Recombinant expression of the voltage-dependent anion channel enhances the transfer of Ca2+ microdomains to mitochondria

Although the physiological relevance of mitochondrial Ca2+ homeostasis is widely accepted, no information is yet available on the molecular identity of the proteins involved in this process. Here we analyzed the role of the voltage-dependent anion channel (VDAC) of the outer mitochondrial membrane in the transmission of Ca2+ signals between the ER and mitochondria by measuring cytosolic and organelle [Ca2+] with targeted aequorins and Ca2+-sensitive GFPs. In HeLa cells and skeletal myotubes, the transient expression of VDAC enhanced the amplitude of the agonist-dependent increases in mitochondrial matrix Ca2+ concentration by allowing the fast diffusion of Ca2+ from ER release sites to the inner mitochondrial membrane. Indeed, high speed imaging of mitochondrial and cytosolic [Ca2+] changes showed that the delay between the rises occurring in the two compartments is significantly shorter in VDAC-overexpressing cells. As to the functional consequences, VDAC-overexpressing cells are more susceptible to ceramide-induced cell death, thus confirming that mitochondrial Ca2+ uptake plays a key role in the process of apoptosis. These results reveal a novel function for the widely expressed VDAC channel, identifying it as a molecular component of the routes for Ca2+ transport across the mitochondrial membranes.     

The effect of extracellular Ca and Mg on mitochondrial ultrastructure in isolated intact neurons

The ultrastructural transformations of mitochondria in isolated crayfish neurons were studied after incubation of the cells in saline media containing different Ca2+ and Mg2+ concentrations. Incubation in a 5-fold higher Ca concentration resulted in the swelling of mitochondria that was prevented by the addition of the calcium channel blocker, verapamil. Exposure of the cells to Mg2+-depleted medium induced swelling of all the mitochondria, followed by substantial shrinkage of most of them. The absence of Ca as well as the presence of verapamil in Mg2+-free medium led to the inhibition of mitochondrial swelling and to a strong contraction of the mitochondria after 1 h incubation. The omission of Ca2+ from the saline medium or the addition of Ca2+-ionophore A23187 in the presence of Ca2+ resulted in strong mitochondrial shrinkage. These structural alterations of mitochondria are interpreted as an osmotic response of the inner mitochondrial membranes to changes in their potassium transport, induced by a disturbance in the cellular and mitochondrial Ca2+-Mg2+ homeostasis.     

The role of Fe2+-induced lipid peroxidation in the initiation of the mitochondrial permeability transition

Iron and iron complexes stimulate lipid peroxidation and formation of malondialdehyde (MDA). We have studied the effects of Fe2+ and ascorbate on mitochondrial permeability transition induced by phosphate and Ca2+. Iron is necessary for detectable MDA formation, but only Ca2+ and phosphate are necessary for the induction of membrane potential loss (Deltapsi) and Ca2+ release. Keeping the iron at a constant concentration and varying the Ca2+ level changed the mitochondrial Ca2+ retention times, but not the amount of MDA formation. The antioxidant butylated hydroxytoluene at low concentrations prevented MDA formation, but not mitochondrial Ca2+ release. Preincubation of mitochondria with Fe2+ decreased Ca2+ retention time in a concentration-dependent manner and facilitated Ca2+-stimulated MDA accumulation. Thus, Ca2+ phosphate-induced mitochondrial permeability transition (MPT) can be separated mechanistically from MDA accumulation. Lipid peroxidation products do not appear to participate in the initial phase of the permeability transition, but sensitize mitochondria toward MPT.     

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.     

The use of the Ca2(+)-sensitive intramitochondrial dehydrogenases and entrapped fura-2 to study Sr2+ and Ba2+ transport across the inner membrane of mammalian mitochondria

In extracts of rat heart mitochondria, Sr2+ mimicked the activatory effects of Ca2+ on the Ca2(+)-sensitive intramitochondrial enzymes, pyruvate dehydrogenase phosphate phosphatase, isocitrate dehydrogenase (NAD+), and 2-oxoglutarate dehydrogenase, but at about tenfold higher concentrations (effective range approximately 1-100 muM) in each case. Ba2+ had no effect on extracted phosphatase, but did mimic the effect of Ca2+ on the other two enzymes with effective concentration ranges similar to those of Sr2+; as with Ca2+ and Sr2+, effective Ba2+ ranges were slightly (2-3-fold) raised by increases in ATP/ADP. In intact uncoupled rat heart mitochondria, the effects of Sr2+ and Ba2+ on the pyruvate and 2-oxoglutarate dehydrogenases were essentially similar to their effects in extracts. In fully coupled rat heart or liver mitochondria, the effective concentration ranges of extramitochondrial Sr2+, leading to activation of the matrix enzymes, were always approximately tenfold higher than those for Ca2+ under all conditions. Ba2+ did not affect pyruvate dehydrogenase in coupled mitochondria, but was shown to activate 2-oxoglutarate dehydrogenase in heart or liver mitochondria, and also isocitrate dehydrogenase (NAD+) in the latter; effective concentration ranges for extramitochondrial Ba2+ were approximately 100-fold greater than those for Ca2+, and like those for Ca2+ and Sr2+, were affected markedly by Mg2+ and spermine (which inhibit and promote mitochondrial Ca2+ uptake, respectively) but, in contrast to Ca2+ and Sr2+, they were hardly affected at all by Na+ (which promotes mitochondrial Ca2+ egress). Ba2+ effects were also blocked by ruthenium red (an inhibitor of mitochondrial Ca2+ uptake), but not so effectively as its blockage of the effects of Sr2+ and Ca2+. Ba2+ and Sr2+ both mimicked the inhibitory effects of extramitochondrial Ca2+ on the Na+/Ca2+ exchanger, but only Sr2+ could mimic Ca2+ in exchanging for internal Ca2+ by this mechanism. Both Sr2+ and Ba2+ changed the fluorescent properties of fura-2 or indo-1 in a similar manner to Ca2+, but with higher kd values. In fura-2-loaded rat heart mitochondria, increases in matrix Sr2+ and Ba2+ and the effects of the transport effectors could be readily demonstrated.     

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

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

On the state of calcium ions in isolated rat liver mitochondria. II. Effects of phosphate and pH on Ca2+-induced Ca2+ release

At high K+ concentration, the effect of phosphate on Ca2+ uptake and release was studied in isolated rat liver mitochondria. Phosphate stimulated uptake at moderately high Ca2+ concentration, and inhibited release at high pH. At low pH, phosphate accelerated Ca2+ release. Ca2+ was released after a lag phase. The time of onset and the velocity of Ca2+ release depended on Ca2+ concentration. Ca2+ release was associated with mitochondrial swelling and destruction of the permeability barrier for sucrose and for chloride. Mg2+ inhibited Ca2+ release and the accompanying events. Ruthenium red and EGTA protected mitochondria from the destructive Ca2+ release and induced an immediate, slow release of Ca2+ and phosphate. Destructive Ca2+ release depended on the time of preincubation of respiration-inhibited mitochondria in the presence of Ca2+, prior to respiration-initiated Ca2+ uptake. The presence of phosphate and mitochondrial energization antagonized the destructive effect of calcium ions. Ca2+ release by acetoacetate also depended on pH. At pH 6.8, phosphate-stimulated Ca2+ release by acetoacetate, while it inhibited the acetoacetate effect at pH 7.6. The results suggest that an essential cause for the destruction of mitochondrial integrity is an increase in the intramitochondrial concentration of free calcium ions under the influence of phosphate.     

Characteristics of the calcium-triggered mitochondrial permeability transition in nonsynaptic brain mitochondria: effect of cyclosporin A and ubiquinone O

The objective of the present study was to assess the capacity of nonsynaptic brain mitochondria to accumulate Ca2+ when subjected to repeated Ca2+ loads, and to explore under what conditions a mitochondrial permeability transition (MPT) pore is assembled. The effects of cyclosporin A (CsA) on Ca2+ accumulation and MPT pore assembly were compared with those obtained with ubiquinone 0 (Ubo), a quinone that is a stronger MPT blocker than CsA, when tested on muscle and liver mitochondria. When suspended in a solution containing phosphate (2 mM) and Mg2+ (1 mM), but no ATP or ADP, the brain mitochondria had a limited capacity to accumulate Ca2+ (210 nmol/mg of mitochondrial protein). Furthermore, when repeated Ca2+ pulses (40 nmol/mg of protein each) saturated the uptake system, the mitochondria failed to release the Ca2+ accumulated. However, in each instance, the first Ca2+ pulse was accompanied by a moderate release of Ca2+, a release that was not observed during the subsequent pulses. The initial release was accompanied by a relatively marked depolarization, and by swelling, as assessed by light-scattering measurements. However, as the swelling was <50% of that observed following addition of alamethicin, it is concluded that the first Ca2+ pulse gives rise to an MPT in a subfraction of the mitochondrial population. CsA, an avid blocker of the MPT pore, only marginally increased the Ca(2+)-sequestrating capacity of the mitochondria. However, CsA eliminated the Ca2+ release accompanying the first Ca2+ pulse. The effects of CsA were shared by Ubo, but when the concentration of Ubo exceeded 20 microM, it proved toxic. The results thus suggest that brain mitochondria are different from those derived from a variety of other sources. The major difference is that a fraction of the brain mitochondria, studied presently, depolarized and showed signs of an MPT. This fraction, but not the remaining ones, contributed to the chemically and electron microscopically verified mitochondrial swelling.     

The activation of adrenal cortex mitochondrial malic enzyme by Ca2+ and Mg2+.

Adrenal cortex mitochondria prepared by a standard method do not exhibit malic enzyme activity. Addition of physiological concentrations of Ca2+ and Mg2+ enables these mitochondria to reduce added NADP+ by malate to form free NADPH. Half-maximum activation of the mitochondrial malic enzyme requires 0.3 mM Ca2+ and 1 mM Mg2+. Solubilized mitochondrial malic enzymes is independent of Ca2+ and has a K M of 0.2 mM for Mg2+. The Ca2+ effect is dependent on an initial period of active Ca2+ uptake which also causes other changes in respiratory properties similar to those observed with mitochondria from other tissues. After Ca2+ accumulation has taken place, free Ca2+, but not additional accumulation, is still required for malic enzyme activity. The requirement for Mg2+ can be met by Mn2+ (1 mM). This concentration of Mn2+ alone yielded only a slight activation of mitochondrial malic enzyme while higher concentrations of Mn2+ alone gave good activation of the mitochondrial malic enzy.e The NADPH generated by the Ca2+-Mg2+ activated malic enzyme effectively supports the 11beta-hydroxylation of deoxycorticosterone, whereas in the presence of malate, or malate plus Mg2+ but absence of Ca2+, the energy linked transhydrogenase supplies all the required NADPH. The activated malic enzyme appears to be more efficient than transhydrogenase in generating NADPH to support 11beta-hydroxylation. Cyanide and azide have been found to inhibit solubilized mitochondrial malic enzyme.     

Role of calcium ions in the regulation of intramitochondrial metabolism. Effects of Na+, Mg2+ and ruthenium red on the Ca2+-stimulated oxidation of oxoglutarate and on pyruvate dehydrogenase activity in intact rat heart mitochondria.

1. In uncoupled rat heart mitochondria, the kinetic parameters for oxoglutarate oxidation were very close to those found for oxoglutarate dehydrogenase activity in extracts of the mitochondria. In particular, Ca2+ greatly diminished the Km for oxoglutarate and the k0.5 value (concentration required for half-maximal effect) for this effect of Ca2+ was close to 1 microM. 2. In coupled rat heart mitochondria incubated with ADP, increases in the extramitochondrial concentration of Ca2+ greatly stimulated oxoglutarate oxidation at low concentrations of oxoglutarate, but not at saturating concentrations of oxoglutarate. The k0.5 value for the activation by extramitochondrial Ca2+ was about 20 nM. In the presence of either Mg2+ or Na+ this value was increased to about 90 nM, and in the presence of both to about 325 nM. 3. In coupled rat heart mitochondria incubated without ADP, increases in the extramitochondrial concentration of Ca2+ resulted in increases in the proportion of pyruvate dehydrogenase in its active non-phosphorylated form. The sensitivity to Ca2+ closely matched that found to affect oxoglutarate oxidation, and Mg2+ and Na+ gave similar effects. 4. Studies of others have indicated that the distribution of Ca2+ across the inner membrane of heart mitochondria is determined by a Ca2+-transporting system which is composed of a separate uptake component (inhibited by Mg2+ and Ruthenium Red) and an efflux component (stimulated by Na+). The present studies are entirely consistent with this view. They also indicate that the intramitochondrial concentration of Ca2+ within heart cells is probably about 2--3 times that in the cytoplasm, and thus the regulation of these intramitochondrial enzymes by Ca2+ is of likely physiological significance. It is suggested that the Ca2+-transporting system in heart mitochondria may be primarily concerned with the regulation of mitochondrial Ca2+ rather than cytoplasmic Ca2+; the possible role of Ca2+ as a mediator of the effects of hormones and neurotransmitters on mammalian mitochondrial oxidative metabolism is discussed.     

Hexose metabolism in pancreatic islets. Participation of Ca2(+)-sensitive 2-ketoglutarate dehydrogenase in the regulation of mitochondrial function

A rise in extracellular D-glucose concentration results in a preferential and Ca2(+)-dependent stimulation of mitochondrial oxidative events in pancreatic islet cells. The possible participation of Ca2(+)-dependent mitochondrial dehydrogenases, especially 2-ketoglutarate dehydrogenase, in such an unusual metabolic situation was explored in intact islets, islet homogenates and isolated islet mitochondria. In intact islets exposed to a high concentration of D-glucose, the removal of extracellular Ca2+ impaired D-[6-14C]glucose oxidation whilst failing to affect the cytosolic or mitochondrial ATP/ADP ratios. In islet homogenates, the activity of 2-ketoglutarate dehydrogenase displayed exquisite Ca2(+)-dependency, the presence of Ca2+ causing a 10-fold increase in affinity for 2-ketoglutarate. In intact islet mitochondria, the oxidation of 2-[1-14C]ketoglutarate also increased as a function of extramitochondrial Ca2+ availability. Moreover, prior stimulation of intact islets by D-glucose resulted in an increased capacity of mitochondria to oxidize 2-[1-14C]ketoglutarate. The absence of extracellular Ca2+ during the initial stimulation of intact islets impaired but did not entirely suppress such a memory phenomenon. It is proposed that the mitochondrial accumulation of Ca2+ in nutrient-stimulated islets indeed accounts, in part at least, for the preferential stimulation of mitochondrial oxidative events in this fuel-sensor organ.     

Brain mitochondria are primed by moderate Ca2+ rise upon hypoxia/reoxygenation for functional breakdown and morphological disintegration

In animal models, brain ischemia causes changes in respiratory capacity, mitochondrial morphology, and cytochrome c release from mitochondria as well as a rise in cytosolic Ca2+ concentration. However, the causal relationship of the cellular processes leading to mitochondrial deterioration in brain has not yet been clarified. Here, by applying various techniques, we used isolated rat brain mitochondria to investigate how hypoxia/reoxygenation and nonphysiological Ca2+ concentrations in the low micromolar range affect active (state 3) respiration, membrane permeability, swelling, and morphology of mitochondria. Either transient hypoxia or a micromolar rise in extramitochondrial Ca2+ concentration, given as a single insult alone, slightly decreased active respiration. However, the combination of both insults caused devastating effects. These implied almost complete loss of active respiration, release of both NADH and cytochrome c, and rupture of mitochondria, as shown by electron microscopy. Mitochondrial respiration deteriorated even in the presence of cyclosporin A, documenting that membrane permeabilization occurred independent of mitochondrial permeability transition pore. Ca2+ has to enter the mitochondrial matrix in order to mediate this mitochondrial injury, because blockade of the mitochondrial Ca2+-transport system by ruthenium red in combination with CGP37157 completely prevented damage. Furthermore, protection of respiration from Ca2+-mediated damage by the adenine nucleotide ADP, but not by AMP, during hypoxia/reoxygenation is consistent with the delayed susceptibility of brain mitochondria to prolonged hypoxia, which is observed in vivo.     

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.     

Mitochondria suppress local feedback activation of inositol 1,4, 5-trisphosphate receptors by Ca2+.

The concerted action of inositol 1,4,5-trisphosphate (IP3) and Ca2+ on the IP3 receptor Ca2+ release channel (IP3R) is a fundamental step in the generation of cytosolic Ca2+ oscillations and waves, which underlie Ca2+ signaling in many cells. Mitochondria appear in close association with regions of endoplasmic reticulum (ER) enriched in IP3R and are particularly responsive to IP3-induced increases of cytosolic Ca2+ ([Ca2+]c). To determine whether feedback regulation of the IP3R by released Ca2+ is modulated by mitochondrial Ca2+ uptake, the interactions between ER and mitochondrial Ca2+ pools were examined by fluorescence imaging of compartmentalized Ca2+ indicators in permeabilized hepatocytes. IP3 decreased luminal ER Ca2+ ([Ca2+]ER), and this was paralleled by an increase in mitochondrial matrix Ca2+ ([Ca2+]m) and activation of Ca2+-sensitive mitochondrial metabolism. Remarkably, the decrease in [Ca2+]ER evoked by submaximal IP3 was enhanced when mitochondrial Ca2+ uptake was blocked with ruthenium red or uncoupler. Moreover, subcellular regions that were relatively deficient in mitochondria demonstrated greater sensitivity to IP3 than regions of the cell with a high density of mitochondria. These data demonstrate that Ca2+ uptake by the mitochondria suppresses the local positive feedback effects of Ca2+ on the IP3R, giving rise to subcellular heterogeneity in IP3 sensitivity and IP3R excitability. Thus, mitochondria can play an important role in setting the threshold for activation and establishing the subcellular pattern of IP3-dependent [Ca2+]c signaling.     

Role of sarcolemmal ATP-sensitive potassium channel in oxidative stress-induced apoptosis: mitochondrial connection

From time of their discovery, sarcolemmal ATP-sensitive K+ (sarcK ATP) channels were thought to have an important protective role in the heart during stress whereby channel opening protects the heart from stress-induced Ca2+ overload and resulting damage. In contrast, some recent studies indicate that sarcK ATP channel closing can lead to cardiac protection. Also, the role of the sarcK ATP channel in apoptotic cell death is unclear. In the present study, the effects of channel inhibition on apoptosis and the specific interaction between the sarcK ATP channel and mitochondria were investigated. Apoptotic cell death of cultured HL-1 and neonatal cardiomyocytes following exposure to oxidative stress was significantly increased in the presence of sarcK ATP channel inhibitor HMR-1098 as evidenced by terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling and caspase-3,7 assays. This was paralleled by an increased release of cytochrome c from mitochondria to cytosol, suggesting activation of the mitochondrial death pathway. sarcK ATP channel inhibition during stress had no effect on Bcl-2, Bad, and phospho-Bad, indicating that the increase in apoptosis cannot be attributed to these modulators of the apoptotic pathway. However, monitoring of mitochondrial Ca2+ with rhod-2 fluorescent indicator revealed that mitochondrial Ca2+ accumulation during stress is potentiated in the presence of HMR-1098. In conclusion, this study provides novel evidence that opening of sarcK ATP channels, through a specific Ca2+-related interaction with mitochondria, plays an important role in preventing cardiomyocyte apoptosis and mitochondrial damage during stress.     

Mitochondrial Ca2+ homeostasis in intact cells

Ca2+ is a key regulator not only of multiple cytosolic enzymes, but also of a variety of metabolic pathways occurring within the lumen of intracellular organelles. Until recently, no technique to selectively monitor the Ca2+ concentration within defined cellular compartments was available. We have recently proposed the use of molecularly engineered Ca(2+)-sensitive photoproteins to obtain such a result and demonstrated the application of this methodology to the study of mitochondrial and nuclear Ca2+ dynamics. We here describe in more detail the use of chimeric recombinant aequorin targeted to the mitochondria. The technique can be applied with equivalent results to different cell models, transiently or permanently transfected. In all the cell types we analyzed, mitochondrial Ca2+ concentration ([Ca2+]m) increases rapidly and transiently upon stimulation with agonists coupled to InsP3 generation. We confirm that the high speed of mitochondrial Ca2+ accumulation with this type of stimuli depends on the generation of local gradients of Ca2+ in the cytosol, close to the channels sensitive to InsP3. In fact, only activation of these channels, but not the simple release from internal stores, as that elicited by blocking the intracellular Ca2+ ATPases, results in a fast mitochondrial Ca2+ accumulation. We also provide evidence in favor of a microheterogeneity among mitochondria of the same cells, about 30% of them apparently sensing the microdomains of high cytosolic Ca2+ concentration ([Ca2+]c). The changes in [Ca2+]m appear sufficiently large to induce a rapid activation of mitochondrial dehydrogenases, which can be followed by monitoring the level of NAD(P)H fluorescence. A general scheme can thus be envisaged by which the triggering of a plasma membrane receptor coupled to InsP3 generation raises the Ca2+ concentration both in the cytoplasm (thereby triggering energy-consuming processes, such as cell proliferation, motility, secretion, etc.) and in the mitochondria, where it activates the metabolic activity according to the increased cell needs.     

Intramitochondrial K+ as activator of carboxyatractyloside-induced Ca2+ release.

The role of intramitochondrial K+ content on the increase in membrane permeability to Ca2+, as induced by carboxyatractyloside was studied. In mitochondria containing a high K+ concentration (83 nmol/mg), carboxyatractyloside induced a fast and extensive mitochondrial Ca2+ release, membrane de-energization, and swelling. Conversely, in K(+)-depleted mitochondria (11 nmol/mg), carboxyatractyloside was ineffective. The addition of 40 mM K+ to K(+)-depleted mitochondria restored the capability of atractyloside to induce an increase in membrane permeability to Ca2+ release. The determination of matrix free Ca2+ concentration showed that, at an external free-Ca2+ concentration of 0.8 microM, control mitochondria contained 3.9 microM of free Ca2+ whereas K(+)-depleted mitochondria contained 0.9 microM free Ca2+. It is proposed that intramitochondrial K+ affects the matrix free Ca2+ concentration required to induce a state of high membrane permeability.     

Biochemical approaches to the study of cytosolic calcium regulation in nerve endings

The nerve ending cytosol is bounded by the plasma membrane, the mitochondrial inner membrane and the endoplasmic reticulum membrane, transport across each of which is capable, in theory, of regulating the cytosolic free Ca2+ concentration. By parallel monitoring of mitochondrial and plasma membrane potentials, ATP levels, Na+ gradients and intrasynaptosomal Ca2+ distribution in preparations of isolated synaptosomes, we conclude the following: (a) mitochondria in situ represent a major Ca2+ pool, regulating the upper steady-state limit of the cytosolic free Ca2+ concentration by sequestering Ca2+ reversibly; (b) this limit is responsive to the cytosolic Na+ concentration, but is below the concentration required for significant exocytosis; (c) plasma membrane Ca2+ transport can be resolved into a constant slow influx, a voltage-dependent and verapamil-sensitive influx and an ATP-dependent efflux, while Ca2+ efflux driven by the sodium electrochemical potential cannot be detected; (d) Ca2+ regulation by intrasynaptosomal endoplasmic reticulum appears to be of minor significance in the present preparation.