The real kinetics of the mitochondrial calcium uniporter of the liver and its role in cell calcium regulation

The activity of the calcium uniporter of rat liver mitochondria, allosterically enhanced by a pulse of calcium, decreases with time and in dependence on extramitochondrial Ca2+ concentration. Therefore, the initial velocity of calcium uptake by mitochondria depends on the extramitochondrial Ca2+ concentration prior to uptake. The allosteric activation by calcium and the hysteretic behaviour of the uniporter are the reasons why the course of calcium distribution between mitochondria and extramitochondrial space is determined for many minutes by the initial extramitochondrial Ca2+ concentration. This dependence and also the independence on the intramitochondrial calcium content are shown in an in vitro system, simulating conditions prevailing in vivo during the action of alpha-adrenergic agonists or vasoactive peptides on liver and during the early phase of carbon tetrachloride intoxication.     

"Allosteric regulation" of calcium-uptake in rat liver mitochondria

During investigations of calcium uptake by rat liver mitochondria, at a buffered free calcium concentration of 2 microM, a considerable acceleration of calcium uptake was occasionally observed. From the following experiments it can be concluded that the acceleration occurred when mitochondria had become anaerobic, and hence deenergized, because they had been stored in the refrigerator for a while. Mitochondria which had become transitorily deenergized by blocking the respiratory chain with KCN, rotenone or antimycin showed an accelerated calcium uptake when the membrane potential necessary for calcium uptake was regenerated. This acceleration of calcium uptake was also seen when a potassium diffusion potential was induced by valinomycin in previously deenergized mitochondria. The velocity of calcium uptake in transitorily deenergized mitochondria increased irrespective of the presence of magnesium in the incubation medium. The activation of the Ca uniporter was reversible, and both processes, activation and deactivation, were time-dependent and developed within a time span of minutes. Oligomycin strongly inhibited the deactivation of the uniporter by ATP, hence the membrane potential is intrinsically effective and does not act via ATP. The altered kinetics of the Ca uniporter were responsible for the acceleration of calcium uptake which was measured at low calcium concentration with previously deenergized mitochondria. The dependence of the rate of calcium uptake on the concentration of calcium in the medium is hyperbolic in transitorily deenergized mitochondria [Km = 6.7 microM; V = 455 nmol/(min X mg protein)] and sigmoidal in normal ones. It is additionally independent of the presence of magnesium ions. We found Hill coefficients of 3.47 and 2.94 in experiments with and without magnesium, respectively. Correspondent kinetics, hyperbolic in deenergized and sigmoidal in normal mitochondria, were obtained when calcium uptake was not driven by the system of respiratory chain, but by the potassium diffusion potential induced by valinomycin. The alteration in the kinetics of the Ca uniporter has consequences in the range of physiological calcium levels, but mainly in pathological states of liver cells. These points are discussed.     

Interactions between spermine and Mg2+ on mitochondrial Ca2+ transport

The effects of the polyamine spermine on the regulation of Ca2+ transport by subcellular organelles from rat liver, heart, and brain were investigated using ion-sensitive minielectrodes and a 45Ca2+ tracer method. Spermine stimulated Ca2+ uptake by mitochondria but not by microsomes. In the presence of spermine, isolated mitochondria could maintain a free extramitochondrial Ca2+ concentration of 0.3-0.2 microM. Stimulation of the initial rates of Ca2+ uptake and 45Ca2+ cycling of mitochondria by spermine shows that this was accomplished through a decrease of the apparent Km for Ca2+ uptake by the Ca2+ uniporter. The half maximally effective concentration of spermine (50 microM) was in the range of physiological concentrations of this polyamine in the cell. Spermidine was five times less effective. Putrescine was ineffective. The stimulation of mitochondrial Ca2+ uptake by spermine was inhibited by Mg2+ in a concentration-dependent manner. However, the diminished contribution of the mitochondria to the regulation of the free extraorganellar Ca2+ concentration could mostly be compensated for by microsomal Ca2+ uptake. Spermine also reversed ruthenium red-induced Ca2+ efflux from mitochondria. It is concluded that spermine is an activator of the mitochondrial Ca2+ uniporter and Mg2+ an antagonist. By this mechanism, the polyamines can confer to the mitochondria an important role in the regulation of the free cytoplasmic Ca2+ concentration in the cell and of the free Ca2+ concentration in the mitochondrial matrix.     

Spermine, another specific allosteric activator of calcium uptake in rat liver mitochondria

Calcium uptake in rat liver mitochondria is accelerated by spermine. At a concentration of 2 microM Ca2+ and 1 mM Mg2+ a maximal, 10-fold activation by 1.2 mM spermidine was obtained; a half-maximal activation was attained with 0.2 mM spermine. Spermidine was far less effective than spermine whereas putrescine was ineffective. The acceleration of Ca uptake at low, physiological Ca2+ concentrations is related to the altered kinetics of the Ca uniporter. Corresponding to the alteration by high Ca2+ concentrations previously described, the kinetics changed from sigmoidal in the absence to nearly hyperbolic in the presence of spermine. Mg2+ behaves as an allosteric inhibitor. This phenomenon of the allosteric activation of Ca uptake could not be observed in heart mitochondria.     

Manganese stimulates calcium flux through the mitochondrial uniporter

Mn2+ alters the balance between the simultaneous uptake and release of Ca2+ across the mitochondrial inner membrane toward a lower external level. Addition of as little as 0.5 microM Mn2+ to energised mitochondria from rat liver, rat heart or guinea-pig brain changed the level at which they buffered Ca2+ in the medium. That extramitochondrial Mn2+ was responsible was suggested by a partial decay in the shift in Ca2+ steady state at a rate similar to the rate at which Mn2+ was accumulated by the mitochondria. The alteration of transmembrane Ca2+ distribution by Mn2+ required that both Mg2+ and Pi be present, and was almost maximal at Mg2+ and Pi levels in the physiological range. Substitution of spermine or Ni2+ for Mg2+, or acetate for Pi, abolished the effect. In contrast to Sr2+, Mn2+ did not inhibit either EGTA- or Ruthenium red-induced release of Ca2+ from the mitochondria. However, when flux through the uniporter was rate-limiting, Mn2+ accelerated Ca2+ uptake. The stimulation showed hyperbolic kinetics, with an element of competition discernible in the Mn2+-Mg2+ interaction. Thus, extramitochondrial Mn2+ at levels occurring in vivo can alter the mitochondrial 'set-point' by stimulating Ca2+ influx through the uniporter.     

EGTA inhibits reverse uniport-dependent Ca2+ release from uncoupled mitochondria. Possible regulation of the Ca2+ uniporter by a Ca2+ binding site on the cytoplasmic side of the inner membrane

When rat liver mitochondria are allowed to accumulate Ca2+, treated with ruthenium red to inhibit reverse activity of the Ca2+ uniporter, and then treated with an uncoupler, they release Ca2+ and endogenous Mg2+ and undergo large amplitude swelling with ultrastructural expansion of the matrix space. These effects are not produced by Ca2+ plus uncoupler alone. Like other "Ca2+-releasing agents" (i.e. N-ethylmaleimide, t-butylhydroperoxide, oxalacetate, etc.), the development of nonspecific permeability produced by ruthenium red plus uncoupler requires accumulated Ca2+ specifically and is antagonized by inhibitors of phospholipase A2. The permeability responses are also antagonized by ionophore A23187, indicating that a rapid pathway for Ca2+ efflux from deenergized mitochondria is necessary to prevent the development of nonspecific permeability. EGTA can be substituted for ruthenium red to produce the nonspecific permeability change in Ca2+-loaded, uncoupler-treated mitochondria. The permeability responses to EGTA plus uncoupler again require accumulated Ca2+ specifically and are antagonized by inhibitors of phospholipase A2 and by ionophore A23187. The equivalent effects of ruthenium red and EGTA on uncoupled, Ca2+-containing mitochondria indicate that reducing the extramitochondrial Ca2+ concentration to the subnanomolar range produces inhibition of reverse uniport activity. It is proposed that inhibition reflect regulation of the uniporter by a Ca2+ binding site which is available from the cytoplasmic side of the inner membrane. EDTA cannot substitute for EGTA to induce nonspecific permeability in Ca2+-loaded, uncoupled mitochondria. Furthermore, EDTA inhibits the response to EGTA with an I50 value of approximately 10 microM. These data suggest that the uniporter regulatory site also binds Mg2+. The data suggest further that Mg2+ binding to the regulatory site is necessary to inhibit reverse uniport activity, even when the site is not occupied by Ca2+.     

Effect of ebselen on Ca2+ transport in mitochondria

The seleno-organic compound ebselen mimics the glutathione-dependent, hydroperoxide reducing activity of glutathione peroxidase. The activity of glutathione peroxidase determines the rate of hydroperoxide-induced Ca2+ release from mitochondria. Ebselen stimulates Ca2+ release from mitochondria, accelerates mitochondrial respiration and uncoupling, and induces mitochondrial swelling, indicating a deterioration of mitochondrial function. These manifestations are abolished by cyclosporine A, a potent inhibitor of the mitochondrial permeability transition. However, when ebselen-induced Ca2+ cycling is prevented with ruthenium red, an inhibitor of the Ca2+ uniporter, or by chelation of extramitochondrial Ca2+ by EGTA, no detectable elevation of swelling or uncoupling is observed. The release of Ca2+ from mitochondria is delayed in the absence of rotenone, i.e. when pyridine nucleotides are maintained in the reduced state due to succinate-driven reversed electron flow. We suggest that ebselen induces Ca2+ release from intact mitochondria via an NAD+ hydrolysis-dependent mechanism.     

Novel role of phospholipase C-delta1: regulation of liver mitochondrial Ca2+ uptake

Mitochondrial Ca2+ (mCa2+) handling is an important regulator of liver cell function that controls events ranging from cellular respiration and signal transduction to apoptosis. Cytosolic Ca2+ enters mitochondria through the ruthenium red-sensitive mCa2+ uniporter, but the mechanisms governing uniporter activity are unknown. Activation of many Ca2+ channels in the cell membrane requires PLC. This activation commonly occurs through phosphitidylinositol-4,5-biphosphate (PIP2) hydrolysis and the production of the second messengers inositol 1,4,5-trisphosphate [I(1,4,5)P3] and 1,2-diacylglycerol (DAG). PIP2 was recently identified in mitochondria. We hypothesized that PLC exists in liver mitochondria and regulates mCa2+ uptake through the uniporter. Western blot analysis with anti-PLC antibodies demonstrated the presence of PLC-delta1 in pure preparations of mitochondrial membranes isolated from rat liver. In addition, the selective PLC inhibitor U-73122 dose-dependently blocked mCa2+ uptake when whole mitochondria were incubated at 37 degrees C with 45Ca2+. Increasing extra mCa2+ concentration significantly stimulated mCa2+ uptake, and U-73122 inhibited this effect. Spermine, a uniporter agonist, significantly increased mCa2+ uptake, whereas U-73122 dose-dependently blocked this effect. The inactive analog of U-73122, U-73343, did not affect mCa2+ uptake in any experimental condition. Membrane-permeable I(1,4,5)P3 receptor antagonists 2-aminoethoxydiphenylborate and xestospongin C also inhibited mCa2+ uptake. Although extra mitochondrial I(1,4,5)P3 had no effect on mCa2+ uptake, membrane-permeable DAG analogs 1-oleoyl-2-acetyl-sn-glycerol and DAG-lactone, which inhibit PLC activity, dose-dependently inhibited mCa2+ uptake. These data indicate that PLC-delta1 exists in liver mitochondria and is involved in regulating mCa2+ uptake through the uniporter.     

Allosteric Activation of Brain Mitochondrial Ca2+ Uptake by Spermine and ty Ca2+: Brain Regional Differences

Analysis of the initial rates of 45Ca2+ uptake by rat brain mitochondria in Ca2+-1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid buffers indicated that nontelencephalic mitochondria exhibited both a much less pronounced stimulatory effect of spermine and significantly more hyperbolic kinetics of Ca2+ uptake than telencephalic mitochondria. Nontelencephalic mitochondria were also markedly less susceptible to a Ca2+-induced hysteretic allosteric activation of the Ca2+ uniporter. A new Ca2+ loading procedure, which strikingly illustrates differences in mitochondrial Ca2+ buffering characteristics, is also described. In this procedure, low concentrations of Ca2+ (1, 2, or 5 microM) were repetitively added to mitochondria every 30 s while changes in free Ca2+ concentration were recorded. Spermine induced a marked attenuation of the rise in free Ca2+ level under these conditions. Steady-state rates of Ca2+ uptake were determined by a quantitative analysis of the buffering of repetitive Ca2+ additions, and, again, brain regional differences were qualitatively similar to those observed in the initial rate kinetics; Ca2+ uptake by nontelencephalic mitochondria in the steady state was markedly less responsive to stimulation by spermine and appeared to have a more hyperbolic dependence on Ca2+ in the absence of spermine. These results also suggest that there is a lag time in the activation of the uniporter by Ca2+, in addition to the hysteresis that has previously been observed in the deactivation of the uniporter.(ABSTRACT TRUNCATED AT 250 WORDS)     

Plasticity of mitochondrial calcium signaling

Evidence is emerging that a quasisynaptic local communication facilitates the calcium signaling between endoplasmic reticulum and mitochondria. However, it remains elusive whether the machinery of mitochondrial calcium signaling displays plasticity similar to the synaptic transmission. Here we studied the relationship between inositol 1,4,5-trisphosphate (IP3)-linked cytosolic [Ca2+] ([Ca2+]c) oscillations and the associated rise in mitochondrial matrix [Ca2+] ([Ca2+]m) in RBL-2H3 mast cells. We observed that the second [Ca2+]c spike is often associated with a larger rise in the [Ca2+]m than the first. It would appear that this phenomenon was not due to a change in the driving force for Ca2+ uptake and therefore must be due to an enhanced Ca2+ permeability of the mitochondrial Ca2+ uptake sites (uniporter). To investigate the activation and deactivation kinetics of the uniporter during IP3 receptor-mediated Ca2+ mobilization, we established novel methods. Using these approaches, we demonstrated that the IP3-induced increase in the permeability of the uniporter lasted longer than the Ca2+ signal. The sustained increase in Ca2+ permeability was bidirectional. Furthermore, the addition of Ca2+ during the decay of the IP3 effect evoked a large further increase in the uniporter permeability. Calmodulin inhibitors did not interfere with the IP3-induced initial activation of the uniporter but inhibited the sustained phase. These results suggest that the uniporter displays a calmodulin-mediated facilitation. This plasticity may allow cooperation among sequential IP3 receptor-mediated [Ca2+] transients in the control of calcium signal propagation to the mitochondria.     

Tissue-specific modulation of the mitochondrial calcium uniporter by magnesium ions

This paper analyzes the kinetics of the Ca2+ uniporter of mitochondria from rat heart, kidney and liver operating in a range of Ca2+ concentrations near the steady-state value (1-4 microM). Heart mitochondria exhibit the lowest activity, and physiological Mg2+ concentrations inhibit the mitochondrial Ca2+ uniporter by approx. 50% in heart and kidney, and by 20% in liver. At physiological Ca2+ and Mg2+ concentrations the external free Ca2+ maintained by respiring mitochondria in vitro is higher in heart and kidney with respect to liver mitochondria. This behaviour could represent an adaptation of different mitochondria to their specific intracellular environment.     

The stoichiometry of the exchange catalysed by the mitochondrial calcium/sodium antiporter.

Rat heart mitochondria respiring on succinate in the presence of Ruthenium Red (to inhibit uptake on the Ca2+ uniporter) released Ca2+ on the calcium/sodium antiporter until a steady state was reached. Addition of the ionophore A23187 (which catalyses Ca2+/2H+ exchange) did not perturb this steady state. Thermodynamic analysis showed that if a Ca2+/nNa+ exchange with any value of n other than 2 was at equilibrium, addition of A23187 would cause an obvious change in extramitochondrial free [Ca2+]. Therefore the endogenous calcium/sodium antiporter of mitochondria catalyses electroneutral Ca2+/2Na+ exchange.     

Ba2+ ions inhibit the release of Ca2+ ions from rat liver mitochondria

The release of Ca2+ from respiring rat liver mitochondria following the addition of either ruthenium red or an uncoupler was measured by a Ca2+-selective electrode or by 45Ca2+ technique. Ba2+ ions are asymmetric inhibitors of both Ca2+ release processes. Ba2+ ions in a concentration of 75 microM inhibited the ruthenium red and the uncoupler induced Ca2+ release by 80% and 50%, respectively. For the inhibition, it was necessary that Ba2+ ions entered the matrix space: Ba2+ ions did not cause any inhibition of Ca2+ release if addition of either ruthenium red or the uncoupler preceded that of Ba2+. The time required for the development of the inhibition of the Ca2+ release and the time course of 140Ba2+ uptake ran in parallel. Ba2+ accumulation is mediated through the Ca2+ uniporter as 140Ba2+ uptake was competitively inhibited by extramitochondrial Ca2+ and prevented by ruthenium red. Due to the inhibition of the ruthenium red insensitive Ca2+ release, Ba2+ shifted the steady-state extramitochondrial Ca2+ concentration to a lower value. Ba2+ is potentially a useful tool to study mitochondrial Ca2+ transport.     

Effects of adrenergic agonists and mitochondrial energy state on the Ca2+ transport systems of mitochondria

This study investigates the effects of adrenergic agonists and mitochondrial energy state on the activities of the Ca2+ transport systems of female rat liver mitochondria. Tissue perfusion with the alpha-adrenergic agonist phenylephrine and with adrenaline, but not with the beta-adrenergic agonist isoprenaline, induced significant activation of the uniporter and the respiratory chain. Uniporter activation was evident under two sets of experimental conditions that excluded influences of delta psi, i.e., at high delta psi, where uniporter activity was delta psi independent, and at low delta psi, where uniporter conductance was measured. Preincubation of mitochondria with extracts from phenylephrine-perfused tissue quantitatively reproduced uniporter activation when comparison was made with mitochondria treated similarly with extracts from tissue perfused without agonist. Similar, but more extensive, data were obtained with heart mitochondria pretreated with extracts from hearts perfused with the alpha-adrenergic agonist methoxamine. Phenylephrine did not affect Ca2+ efflux mediated by the Na+-Ca2+ carrier or the Na+-independent system. In contrast, the liver mitochondrial Na+-Ca2+ carrier was activated by tissue perfusion with isoprenaline; the Na+-independent system was unaffected. Na+-Ca2+ carrier activation was not associated with any change in a number of basic bioenergetic parameters. It is concluded that the Ca2+ transport systems of liver mitochondria may be controlled in an opposing manner by alpha-adrenergic agonists (promotion of Ca2+ influx) and beta-adrenergic agonists (promotion of Ca2+ efflux). At delta psi values greater than 110 mV, the Na+-independent system was activated by increase in delta psi; the uniporter and Na+-Ca2+ carrier activities were insensitive to delta psi changes in this range.(ABSTRACT TRUNCATED AT 250 WORDS)     

The regulation of brain mitochondrial calcium-ion transport. The role of ATP in the discrimination between kinetic and membrane-potential-dependent calcium-ion efflux mechanisms.

Mitochondria from guinea-pig cerebral cortex incubated in the presence of Pi or acetate are unable to regulate the extramitochondrial free Ca2+ at a steady-state which is independent of the Ca2+ accumulated in the matrix. This is due to the superimposition on kinetically regulated Ca2+ cycling of a membrane-potential-dependent reversal of the Ca2+ uniporter. The latter efflux is a consequence of a low membrane potential, which correlates with a loss of adenine nucleotide loss from the matrix, enable the mitochondria to maintain a high membrane potential and allow the mitochondria to buffer the extramitochondrial free Ca2+ precisely when up to 200 nmol of Ca2+/mg of protein is accumulated in the matrix. The steady-state extramitochondrial free Ca2+ is maintained as low as 0.3 microM. The Na+-activated efflux pathway is functional in the presence of ATP and oligomycin and accounts precisely for the change in steady-state free Ca2+ induced by Na+ addition. The need to distinguish carefully between kinetic and membrane-potential-dependent efflux pathways is emphasized and the competence of brain mitochondria to regulate cytosolic free Ca2+ concentrations in vivo is discussed.     

Inhibition of ruthenium red-induced Ca2+ efflux from liver mitochondria by the antibiotic X-537A

It has been reported (Becker, G.L., Fiskum, G. and Lehninger, A.L. (1980) J. Biol. Chem. 255, 9009-9012) that respiring rat liver mitochondria suspended in KC1 medium containing ATP, Mg2+ and phosphate, maintain a steady state extramitochondrial free Ca2+ concentration of about 0.5 microM. The results reported here show that the addition of the antibiotic X-537A, at concentrations far below those required for ionophorous activity, caused a perturbation in this steady state, lowering the extramitochondrial free Ca2+ concentration by about 0.20 microM. This shift in steady state was clarified by a study of X-537A inhibition of the Ca2+ efflux induced by ruthenium red; a half-maximum effect was observed at approximately 25 nM X-537A. No effect on Ca2+ transport through the influx uniporter was observed. The possibility of a generalized stabilizing action of the antibiotic on the mitochondrial membrane seems to be ruled out by its effectiveness at very low concentrations.     

pH regulation of calcium efflux by turtle liver mitochondria

1. Turtle liver mitochondria are capable of taking up calcium in a pattern similar to that described from rat liver mitochondria. 2. Turtle liver mitochondria also possess a system for calcium efflux which is extremely sensitive to changes in extramitochondrial pH. A decrease in extramitochondrial pH by addition of HCl or by gassing with CO2 caused a rapid release of calcium. 3. The profound changes in pH and pCO2 during deep diving likely affects Ca efflux from mitochondria in the turtle liver.     

The regulation of extramitochondrial steady state free Ca2+ concentration by rat insulinoma mitochondria

For the study of Ca2+ handling by mitochondria of an insulin secretory tissue, a method for the isolation of functionally intact insulinoma mitochondria is described. The mitochondria had a respiratory control ratio of 6.3 +/- 0.3 with succinate as a substrate. The regulation of extramitochondrial [Ca2+]o concentration by suspensions of insulinoma mitochondria was studied using Ca2+-selective minielectrodes. The mitochondria were found to maintain an ambient free Ca2+ concentration of about 0.3 and 0.9 microM in the absence or presence of Mg2+ (1 mM), respectively. The addition of Na+ resulted in a dose-dependent (half-maximal 4 mM Na+) increase in steady state [Ca2+]o. Na+ accelerated the ruthenium red-induced Ca2+ efflux, suggesting the existence of a Ca2+/2Na+ antiporter, as described in mitochondria of excitable tissues. Experiments were performed to study the effects of various agents on the steady state extramitochondrial free Ca2+. cAMP, 3-isobutyl-1-methylxanthine, and NADH were found to have no effect, whereas phosphoenolpyruvate induced a net Ca2+ efflux, the kinetic of which suggests deleterious effects on mitochondrial functions. A small decrease in pH (0.1 unit) of the incubation buffer resulted in an increase of the extramitochondrial Ca2+ steady state that was reversible upon restoration of the pH to its initial value. In conclusion, insulinoma mitochondria were able to maintain an extramitochondrial [Ca2+]o steady state in the submicromolar range that was markedly influenced by the ionic composition of the incubation medium. Thus, mitochondria may play a role in the regulation of cellular calcium homeostasis and insulin release.     

Electroneutral efflux of Ca2+ from liver mitochondria.

Respiring liver mitochondria were allowed to export Ca2+ on the endogenous Ca2+/nH+ antiporter in the presence of Ruthenium Red (to inhibit uptake on the Ca2+ uniporter) until a steady state was reached. Addition of sufficient of the ionophore A23187 (which catalyses Ca2+/2H+ exchange) to bring the Ca2+ and H+ gradients into equilibrium did not alter the steady state. Thermodynamic analysis showed that if a Ca2+/nH+ exchange with any value of n other than 2 was at equilibrium, addition of A23187 would have caused an easily measurable change in extramitochondrial free [Ca2+]. Therefore, the endogenous carrier of liver mitochondria catalyses electroneutral Ca2+/2H+ antiport.     

Reversibility of energy-dependent Ca2+ accumulation in mitochondria

Ca2+ accumulation in energized rat liver mitochondria has been studied after the blockage of mitochondrial permeability transition pore (MPTP) by cyclosporin A. It is shown that Ca2+ transport is coupled to the countertransport of protons: from the matrix of mitochondria in the medium in the course of Ca2+ accumulation, and, on the contrary, from the medium to mitochondrial matrix after membrane depolarization. In standard incubation medium containing K+, Cl-, oxidation substrate (glutamate) and inorganic phosphate (H2PO4(-)) the observed stoichiometry of the exchange is 1Ca2+ : 1H+. In accordance with this exchange ratio, proton, as well as cation, transport follows the same first-order kinetics, which is characterized in both cases by very close values of reaction half-times and rate constants. It is shown that reversion of Ca2+ -uniporter, sensitive to ruthenium red, is necessary for Ca2+ - efflux from the matrix ofdeenergized mitochondria when MPTP is blocked by cyclosporin A. It is also shown that Ca2+ -uniporter reversion takes place only after membrane depolarization and permeabilization by protonophore CCCP. Calcium release from mitochondria in the presence of CCCP is accompanied by proton flow into the matrix. Both calcium and proton fluxes are sensitive to Ca2+ uniporter blocker, ruthenium red, which gives the evidence of the identity of Ca2+ -efflux and influx pathways. The data obtained lead to the conclusion that calcium-proton exchange is necessary for Ca2+ -uniporter reversion and the reversibility of energy-dependent Ca2+ -uptake in mitochondria.