxidase subunit I.     

New control of mitochondrial membrane potential and ROS formation--a hypothesis

A new control of mitochondrial membrane potential delta(psi)m and formation of reactive oxygen species (ROS) is presented, based on allosteric ATP-inhibition of cytochrome c oxidase at high intramitochondrial ATP/ADP ratios. Since the rate of ATP synthesis by the ATP synthase is already maximal at low membrane potentials (100-120 mV), the ATP/ADP ratio will also be maximal at this delta(psi)m (at constant rate of ATP consumption). Therefore the control of respiration by the ATP/ADP-ratio keeps delta(psi)m low. In contrast, the known 'respiratory control' leads to an inhibition of respiration only at high delta(psi)m values (150-200 mV) which cause ROS formation. ATP-inhibition of cytochrome c oxidase is switched on and off by reversible phosphorylation (via cAMP and calcium, respectively). We propose that 'stress hormones' which increase intracellular [Ca2+] also increase delta(psi)m and ROS formation, which promote degenerative diseases and accelerate aging.     

The allosteric ATP-inhibition of cytochrome c oxidase activity is reversibly switched on by cAMP-dependent phosphorylation

In previous studies the allosteric inhibition of cytochrome c oxidase at high intramitochondrial ATP/ADP-ratios via binding of the nucleotides to the matrix domain of subunit IV was demonstrated. Here we show that the allosteric ATP-inhibition of the isolated bovine heart enzyme is switched on by cAMP-dependent phosphorylation with protein kinase A of subunits II (and/or III) and Vb, and switched off by subsequent incubation with protein phosphatase 1. It is suggested that after cAMP-dependent phosphorylation of cytochrome c oxidase mitochondrial respiration is controlled by the ATP/ADP-ratio keeping the proton motive force Deltap low, and the efficiency of energy transduction high. After Ca(2+)-induced dephosphorylation this control is lost, accompanied by increase of Deltap, slip of proton pumping (decreased H(+)/e(-) stoichiometry), and increase of the rate of respiration and ATP-synthesis at a decreased efficiency of energy transduction.     

The possible role of cytochrome c oxidase in stress-induced apoptosis and degenerative diseases

Apoptotic cell death can occur by two different pathways. Type 1 is initiated by the activation of death receptors (Fas, TNF-receptor-family) on the plasma membrane followed by activation of caspase 8. Type 2 involves changes in mitochondrial integrity initiated by various effectors like Ca(2+), reactive oxygen species (ROS), Bax, or ceramide, leading to the release of cytochrome c and activation of caspase 9. The release of cytochrome c is followed by a decrease of the mitochondrial membrane potential DeltaPsi(m). Recent publications have demonstrated, however, that induction of apoptosis by various effectors involves primarily a transient increase of DeltaPsi(m) for unknown reason. Here we propose a new mechanism for the increased DeltaPsi(m) based on experiments on the allosteric ATP-inhibition of cytochrome c oxidase at high matrix ATP/ADP ratios, which was concluded to maintain low levels of DeltaPsi(m) in vivo under relaxed conditions. This regulatory mechanism is based on the potential-dependency of the ATP synthase, which has maximal activity at DeltaPsi(m)=100-120 mV. The mechanism is turned off either through calcium-activated dephosphorylation of cytochrome c oxidase or by 3,5-diiodo-L-thyronine, palmitate, and probably other so far unknown effectors. Consequently, energy metabolism changes to an excited state. We propose that this change causes an increase in DeltaPsi(m), a condition for the formation of ROS and induction of apoptosis.     

Mitochondrial respiration and membrane potential are regulated by the allosteric ATP-inhibition of cytochrome c oxidase

This paper describes the problems of measuring the allosteric ATP-inhibition of cytochrome c oxidase (CcO) in isolated mitochondria. Only by using the ATP-regenerating system phosphoenolpyruvate and pyruvate kinase full ATP-inhibition of CcO could be demonstrated by kinetic measurements. The mechanism was proposed to keep the mitochondrial membrane potential (?Ψ_m) in living cells and tissues at low values (100-140 mV), when the matrix ATP/ADP ratios are high. In contrast, high ?Ψ_m values (180-220 mV) are generally measured in isolated mitochondria. By using a tetraphenyl phosphonium electrode we observed in isolated rat liver mitochondria with glutamate plus malate as substrates a reversible decrease of ?Ψ_m from 233 to 123 mV after addition of phosphoenolpyruvate and pyruvate kinase. The decrease of ?Ψ_m is explained by reversal of the gluconeogenetic enzymes pyruvate carboxylase and phosphoenolpyruvate carboxykinase yielding ATP and GTP, thus increasing the matrix ATP/ADP ratio. With rat heart mitochondria, which lack these enzymes, no decrease of ?Ψ_m was found. From the data we conclude that high matrix ATP/ADP ratios keep ?Ψ_m at low values by the allosteric ATP-inhibition of CcO, thus preventing the generation of reactive oxygen species which could generate degenerative diseases. It is proposed that respiration in living eukaryotic organisms is normally controlled by the ?Ψ_m-independent “allosteric ATP-inhibition of CcO.” Only when the allosteric ATP-inhibition is switched off under stress, respiration is regulated by “respiratory control,” based on ?Ψ_m according to the Mitchell Theory.     

During the stationary growth phase, Yarrowia lipolytica prevents the overproduction of reactive oxygen species by activating an uncoupled mitochondrial respiratory pathway

In the branched mitochondrial respiratory chain from Yarrowia lipolytica there are two alternative oxido-reductases that do not pump protons, namely an external type II NADH dehydrogenase (NDH2e) and the alternative oxidase (AOX). Direct electron transfer between these proteins is not coupled to ATP synthesis and should be avoided in most physiological conditions. However, under low energy-requiring conditions an uncoupled high rate of oxygen consumption would be beneficial, as it would prevent overproduction of reactive oxygen species (ROS). In mitochondria from high energy-requiring, logarithmic-growth phase cells, most NDH2e was associated to cytochrome c oxidase and electrons from NADH were channeled to the cytochromic pathway. In contrast, in the low energy requiring, late stationary-growth phase, complex IV concentration decreased, the cells overexpressed NDH2e and thus a large fraction of this enzyme was found in a non-associated form. Also, the NDH2e-AOX uncoupled pathway was activated and the state IV external NADH-dependent production of ROS decreased. Association/dissociation of NDH2e to/from complex IV is proposed to be the switch that channels electrons from external NADH to the coupled cytochrome pathway or allows them to reach an uncoupled, alternative, ΔΨ-independent pathway.     

The mitochondrial bioenergetic phenotype for protection from cardiac ischemia in SUR2 mutant mice

The sulfonylurea receptor-2 (SUR2) is a subunit of ATP-sensitive potassium channels (K(ATP)) in heart. Mice with the SUR2 gene disrupted (SUR2m) are constitutively protected from ischemia-reperfusion (I/R) cardiac injury. This was surprising because K(ATP), either sarcolemmal or mitochondrial or both, are thought to be important for cardioprotection. We hypothesized that SUR2m mice have an altered mitochondrial phenotype that protects against I/R. Mitochondrial membrane potential (ΔΨ(m)), tolerance to Ca(2+) load, and reactive oxygen species (ROS) generation were studied by fluorescence-based assays, and volumetric changes in response to K(+) were measured by light scattering in isolated mitochondria. For resting SUR2m mitochondria compared with wild type, the ΔΨ(m) was less polarized (46.1 ± 0.4 vs. 51.9 ± 0.6%), tolerance to Ca(2+) loading was increased (163 ± 2 vs. 116 ± 2 μM), and ROS generation was enhanced with complex I [8.5 ± 1.2 vs. 4.9 ± 0.2 arbitrary fluorescence units (afu)/s] or complex II (351 ± 51.3 vs. 166 ± 36.2 afu/s) substrates. SUR2m mitochondria had greater swelling in K(+) medium (30.2 ± 3.1%) compared with wild type (14.5 ± 0.6%), indicating greater K(+) influx. Additionally, ΔΨ(m) decreased and swelling increased in the absence of ATP in SUR2m, but the sensitivity to ATP was less compared with wild type. When the mitochondria were subjected to hypoxia-reoxygenation, the decrease in respiration rates and respiratory control index was less in SUR2m. ΔΨ(m) maintenance in the SUR2m intact myocytes was also more tolerant to metabolic inhibition. In conclusion, the cardioprotection observed in the SUR2m mice is associated with a protected mitochondrial phenotype resulting from enhanced K(+) conductance that partially dissipated ΔΨ(m). These results have implications for possible SUR2 participation in mitochondrial K(ATP).     

Regulation of mitochondrial respiration and apoptosis through cell signaling: cytochrome c oxidase and cytochrome c in ischemia/reperfusion injury and inflammation

Cytochrome c (Cytc) and cytochrome c oxidase (COX) catalyze the terminal reaction of the mitochondrial electron transport chain (ETC), the reduction of oxygen to water. This irreversible step is highly regulated, as indicated by the presence of tissue-specific and developmentally expressed isoforms, allosteric regulation, and reversible phosphorylations, which are found in both Cytc and COX. The crucial role of the ETC in health and disease is obvious since it, together with ATP synthase, provides the vast majority of cellular energy, which drives all cellular processes. However, under conditions of stress, the ETC generates reactive oxygen species (ROS), which cause cell damage and trigger death processes. We here discuss current knowledge of the regulation of Cytc and COX with a focus on cell signaling pathways, including cAMP/protein kinase A and tyrosine kinase signaling. Based on the crystal structures we highlight all identified phosphorylation sites on Cytc and COX, and we present a new phosphorylation site, Ser126 on COX subunit II. We conclude with a model that links cell signaling with the phosphorylation state of Cytc and COX. This in turn regulates their enzymatic activities, the mitochondrial membrane potential, and the production of ATP and ROS. Our model is discussed through two distinct human pathologies, acute inflammation as seen in sepsis, where phosphorylation leads to strong COX inhibition followed by energy depletion, and ischemia/reperfusion injury, where hyperactive ETC complexes generate pathologically high mitochondrial membrane potentials, leading to excessive ROS production. Although operating at opposite poles of the ETC activity spectrum, both conditions can lead to cell death through energy deprivation or ROS-triggered apoptosis.     

Interplay between Ca2+ cycling and mitochondrial permeability transition pores promotes reperfusion-induced injury of cardiac myocytes

Uncontrolled release of Ca(2+) from the sarcoplasmic reticulum (SR) contributes to the reperfusion-induced cardiomyocyte injury, e.g. hypercontracture and necrosis. To find out the underlying cellular mechanisms of this phenomenon, we investigated whether the opening of mitochondrial permeability transition pores (MPTP), resulting in ATP depletion and reactive oxygen species (ROS) formation, may be involved. For this purpose, isolated cardiac myocytes from adult rats were subjected to simulated ischemia and reperfusion. MPTP opening was detected by calcein release and by monitoring the ΔΨ(m). Fura-2 was used to monitor cytosolic [Ca(2+)](i) or mitochondrial calcium [Ca(2+)](m), after quenching the cytosolic compartment with MnCl(2). Mitochondrial ROS [ROS](m) production was detected with MitoSOX Red and mag-fura-2 was used to monitor Mg(2+) concentration, which reflects changes in cellular ATP. Necrosis was determined by propidium iodide staining. Reperfusion led to a calcein release from mitochondria, ΔΨ(m) collapse and disturbance of ATP recovery. Simultaneously, Ca(2+) oscillations occurred, [Ca(2+)](m) and [ROS](m) increased, cells developed hypercontracture and underwent necrosis. Inhibition of the SR-driven Ca(2+) cycling with thapsigargine or ryanodine prevented mitochondrial dysfunction, ROS formation and MPTP opening. Suppression of the mitochondrial Ca(2+) uptake (Ru360) or MPTP (cyclosporine A) significantly attenuated Ca(2+) cycling, hypercontracture and necrosis. ROS scavengers (2-mercaptopropionyl glycine or N-acetylcysteine) had no effect on these parameters, but reduced [ROS](m). In conclusion, MPTP opening occurs early during reperfusion and is due to the Ca(2+) oscillations originating primarily from the SR and supported by MPTP. The interplay between Ca(2+) cycling and MPTP promotes the reperfusion-induced cardiomyocyte hypercontracture and necrosis. Mitochondrial ROS formation is a result rather than a cause of MPTP opening.     

Potassium channel-oxidative phosphorylation relationship in durum wheat mitochondria from control and hyperosmotic-stressed seedlings

Durum wheat mitochondria (DWM) possess an ATP-inhibited K(+) channel, the plant mitoK(ATP) (PmitoK(ATP) ), which is activated under environmental stress to control mitochondrial ROS production. To do this, PmitoK(ATP) collapses membrane potential (ΔΨ), thus suggesting mitochondrial uncoupling. We tested this point by studying oxidative phosphorylation (OXPHOS) in DWM purified from control seedlings and from seedlings subjected both to severe mannitol and NaCl stress. In severely-stressed DWM, the ATP synthesis via OXPHOS, continuously monitored by a spectrophotometric assay, was about 90% inhibited when the PmitoK(ATP) was activated by KCl. Contrarily, in control DWM, although PmitoK(ATP) collapsed ΔΨ, ATP synthesis, as well as coupling [respiratory control (RC) ratio and ratio between phosphorylated ADP and reduced oxygen (ADP/O)] checked by oxygen uptake experiments, were unaffected. We suggest that PmitoK(ATP) may play an important defensive role at the onset of the environmental/oxidative stress by preserving energy in a crucial moment for cell and mitochondrial bioenergetics. Consistently, under moderate mannitol stress, miming an early stress condition, the channel may efficiently control reactive oxygen species (ROS) generation (about 35-fold from fully open to closed state) without impairing ATP synthesis. Anyway, if the stress significantly proceeds, the PmitoK(ATP) becomes fully activated by decrease of ATP concentration (25-40%) and increase of activators [free fatty acids (FFAs) and superoxide anion], thus impairing ATP synthesis.     

Modeling Cardiac Action Potential Shortening Driven by Oxidative Stress-Induced Mitochondrial Oscillations in Guinea Pig Cardiomyocytes

Ischemia-induced shortening of the cardiac action potential and its heterogeneous recovery upon reperfusion are thought to set the stage for reentrant arrhythmias and sudden cardiac death. We have recently reported that the collapse of mitochondrial membrane potential (ΔΨ_m) through a mechanism triggered by reactive oxygen species (ROS), coupled to the opening of sarcolemmal ATP-sensitive potassium (K_ATP) channels, contributes to electrical dysfunction during ischemia-reperfusion. Here we present a computational model of excitation-contraction coupling linked to mitochondrial bioenergetics that incorporates mitochondrial ROS-induced ROS release with coupling between the mitochondrial energy state and electrical excitability mediated by the sarcolemmal K_ATP current (I_K,ATP). Whole-cell model simulations demonstrate that increasing the fraction of oxygen diverted from the respiratory chain to ROS production triggers limit-cycle oscillations of ΔΨ_m, redox potential, and mitochondrial respiration through the activation of a ROS-sensitive inner membrane anion channel. The periods of transient mitochondrial uncoupling decrease the cytosolic ATP/ADP ratio and activate I_K,ATP, consequently shortening the cellular action potential duration and ultimately suppressing electrical excitability. The model simulates emergent behavior observed in cardiomyocytes subjected to metabolic stress and provides a new tool for examining how alterations in mitochondrial oxidative phosphorylation will impact the electrophysiological, contractile, and Ca²⁺ handling properties of the cardiac cell. Moreover, the model is an important step toward building multiscale models that will permit investigation of the role of spatiotemporal heterogeneity of mitochondrial metabolism in the mechanisms of arrhythmogenesis and contractile dysfunction in cardiac muscle.     

Selenoprotein S silencing triggers mouse hepatoma cells apoptosis and necrosis involving in intracellular calcium imbalance and ROS-mPTP-ATP

Selenoprotein S (SelenoS) is one of the cellular endoplasmic reticulum (ER) and membrane located selenoproteins, and it has the main functions of anti-oxidation, anti-apoptosis and anti-ER stress. To investigate the effect of SelenoS silencing on mouse hepatoma cell death and the intracellular biological function of SelenoS, we knocked down SelenoS in Hepa1-6 cells, and detected ER stress, intracellular calcium homeostasis, mitochondrial dynamics, apoptosis and necrosis. To further explore whether reactive oxygen species (ROS) has an effect on apoptosis and necrosis under SelenoS silencing, we used NAC (2.5?mM) to pretreat cells, and detected ΔΨm, ATP, and apoptosis and necrosis rates. SelenoS silencing broke the intracellular calcium homeostasis, induced mitochondrial dynamic disorder, ROS accumulation, loss of ΔΨm and ATP, and triggered apoptosis and necrosis in mouse hepatoma cells. The clearance of ROS alleviated the loss of ΔΨm and ATP caused by silencing of SelenoS, reduced cell necrosis and increased apoptosis. However, SelenoS silencing did not cause ER stress in Hepa1-6 cells. These results indicate that SelenoS silencing triggers mouse hepatoma cells apoptosis and necrosis through affecting intracellular calcium homeostasis and ROS-mPTP-ATP participates in cell death transformation from apoptosis to necrosis to rise damage.