Cd2+ versus Ca2+-produced mitochondrial membrane permeabilization: a proposed direct participation of respiratory complexes I and III

A comparison of Cd²⁺ and Ca²⁺ effects on in vitro rat liver mitochondria function and a further study of their interaction were conducted. Similarity and distinction in action of rotenone, oligomycin, N-ethylmaleimide, dithiothreitol, catalase, dibucaine, ruthenium red, cyclosporin A (CsA), and ADP on Cd²⁺ and/or Ca²⁺-induced mitochondrial dysfunction were revealed. We found that rotenone exerted a strong protective action both against Ca²⁺ and Cd²⁺-produced mitochondrial membrane permeabilization (MMP). In contrast to Ca²⁺, catalase and dibucaine did not influence on main Cd²⁺ effects. In NH₄NO₃ medium N-ethylmaleimide (NEM) at low concentrations increased markedly Cd²⁺-produced swelling of non-energized mitochondria, whereas it exhibited a partial reversal effect following energization. In sucrose medium low [NEM] did not change Cd²⁺-produced mitochondrial swelling. High [NEM] promoted synergistic increase of the Cd²⁺-produced swelling in NH₄NO₃ medium; all above effects were reversed (and prevented) by dithiothreitol, DTT. We shown also that when exogenous Ca²⁺ and P_i were simultaneously present in NH₄NO₃ medium, DTT reversed only partially Cd²⁺-produced swelling of succinate plus rotenone-energized mitochondria, while DTT recovery action was complete when either Ca²⁺ or P_i were separately administered to the Cd²⁺-treated mitochondria. Besides, DTT added following a low Cd²⁺ pulse in KCl medium containing exogenous Ca²⁺ induced a substantial enhancing of sustained Cd²⁺ stimulation of mitochondrial basal respiration and the stimulation was CsA-sensitive, while the activation promoted by low [Cd²⁺] alone was totally eliminated by DTT supplement. We observed the similar respiratory activation earlier when high concentrations of Cd²⁺ in the absence of added Ca²⁺ were used but it was completely CsA-insensitive. A possible involvement of respiratory chain components, namely complex I (P-site) and complex III (S-site) in Cd²⁺and/or Ca²⁺-produced MMP was discussed.     

Characteristics and possible functions of mitochondrial Ca transport mechanisms

Mitochondria produce around 92% of the ATP used in the typical animal cell by oxidative phosphorylation using energy from their electrochemical proton gradient. Intramitochondrial free Ca²⁺ concentration ([Ca²⁺]_m) has been found to be an important component of control of the rate of this ATP production. In addition, [Ca²⁺]_m also controls the opening of a large pore in the inner mitochondrial membrane, the permeability transition pore (PTP), which plays a role in mitochondrial control of programmed cell death or apoptosis. Therefore, [Ca²⁺]_m can control whether the cell has sufficient ATP to fulfill its functions and survive or is condemned to death. Ca²⁺ is also one of the most important second messengers within the cytosol, signaling changes in cellular response through Ca²⁺ pulses or transients. Mitochondria can also sequester Ca²⁺ from these transients so as to modify the shape of Ca²⁺ signaling transients or control their location within the cell. All of this is controlled by the action of four or five mitochondrial Ca²⁺ transport mechanisms and the PTP. The characteristics of these mechanisms of Ca²⁺ transport and a discussion of how they might function are described in this paper.     

The irreversibility of inner mitochondrial membrane permeabilization by Ca2+ plus prooxidants is determined by the extent of membrane protein thiol cross-linking

We have previously shown that mitochondrial membrane potential () drop promoted by prooxidants and Ca²⁺ can be reversed but not sustained by ethylene glycol-bis(-aminoethylether)-N,N,N,N-tetraacetic acid (EGTA) unless dithiothreitol (DTT), a disulfide reductant, is also added [Valle, V. G. R., Fagian, M. M., Parentoni, L. S., Meinicke, A. R., and Vercesi, A. E. (1993).Arch. Biochem. Biophys. 307, 1–7]. In this study we show that catalase or ADP are also able to potentiate this EGTA effect. When EGTA is added long after (12 min) the completion of swelling or elimination, no membrane resealing occurs unless the EGTA addition was preceded by the inclusion of DTT, ADP, or catalase soon after was collapsed. Total recovery by EGTA is obtained only in the presence of ADP. The sensitivity of the ADP effect to carboxyatractyloside strongly supports the involvement of the ADP/ATP carrier in this mechanism. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of solubilized membrane proteins shows that protein aggregation due to thiol cross-linkage formed during drop continues even after is already eliminated. Titration with 5,5-dithio-bis(2-nitrobenzoic acid) supports the data indicating that the formation of protein aggregates is paralleled by a decrease in the content of membrane protein thiols. Since the presence of ADP and EGTA prevents the progress of protein aggregation, we conclude that this process is responsible for both increased permeability to larger molecules and the irreversibility of drop. The protective effect of catalase suggests that the continuous production of protein thiol cross-linking is mediated by mitochondrial generated reactive oxygen species.     

A permeability transition in liposomes induced by the formation of Ca/palmitic acid complexes

Formation of palmitic acid/Ca²⁺ (PA/Ca²⁺) complexes was suggested to play a key role in the non-classical permeability transition in mitochondria (NCPT), which seems to be involved in the PA-induced apoptosis of cardiomyocytes. Our previous studies of complexation of free fatty acids (FFA) with Ca²⁺ showed that long-chain (C:16-C:22) saturated FFA had an affinity to Ca²⁺, which was much higher than that of other FFA and lipids. The formation of FFA/Ca²⁺ complexes in the black-lipid membrane (BLM) was demonstrated to induce a nonspecific ion permeability of the membrane. In the present work, we have found that binding of Ca²⁺ to PA incorporated into the membrane of sulforhodamine B (SRB)-loaded liposomes results in an instant release of a part of SRB, with the quantity of SRB released depending on the concentration of PA and Ca²⁺. The pH-optimum of this phenomenon, similar to that of PA/Ca²⁺ complexation, is in the alkaline range. The same picture of SRB release has been revealed for stearic, but not for linoleic acid. Along with Ca²⁺, some other bivalent cations (Ba²⁺, Sr²⁺, Mn²⁺, Ni²⁺, Co²⁺) also induce SRB release upon binding to PA-containing liposomes, while Mg²⁺ turns out to be relatively ineffective. As revealed by fluorescence correlation spectroscopy, the apparent size of liposomes does not alter after the addition of PA, Ca²⁺ or their combination. So it has been supposed that the cause of SRB release from liposomes is the formation of lipid pores. The effect of FFA/Ca²⁺-induced permeabilization of liposomal membranes has several analogies with NCPT, suggesting that both these phenomena are of similar nature.     

On the mechanism of palmitic acid-induced apoptosis: the role of a pore induced by palmitic acid and Ca2+ in mitochondria

Palmitic acid (Pal) is known to promote apoptosis (Sparagna G et al (2000) Am J Physiol Heart Circ Physiol 279: H2124–H2132) and its amount in blood and mitochondria increases under some pathological conditions. Yet, the mechanism of the proapoptotic action of Pal has not been elucidated. We present evidence for the involvement of the mitochondrial cyclosporin A-insensitive pore induced by Pal/Ca²⁺ complexes in the apoptotic process. Opening of this pore led to a fall of the mitochondrial membrane potential and the release of the proapoptotic signal cytochrome c. The addition of cytochrome c prevented these effects and recovered membrane potential, which is in contrast to the cyclosporin A-sensitive mitochondrial permeability transition pore. Oleic and linoleic acids prevented the Pal/Ca²⁺-induced pore opening in the intact mitochondria, this directly and significantly correlating with the effect of these fatty acids on Pal-induced apoptosis in cells (Hardy S et al (2003) J Biol Chem 278: 31861–31870). The specific probe for cardiolipin, 10-N-nonyl acridine orange, inhibited formation of this pore.     

Mn2+ prevents the Ca2+-induced inhibition of ATP synthesis in brain mitochondria

The differential scanning microcalorimetry and fluorescence methods, using probes ANS and pyrene, have been employed to study thermotropic behaviour of rat liver microsomes in the presence and absence of Mg2+. Addition of Mg2+ yields three partially reversible phase transitions at 18, 27 and 32 degrees C, respectively. A character of Mg2+-induced rearrangements in a membrane and their relation to a catalytic function of a cytochrome P-450-dependent enzymatic system is discussed.     

F-ATPase of Drosophila melanogaster Forms 53-Picosiemen (53-pS) Channels Responsible for Mitochondrial Ca2+-induced Ca2+ Release

Mitochondria of Drosophila melanogaster undergo Ca²⁺-induced Ca²⁺ release through a putative channel (mCrC) that has several regulatory features of the permeability transition pore (PTP). The PTP is an inner membrane channel that forms from F-ATPase, possessing a conductance of 500 picosiemens (pS) in mammals and of 300 pS in yeast. In contrast to the PTP, the mCrC of Drosophila is not permeable to sucrose and appears to be selective for Ca²⁺ and H⁺. We show (i) that like the PTP, the mCrC is affected by the sense of rotation of F-ATPase, by Bz-423, and by Mg²⁺/ADP; (ii) that expression of human cyclophilin D in mitochondria of Drosophila S₂R⁺ cells sensitizes the mCrC to Ca²⁺ but does not increase its apparent size; and (iii) that purified dimers of D. melanogaster F-ATPase reconstituted into lipid bilayers form 53-pS channels activated by Ca²⁺ and thiol oxidants and inhibited by Mg²⁺/γ-imino ATP. These findings indicate that the mCrC is the PTP of D. melanogaster and that the signature conductance of F-ATPase channels depends on unique structural features that may underscore specific roles in different species.     

Regulation of Ca2+ fluxes in rat liver mitochondria by Ca2+. Effects on Ca2+ distribution

Rat liver mitochondria are able to temporarily lower the steady-state concentration of external Ca²⁺ after having accumulated a pulse of added Ca²⁺. This has been attributed to inhibition of a putative -modulated efflux pathway [Bernardi, P. (1984)Biochim. Biophys. Acta 766, 277–282]. On the other hand, the rebounding could be due to stimulation of the uniporter by Ca²⁺ [Kröner, H. (1987)Biol. Chem. Hoppe-Seyler 369, 149–155]. By measuring unidirectional Ca²⁺ fluxes, it was found that the uniporter was stimulated during the rebounding peak both under Bernardi's and Kröner's conditions, while no effects on the efflux could be demonstrated. The rate of unidirectional efflux of Ca²⁺ was not affected by inhibition of the uniporter. It appears likely that the rebounding is due to stimulation of the uniporter rather than inhibition of efflux.     

Fatty acid-promoted mitochondrial permeability transition by membrane depolarization and binding to the ADP/ATP carrier

The mechanism by which non-esterified long-chain fatty acids (FFA) promote mitochondrial permeability transition (MPT) is not clear. We examined with energized rat liver mitochondria the role of two possible actions of FFA in MPT, (i) the reduction of the transmembrane potential (Δψ) and (ii) the increase of the negative surface charge of the inner mitochondrial membrane [Broekemeier, K.M. and Pfeiffer, D.G., Biochemistry 43, (1995) 16440–16449]. It was found that the ability of FFA to stimulate large amplitude swelling is clearly related to their uncoupling activity. Moreover, compared with classical protonophores (FCCP) FFA increase the sensitivity of the pore opening process to Δψ changes. In addition, FFA interact like their thioester derivatives in a structure-dependent manner with the ADP/ATP carrier (measured as inhibition of [³H]atractyloside binding to the AAC protein). It is suggested that not only the protonophoric action of FFA, but also a presumable stabilization of the ‘cytosolic' conformation of AAC contribute to the FFA-promoted MPT.     

New concepts on the role of ubiquinone in the mitochondrial respiratory chain

Ubiquinone participates in the oxidation-reduction reactions of the mitochondrial respiratory chain. In addition, this molecule possesses the necessary properties to function as a hydrogen carrier, thereby stoichiometrically coupling proton translocation to respiration by a direct chemiosmotic mechanism. This review discusses recent experimental evidence and new concepts relating to ubiquinone function in the mitochondrial respiratory chain. Emphasis is placed on possible protonmotive mechanisms of ubiquinone function, recent evidence implicating stable forms of ubisemiquinone in the respiratory chain, and properties of the ubiquinone molecule which may relate to its biological function.     

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

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

Measurements of mitochondrial pH in cultured cortical neurons clarify contribution of mitochondrial pore to the mechanism of glutamate-induced delayed Ca2+ deregulation

To clarify the role of the mitochondrial permeability transition pore (MPT) in the mechanism of the glutamate-induced delayed calcium deregulation (DCD) and mitochondrial depolarization (MD), we studied changes in cytosolic (pH(c)) and mitochondrial pH (pH(m)) induced by glutamate in cultured cortical neurons expressing pH-sensitive fluorescent proteins. We found that DCD and MD were associated with a prominent pH(m) decrease which presumably resulted from MPT opening. This pH(m) decrease occurred with some delay after the onset of DCD and MD. This argued against the hypothesis that MPT opening plays a dominant role in triggering of DCD. This conclusion was also supported by experiments in which Ca(2+) was replaced with antagonist of MPT opening Sr(2+). We found that in Sr(2+)-containing medium glutamate-induced delayed strontium deregulation (DSD), similar to DCD, which was accompanied by a profound MD. Analysis of the changes in pH(c) and pH(m) associated with DSD led us to conclude that MD in Sr(2+)-containing medium occurred without involvement of the pore. In contrast, in Ca(2+)-containing medium such "non-pore mechanism" was responsible only for MD initiation while in the final stages of MD development the MPT played a major role.     

Mitochondrial Ca2+ transport and permeability transition pore opening and mitochondrial energetic status

The relationship between mitochondrial Ca2+ transport and permeability transition pore (PTP) opening as well as the effects of mitochondrial energetic status on mitochondrial Ca2+ transport and PTP opening were studied. The results showed that the calcium-induced calcium release from mitochondria (mCICR) induced PTP opening. Inhibitors for electron transport of respiratory chain inhibited mCICR and PTP opening. Partial recovery of electron transport in respiratory chain resulted in partial recovery of mCICR and PTP opening. mCICR and PTP opening were also inhibited by CCCP which eliminated transmembrane proton gradient. The results indicated that mitochondrial Ca2+ transport and PTP opening are largely dependent on electron transport and energy coupling.     

beta-Amyloid increases dendritic Ca2+ influx by inhibiting the A-type K+ current in hippocampal CA1 pyramidal neurons

Accumulation of the beta-amyloid peptide (Abeta) is a primary event in the pathogenesis of Alzheimer's disease (AD). However, the mechanisms by which Abeta mediates neurotoxicity and initiates the degenerative processes of AD are still not clear. Recent evidence shows that voltage-gated K+ channels may be involved in Abeta-induced neurodegenerative processes. In particular, a transient A-type K+ current, with a linear increase in its density with distance from soma to distal dendrites in hippocampal CA1 pyramidal neurons, has been shown to contribute to dendritic membrane excitability. Here, I report that Abeta (1-42) inhibits the dendritic A-type K+ current in hippocampal CA1 pyramidal neurons, and this inhibition causes increases in back-propagating dendritic action potential amplitude and associated Ca2+ influx. These results suggest that the persistent inhibition of the A-type K+ current resulting from deposition of Abeta in dendritic arborization will induce a sustained increase in dendritic Ca2+ influx and lead to loss of Ca2+ homeostasis. This may be a component of the events that cause synaptic failure and initiate neuronal degenerative processes in the hippocampus.     

Mitochondrial Ca2+ transport and permeability transition pore opening and mitochondrial energetic status

The relationship between mitochondrial Ca2 transport and permeability transition pore (PTP) opening as well as the effects of mitochondrial energetic status on mitochondrial Ca2 transport and PTP opening were studied. The results showed that the calcium-induced calcium release from mitochondria (mClCR) induced PTP opening. Inhibitors for electron transport of respiratory chain inhibited mClCR and PTP opening. Partial recovery of electron transport in respiratory chain resulted in partial recovery of mClCR and PTP opening. mClCR and PTP opening were also inhibited by CCCP which eliminated transmembrane proton gradient. The results indicated that mitochondrial Ca2 transport and PTP opening are largely dependent on electron transport and energy coupling.     

Competitive modulation of Ca2+ release-activated Ca2+ channel gating by STIM1 and 2-aminoethyldiphenyl borate

Activation of Ca(2+) release-activated Ca(2+) channels by depletion of intracellular Ca(2+) stores involves physical interactions between the endoplasmic reticulum Ca(2+) sensor, STIM1, and the channels composed of Orai subunits. Recent studies indicate that the Orai3 subtype, in addition to being store-operated, is also activated in a store-independent manner by 2-aminoethyldiphenyl borate (2-APB), a small molecule with complex pharmacology. However, it is unknown whether the store-dependent and -independent activation modes of Orai3 channels operate independently or whether there is cross-talk between these activation states. Here we report that in addition to causing direct activation, 2-APB also regulates store-operated gating of Orai3 channels, causing potentiation at low doses and inhibition at high doses. Inhibition of store-operated gating by 2-APB was accompanied by the suppression of several modes of Orai3 channel regulation that depend on STIM1, suggesting that high doses of 2-APB interrupt STIM1-Orai3 coupling. Conversely, STIM1-bound Orai3 (and Orai1) channels resisted direct gating by high doses of 2-APB. The rate of direct 2-APB activation of Orai3 channels increased linearly with the degree of STIM1-Orai3 uncoupling, suggesting that 2-APB has to first disengage STIM1 before it can directly gate Orai3 channels. Collectively, our results indicate that the store-dependent and -independent modes of Ca(2+) release-activated Ca(2+) channel activation are mutually exclusive: channels bound to STIM1 resist 2-APB gating, whereas 2-APB antagonizes STIM1 gating.     

Ca2+ Signals Generated by CatSper and Ca2+ Stores Regulate Different Behaviors in Human Sperm

[Ca²⁺]_i signaling regulates sperm motility, enabling switching between functionally different behaviors that the sperm must employ as it ascends the female tract and fertilizes the oocyte. We report that different behaviors in human sperm are recruited according to the Ca²⁺ signaling pathway used. Activation of CatSper (by raising pH_i or stimulating with progesterone) caused sustained [Ca²⁺]_i elevation but did not induce hyperactivation, the whiplash-like behavior required for progression along the oviduct and penetration of the zona pellucida. In contrast, penetration into methylcellulose (mimicking penetration into cervical mucus or cumulus matrix) was enhanced by activation of CatSper. NNC55-0396, which abolishes CatSper currents in human sperm, inhibited this effect. Treatment with 5 μm thimerosal to mobilize stored Ca²⁺ caused sustained [Ca²⁺]_i elevation and induced strong, sustained hyperactivation that was completely insensitive to NNC55-0396. Thimerosal had no effect on penetration into methylcellulose. 4-Aminopyridine, a powerful modulator of sperm motility, both raised pH_i and mobilized Ca²⁺ stored in sperm (and from microsomal membrane preparations). 4-Aminopyridine-induced hyperactivation even in cells suspended in Ca²⁺-depleted medium and also potentiated penetration into methylcellulose. The latter effect was sensitive to NNC55-039, but induction of hyperactivation was not. We conclude that these two components of the [Ca²⁺]_i signaling apparatus have strikingly different effects on sperm motility. Furthermore, since stored Ca²⁺ at the sperm neck can be mobilized by Ca²⁺-induced Ca²⁺ release, we propose that CatSper activation can elicit functionally different behaviors according to the sensitivity of the Ca²⁺ store, which may be regulated by capacitation and NO from the cumulus.     

Connexin 43 hemichannels contribute to cytoplasmic Ca2+ oscillations by providing a bimodal Ca2+-dependent Ca2+ entry pathway

Many cellular functions are driven by changes in the intracellular Ca(2+) concentration ([Ca(2+)](i)) that are highly organized in time and space. Ca(2+) oscillations are particularly important in this respect and are based on positive and negative [Ca(2+)](i) feedback on inositol 1,4,5-trisphosphate receptors (InsP(3)Rs). Connexin hemichannels are Ca(2+)-permeable plasma membrane channels that are also controlled by [Ca(2+)](i). We aimed to investigate how hemichannels may contribute to Ca(2+) oscillations. Madin-Darby canine kidney cells expressing connexin-32 (Cx32) and Cx43 were exposed to bradykinin (BK) or ATP to induce Ca(2+) oscillations. BK-induced oscillations were rapidly (minutes) and reversibly inhibited by the connexin-mimetic peptides (32)Gap27/(43)Gap26, whereas ATP-induced oscillations were unaffected. Furthermore, these peptides inhibited the BK-triggered release of calcein, a hemichannel-permeable dye. BK-induced oscillations, but not those induced by ATP, were dependent on extracellular Ca(2+). Alleviating the negative feedback of [Ca(2+)](i) on InsP(3)Rs using cytochrome c inhibited BK- and ATP-induced oscillations. Cx32 and Cx43 hemichannels are activated by <500 nm [Ca(2+)](i) but inhibited by higher concentrations and CT9 peptide (last 9 amino acids of the Cx43 C terminus) removes this high [Ca(2+)](i) inhibition. Unlike interfering with the bell-shaped dependence of InsP(3)Rs to [Ca(2+)](i), CT9 peptide prevented BK-induced oscillations but not those triggered by ATP. Collectively, these data indicate that connexin hemichannels contribute to BK-induced oscillations by allowing Ca(2+) entry during the rising phase of the Ca(2+) spikes and by providing an OFF mechanism during the falling phase of the spikes. Hemichannels were not sufficient to ignite oscillations by themselves; however, their contribution was crucial as hemichannel inhibition stopped the oscillations.     

Identification of putative voltage-dependent Ca2+-permeable channels involved in cryptogein-induced Ca2+ transients and defense responses in tobacco BY-2 cells

Ca(2+) is the pivotal second messenger for induction of defense responses induced by treatment of pathogen-derived elicitor or microbial infection in plants. However, molecular bases for elicitor-induced generation of Ca(2+) signals (Ca(2+) transients) are largely unknown. We here identified cDNAs for putative voltage-dependent Ca(2+)-permeable channels, NtTPC1A and NtTPC1B, that are homologous to TPC1 (two pore channel) from suspension-cultured tobacco BY-2 cells. NtTPC1s complemented the growth of a Saccharomyces cerevisiae mutant defective in CCH1, a putative Ca(2+) channel, in a low Ca(2+) medium, suggesting that both products permeate Ca(2+) through the plasma membrane. Cosuppression of NtTPC1s in apoaequorin-expressing BY-2 cells resulted in inhibition of rise in cytosolic free Ca(2+) concentration ([Ca(2+)](cyt)) in response to sucrose and a fungal elicitor cryptogein, while it did not affect hypoosmotic shock-induced [Ca(2+)](cyt) increase. Cosuppression of NtTPC1s also caused suppression of cryptogein-induced programmed cell death and defense-related gene expression. These results suggest that NtTPC1s are involved in Ca(2+) mobilization induced by the cryptogein and sucrose, and have crucial roles in cryptogein-induced signal transduction pathway.