Ca binding to c-state of adenine nucleotide translocase (ANT)-surrounding cardiolipins enhances (ANT)-Cys relative mobility: A computational-based mitochondrial permeability transition study

The oxidation of critical cysteines/related thiols of adenine nucleotide translocase (ANT) is believed to be an important event of the Ca²⁺-induced mitochondrial permeability transition (MPT), a process mediated by a cyclosporine A/ADP-sensitive permeability transition pores (PTP) opening. We addressed the ANT-Cys⁵⁶ relative mobility status resulting from the interaction of ANT/surrounding cardiolipins with Ca²⁺ and/or ADP by means of computational chemistry analysis (Molecular Interaction Fields and Molecular Dynamics studies), supported by classic mitochondrial swelling assays. The following events were predicted: (i) Ca²⁺ interacts preferentially with the ANT surrounding cardiolipins bound to the H4 helix of translocase, (ii) weakens the cardiolipins/ANT interactions and (iii) destabilizes the initial ANT-Cys⁵⁶ residue increasing its relative mobility. The binding of ADP that stabilizes the conformation “m” of ANT and/or cardiolipin, respectively to H5 and H4 helices, could stabilize their contacts with the short helix h56 that includes Cys⁵⁶, accounting for reducing its relative mobility. The results suggest that Ca²⁺ binding to adenine nucleotide translocase (ANT)-surrounding cardiolipins in c-state of the translocase enhances (ANT)-Cys⁵⁶ relative mobility and that this may constitute a potential critical step of Ca²⁺-induced PTP opening.     

Ca<Superscript>2+</Superscript> release from intracellular stores of pig oocytes at different stages of growth

Ca²⁺ release from intracellular stores of pig oocytes was investigated using the Ca²⁺-sensitive fluorescent dye chlorotetracycline. Oocytes were divided into growing ones and those that completed their growth using brilliant cresyl blue (BCB) staining. The stained oocytes (BCB “+”) were determined as the ones that completed their growth, while the stainless ones (BCB “−”) were determined as those in the final stages of growth. In the BCB “+” and BCB “−” oocytes, prolactin, theophylline, GTP, and GDP cause Ca²⁺ to exit intracellular stores. In the oocytes that completed their growth, joint action of prolactin and GTP activates additional release of Ca²⁺, in which protein kinase C takes part. In growing oocytes, joint action of prolactin and GTP does not lead to additional release of Ca2⁺. Joint action of theophylline and GDP in growing oocytes and oocytes that completed the growth stage promotes additional Ca²⁺ exit from intracellular stores. This exit is regulated by protein kinase A. The obtained data show that there various routes of Ca²⁺ release from intracellular stores in growing and grown pig oocytes.     

Preload-induced changes in isometric tension and [Ca<Superscript>2+</Superscript>]<Subscript>i</Subscript> in rat myocardium

Preload-induced changes of active tension and [Ca²⁺]i are “dissociated” in mammalian myocardium. This study aimed to describe the distinct effects of preload at low and physiological [Ca²⁺]_o. Rat RV papillary muscles were studied in isometric conditions at 25‡C and 0.33 Hz at 1 mM (hypo-Ca group) and 2.5 mM [Ca²⁺]_o (normal-Ca group). [Ca²⁺]_i was monitored with fura-2/AM. Increase of preload caused a rise of active tension in hypo-Ca and normal-Ca groups whereas peak fluorescence rose significantly only at low [Ca²⁺]_o. End-diastolic tension, end-diastolic level of fluorescence, time-to-peak tension, but not time-to-peak of Ca²⁺ transient, progressively increased with preload. Mechanical relaxation decelerated with preload while Ca²⁺ transient decay time decreased in the initial phase and increased in the late phase, resulting in a prominent “bump” configuration. The “bump” was assessed as a ratio of its area to the fluorescence trace area. It was a new finding that the preload-induced rise of this ratio was twice as large in hypo-Ca. Our results indicate that preload-induced changes in active tension and [Ca²⁺]_i are “dissociated” in rat myocardium, with relatively higher expression at low [Ca²⁺]_o. Ca-dependence of Ca-TnC association/dissociation kinetics is thought to be a main contributor to these preload-induced effects.     

Genetic Dissection of the Permeability Transition Pore

The permeability transition pore (PTP) regulates the structural re-organization of mitochondria in response to changes in cellular Ca²⁺ and is thought to be an important participant in mitochondrial responses to cell death signals. Although the proteins forming the PTP have yet to be rigorously identified, recent examination of the response of mitochondria, cells and tissues lacking putative components of the PTP have been reported. Studies on mitochondria lacking cyclophilin D (CyP-D) have proved that this protein is the target for PTP inhibition by CsA; yet they have also unequivocally demonstrated that the PTP can form and open in the absence of CyP-D. Likewise, studies in mice lacking the two adenine nucleotide translocators expressed in this species have shown that a functional PTP can form in the absence of these proteins. Thus, the inner mitochondrial membrane components of the PTP remain to be identified, and the absence of CyP-D may not preclude PTP opening in vivo – a finding that questions the conclusion that the PTP participates in cell death pathways only in response to a restricted set of challenges.     

Study on the relationship between calcium-induced calcium release from mitochondria and PTP opening

The permeability transition pore (PTP) is central for mitochondria function. PTP either open in low-conductance state to carry out mCICR (Ca²⁺-induced Ca²⁺ release from mitochondria) and play roles in cell phsyiological activities or open in high-conductance conformation to release harmful substances and play important roles in cell pathological responses and apoptosis. The results of study on the relationship between mCICR and PTP opening show Ca²⁺ concentrations but not the Ca²⁺ delivery mode determined the occurrence of mCICR or PTP opening. Ca²⁺-induced PTP opening began with and depended on mCICR. mCICR was a prerequisite for H₂O₂ and As₂O₃-induced PTP opening. The results indicated that the PTP opening was determined by Ca²⁺ stimulation intensity but not mode. PTP could switch from low- to high-conductance conformation and the PTP open by high-conductance began with low-conductance state. mCICR is necessary for Ca²⁺-dependent PTP opening. Our data suggested also that it would be possible to control cellular responses first by modulating mCICR, then by regulating PTP opening.     

Properties of the permeability transition pore in mitochondria devoid of Cyclophilin D

We have studied the properties of the permeability transition pore (PTP) in mitochondria from the liver of mice where the Ppif gene encoding for mitochondrial Cyclophilin D (CyP-D) had been inactivated. Mitochondria from Ppif-/- mice had no CyP-D and displayed a striking desensitization of the PTP to Ca2+, in that pore opening required about twice the Ca2+ load necessary to open the pore in strain-matched, wild-type mitochondria. Mitochondria lacking CyP-D were insensitive to Cyclosporin A (CsA), which increased the Ca2+ retention capacity only in mitochondria from wild-type mice. The PTP response to ubiquinone 0, depolarization, pH, adenine nucleotides, and thiol oxidants was similar in mitochondria from wild-type and Ppif-/- mice. These experiments demonstrate that (i) the PTP can form and open in the absence of CyP-D, (ii) that CyP-D represents the target for PTP inhibition by CsA, and (iii) that CyP-D modulates the sensitivity of the PTP to Ca2+ but not its regulation by the proton electrochemical gradient, adenine nucleotides, and oxidative stress. These results have major implications for our current understanding of the PTP and its modulation in vitro and in vivo.     

Control of the Mitochondrial Permeability Transition Pore by High-Affinity ADP Binding at the ADP/ATP Translocase in Permeabilized Mitochondria

Low levels of ADP binding at the ADP/ATP translocase caused inhibition of the Ca²⁺-inducedpermeability transition of the mitochondrial inner membrane, when measured using the shrinkage assay on mitochondria, which have already undergone a transition. Inhibition was preventedby carboxyatractyloside, but potentiated by bongkrekic acid, which increased the affinity forinhibition by ADP. This suggests that inhibition was related to the conformation of thetranslocase. Ca²⁺ addition was calculated to remove most of the free ADP. Ca²⁺ added after ADPinduced a slow decay of the inhibition, which probably reflected the dissociation of ADP fromthe translocator. We conclude that the probability of forming a permeability transition pore(PTP) is much greater when the translocase is in the CAT conformation than in the BKAconformation, and, in the absence of CAT and BKA, the translocator is shifted between theBKA and CAT conformations by ADP binding and removal, even in deenergized mitochondria with no nucleotide gradients.     

Regulation of total mitochondrial Ca2+ in perfused liver is independent of the permeability transition pore

Triggering ofthe permeability transition pore (PTP) in isolated mitochondria causesrelease of matrix Ca²⁺, ions, andmetabolites, and it has been proposed that the PTP mediatesmitochondrial Ca²⁺ release inintact cells. To study the role of the PTP in mitochondrial energymetabolism, the mitochondrial content ofCa²⁺,Mg²⁺, ATP, and ADP was determinedin hormonally stimulated rat livers perfused with cyclosporin A (CsA).Stimulation of livers perfused in the absence of CsA with glucagon andphenylephrine induced an extensive uptake ofCa²⁺,Mg²⁺, and ATP plus ADP by themitochondria, followed by a release on omission of hormones. In thepresence of CsA, the PTP was fully inhibited, but neither thehormone-induced uptake of Ca²⁺,ATP, or ADP by mitochondria nor their release after washout of hormoneswas significantly changed. We conclude that the regulation of sustainedchanges in mitochondrial Ca²⁺content induced by hormonal stimulation is independent of the PTP.

     

Sanglifehrin A acts as a potent inhibitor of the mitochondrial permeability transition and reperfusion injury of the heart by binding to cyclophilin-D at a different site from cyclosporin A

Cyclosporin A (CsA) inhibits opening of the mitochondrial permeability transition pore (MPTP), a critical event in some forms of necrotic and apoptotic cell death, by binding to cyclophilin D (CyP-D) and inhibiting its peptidyl-prolyl cis-trans isomerase (PPIase) activity. Sanglifehrin A (SfA), like CsA, exerts its immunosuppressive action by binding to cyclophilin A but at a different site from CsA, and unlike the latter, SfA does not inhibit calcineurin activity. Here we demonstrate that SfA inhibits the PPIase activity of CyP-D (K(0.5) 2 nm) and acts as a potent inhibitor of MPTP opening under both energized and de-energized conditions. However, unlike CsA, the dose-response curve for inhibition by SfA is sigmoidal rather than hyperbolic, suggesting a multimeric structure for the MPTP with cooperativity between subunits. Furthermore, SfA does not prevent CyP-D binding to submitochondrial particles or detergent-solubilized adenine nucleotide translocase (ANT), implying that CyP-D binding to the ANT does not require PPIase activity but pore opening does. Once bound to the MPTP, SfA is not readily dissociated, and inhibition of pore opening is maintained following extensive washing. To investigate the potential of SfA as an inhibitor of cell death in vivo, we used the Langendorff perfused rat heart. SfA caused a time-dependent inhibition of the MPTP that was maintained on mitochondrial isolation to a greater extent than was CsA inhibition. We demonstrate that SfA, like CsA, improves the recovery of left ventricular developed pressure during reperfusion after 30 min of global ischemia and greatly reduces lactate dehydrogenase release, implying inhibition of necrotic damage. Because SfA does not inhibit calcineurin activity, our data suggest that it may be more desirable than CsA for protecting tissues recovering from ischemic episodes and for studying the role of the MPTP in cell death.     

Influence of cholesterol on the formation of palmitate/Ca<Superscript>2+</Superscript>-activated pores in mitochondria and liposomes

The influence of cholesterol on the formation of a mitochondrial cyclosporin A-insensitive palmitate/Ca²⁺-activated pore has been studied. Loading of mitochondrial membranes with cholesterol increases the rate of mitochondrial swelling induced by palmitic acid (≥20 μM) and Ca²⁺ (30 μM). This effect is not related to changes in the functional activity of organelles, since cholesterol does not influence the mitochondrial respiration in different metabolic states. At the same time, palmitate/Ca²⁺-induced permeabilization of azolectin/cholesterol liposomes is more pronounced than that of azolectin liposomes. In the liposomal membrane, Ca²⁺ induces phase separation of palmitic acid into distinct membrane domains; the presence of cholesterol in membranes enhances this effect.     

Butylated hydroxytoluene and inorganic phosphate plus Ca2+ increase mitochondrial permeability via mutually exclusive mechanisms

Mitochondria undergo a permeability transition (PT), i.e., become nonselectively permeable to small solutes, in response to a wide range of conditions/compounds. In general, opening of the permeability transition pore (PTP) is Ca²⁺- and P_i-dependent and is blocked by cyclosporin A (CsA), trifluoperazine (TFP), ADP, and butylated hydroxytoluene (BHT). Gudz and coworkers have reported [7th European Bioenergetics Conference, EBEC Short Reports (1992)7, 125], however, that, under some conditions, BHT increases mitochondrial permeability via a process that may not share all of these characteristics. Specifically, they determined that the BHT-induced permeability transition was independent of Ca²⁺ and was insensitive to CsA. We have used mitochondrial swelling to compare in greater detail the changes in permeability induced by BHT and by Ca²⁺ plus P_i with the following results. (1) The dependence of permeability on BHT concentration is triphasic: there is a threshold BHT concentration (ca. 60 nmol BHT/ mg mitochondrial protein) below which no increase occurs; BHT enhances permeability in an intermediate concentration range; and at high BHT concentrations (> 120 nmol/mg) permeability is again reduced. (2) The effects of BHT depend on the ratio of BHT to mitochondrial protein. (3) Concentrations of BHT too low to induce swelling block the PT induced by Ca²⁺ and P_i. (4) The dependence of the Ca²⁺-triggered PT on P_i concentration is biphasic. Below a threshold of 50–100 M, no swelling occurs. Above this threshold swelling increases rapidly. (5) P_i levels too low to support the Ca²⁺-induced PT inhibit BHT-induced swelling. (6) Swelling induced by BHT can bestimulated by agents and treatments that block the PT induced by Ca²⁺ plus P_i. These data suggest that BHT and Ca²⁺ plus P_i, increase mitochondrial permeability via two mutually exclusive mechanisms.     

Binding of Ca<Superscript>2+</Superscript> and Zn<Superscript>2+</Superscript> to factor IX/X-binding protein from venom of <Emphasis Type="Italic">Agkistrodon halys</Emphasis> Pallas: stabilization of the structure during GdnHCl-induced and thermally induced denaturation

Coagulation factor IX/coagulation factor X binding protein from the venom of Agkistrodon halys Pallas (AHP IX/X-bp) is a unique coagulation factor IX/coagulation factor X binding protein (IX/X-bp). Among all IX/X-bps identified, only AHP IX/X-bp is a Ca²⁺- and Zn²⁺-binding protein. The binding properties of Ca²⁺ and Zn²⁺ ions binding to apo-AHP IX/X-bp and their effects on the stability of the protein have been investigated by isothermal titration calorimetry, fluorescence spectroscopy, and differential scanning calorimetry. The results show that AHP IX/X-bp has two metal binding sites, one specific for Ca²⁺ with lower affinity for Zn²⁺ and one specific for Zn²⁺ with lower affinity for Ca²⁺. The bindings of Ca²⁺ and Zn²⁺ in the two sites are entropy- and enthalpy-driven. The binding affinity of AHP IX/X-bp for Zn²⁺ is 1 order of magnitude higher than for Ca²⁺ for either high-affinity binding or low-affinity binding, which accounts for the existence of one Zn²⁺ in the purified AHP IX/X-bp. Guanidine hydrochloride (GdnHCl)-induced and thermally induced denaturations of Ca²⁺–Ca²⁺-AHP IX/X-bp, Zn²⁺–Zn²⁺-AHP IX/X-bp, and Ca²⁺–Zn²⁺-AHP IX/X-bp are all a two-state processes with no detectable intermediate state(s), indicating the Ca²⁺/Zn²⁺-induced tight packing of the protein. Ca²⁺ and Zn²⁺ increase the structural stability of AHP IX/X-bp against GdnHCl or thermal denaturation to a similar extent. Although Ca²⁺ and Zn²⁺ have no obvious effect on the secondary structure of AHP IX/X-bp, they induce different rearrangements in local conformation. The Zn²⁺-stabilized specific conformation of AHP IX/X-bp may be helpful to its recognition of the structure of coagulation factor IX. This work suggests that in vitro, Ca²⁺ plays a structural rather than an active role in the anticoagulation of AHP IX/X-bp, whereas Zn²⁺ plays both structural and active roles in the anticoagulation. In blood, Ca²⁺ binds to AHP IX/X-bp and stabilizes its structure, whereas Zn²⁺ cannot bind to AHP IX/X-bp owing to the low Zn²⁺ concentration. AHP IX/X-bp prolongs the clotting time in vivo through its binding only with coagulation factor X/activated coagulation factor X.     

Duality of effect of La3+ on mitochondrial permeability transition pore depending on the concentration

In order to explore the role of mitochondria in proliferation promotion and/or apoptosis induction of lanthanum, the mutual influences between La³⁺ and Ca²⁺ on mitochondrial permeability transition pore (PTP) opening were investigated with isolated mitochondria from rat liver. The experimental results revealed that La³⁺ influence the state of mitochondria in a concentration-dependent biphasic manner. La³⁺ in nanomolar concentrations, acting as a Ca²⁺ analog, entered mitochondrial matrix via the RuR sensitive Ca²⁺ channel and elevated ROS level, leading to opening of PTP indicated by mitochondrial swelling, reduction of ΔΨ_m and cytochrome c release. Inhibition of PTP with 10 μM CsA attenuated the effects of La³⁺. However, micromolar concentrations La³⁺ acted mainly as a Ca²⁺ antagonist, inhibiting PTP opening induced by Ca²⁺. We postulated that this action of La³⁺ on mitochondria through interaction with Ca²⁺ might be involved in the proliferation-promoting and apoptosis induction by La³⁺.     

The yeast mitochondrial permeability transition is regulated by reactive oxygen species,endogenous Ca2+ and Cpr3, mediating cell death

We investigated the properties of the permeability transition pore (PTP) in Saccharomyces cerevisiae in agar-embedded mitochondria (AEM) and agar-embedded cells (AEC) and its role in yeast death. In AEM, ethanol-induced pore opening, as indicated by the release of calcein and mitochondrial membrane depolarization, can be inhibited by CsA, by Cpr3 deficiency, and by the antioxidant glutathione. Notably, the pore opening is inhibited, when mitochondria are preloaded by EGTA or Fluo3 to chelate matrix Ca²⁺, or are pretreated with 4-Br A23187 to extract matrix Ca²⁺, prior to agar-embedding, or when pore opening is induced in the presence of EGTA; opened pores are re-closed by sequential treatment with CsA, 4-Br A23187 plus EGTA and NADH, indicating endogenous matrix Ca²⁺ involvement. CsA also inhibits the pore opening with low conductance triggered by exogenous Ca²⁺ transport with ETH129. In AEC, the treatment of tert-butylhydroperoxide, a pro-oxidant that triggers transient pore opening in high conductance in AEM, induces yeast death, which is also dependent on CsA and Cpr3. Furthermore, AEMs from mutants lacking three ADP/ATP carrier (AAC) isoforms and with defective ATP synthase dimerization exhibit high and low conductance pore openings with CsA sensitivity, respectively. Collectively, these data show that the yeast PTP is regulated by Cpr3, endogenous matrix Ca²⁺, and reactive oxygen species, and that it is involved in yeast death; furthermore, ATP synthase dimers play a key role in CsA-sensitive pore formation, while AACs are dispensable.     

Low voltage-activated Ca2+ conductances in thalamic neurons

Low voltage-activated (LVA) Ca²⁺ conductances were characterized in the neurons of the associative laterodorsal (LD) thalamic nucleus in rat brain slices and in enzymatically isolated thalamic units using electrophysiological techniques. Voltage dependence, kinetics of inactivation, pharmacology, and selectivity of the LVA current in the thalamic neurons from animals older than 14 postnatal days were consistent with the existence of two, “fast” and “slow,” subtypes of LVA Ca²⁺ channels. “Slow” LVA current in enzymatically isolated thalamic neurons was much less prominent, compared with that in slice neurons, suggesting that respective channels are predominatly located on the distal dendrites. “Fast” Ca²⁺ channels were sensitive to nifedipine (K _d−2.6 μM) and La³⁺ (K _d−1.0 mM), whereas “slow” Ca²⁺ channels were sensitive to Ni²⁺ (25 μM). Selectivity of the “fast” Ca²⁺ channels was similar to that found for the LVA Ca²⁺ channels in other preparations (I _Ca:I _Sr:I _Ba−1.0: 1.23: 0.94), while selectivity of the “slow” Ca²⁺ channels more resembled selectivity of the HVA Ca²⁺ channels (I _Ca:I _Sr:I _Ba−1.0: 2.5: 3.4).     

Calcium Inhibits Dihydropyridine-Stimulated Increases in Opening and Unitary Conductance of a Plant Ca<Superscript>2+</Superscript> Channel

We have previously characterized the “RCA” channel (root Ca²⁺ channel), a voltage-dependent, Ca²⁺-permeable channel found in plasma membrane-enriched vesicles from wheat roots incorporated into artificial planar lipid bilayers. Earlier work indicated that this channel was insensitive to 1,4-dihydropyridines (DHPs, such as nifedipine and 202–791). However, the present study shows that this channel is sensitive to DHPs, but only with submillimolar Ca²⁺, when the probability of channel opening is reduced, with flickery closures becoming increasingly evident as Ca²⁺ activity decreases. Under these ionic conditions, addition of nanomolar concentrations of (+) 202–791 or nifedipine caused an increase in both the probability of channel opening and the unitary conductance. It is proposed that there is a competitive interaction between Ca²⁺ and DHPs at one of the Ca²⁺-binding sites involved in Ca²⁺ permeation and that binding of a DHP to one of the Ca²⁺-permeation sites facilitates movement of other calcium ions through the channel. The present study shows that higher plant Ca²⁺-permeable channels can be greatly affected by very low concentrations of DHPs and that channel sensitivity may vary with the ionic conditions of the experiment. The results also indicate interesting structural and functional differences between plant and animal Ca²⁺-permeable channels.     

Inhibition of Ca<Superscript>2+</Superscript> Channels and Adrenal Catecholamine Release by G Protein Coupled Receptors

Catecholamines and other transmitters released from adrenal chromaffin cells play central roles in the “fight-or-flight” response and exert profound effects on cardiovascular, endocrine, immune, and nervous system function. As such, precise regulation of chromaffin cell exocytosis is key to maintaining normal physiological function and appropriate responsiveness to acute stress. Chromaffin cells express a number of different G protein coupled receptors (GPCRs) that sense the local environment and orchestrate this precise control of transmitter release. The primary trigger for catecholamine release is Ca²⁺ entry through voltage-gated Ca²⁺ channels, so it makes sense that these channels are subject to complex regulation by GPCRs. In particular G protein βγ heterodimers (Gβγ) bind to and inhibit Ca²⁺ channels. Here I review the mechanisms by which GPCRs inhibit Ca²⁺ channels in chromaffin cells and how this might be altered by cellular context. This is related to the potent autocrine inhibition of Ca²⁺ entry and transmitter release seen in chromaffin cells. Recent data that implicate an additional inhibitory target of Gβγ on the exocytotic machinery and how this might fine tune neuroendocrine secretion are also discussed.     

Studies elucidating the mechanisms of calcium induced mitochondrial depolarization in individual isolated brain mitochondria

Among other mitochondrial functions, energy production and Ca²⁺ uptake are crucial for maintaining neuronal viability. Both of these functions are critically dependent on mitochondrial membrane potential (ΔΨ_m). Mitochondrial Ca²⁺ overload causing a dissipation of ΔΨ_m is a key component of several neuronal pathologies. However, the mechanism of Ca²⁺-induced depolarization in neuronal mitochondria remains unclear. Typically, ΔΨ_m has been evaluated as a single overall estimate from all mitochondria present in a given cell or tissue. However, recent data showed that the population of mitochondria isolated from tissues is not homogeneous, and averaged parameters from the whole population do not necessarily reflect the processes taking place in a single organelle. This review summarizes our recent studies of Ca²⁺-induced depolarization in individual mitochondria isolated from rat forebrain and immobilized to coverslips. Fluorescence imaging techniques and potentiometric fluorescent dyes were effectively used to study ΔΨ_m changes. The data have shown that Ca²⁺ triggers ΔΨ_m oscillations in brain mitochondria followed by a complete depolarization. Further investigation of this phenomenon led us to suggest that Ca²⁺-induced ΔΨ_m oscillations can represent an intermediate unstable state that may lead to irreversible mitochondrial dysfunction. Therefore, further study of this phenomenon would help to understand what causes the irreversible damage of mitochondria during cytosolic/mitochondrial Ca²⁺ overload. Here we discuss the effects of different modulators of the mitochondrial permeability transition pore on Ca²⁺-induced depolarization in brain mitochondria and in liver mitochondria, where the mechanism of Ca²⁺-depolarization is better understood. A comparison of these effects in brain and liver mitochondria led us to conclude that Ca²⁺ can induce reversible “low conductance” permeability transition in brain mitochondria, the phenomenon which requires a transient conformational change of the adenine nucleotide translocator from a specific transporter to a non-specific pore. The article is published in the original.     

The Cratylia mollis Seed Lectin Induces Membrane Permeability Transition in Isolated Rat Liver Mitochondria and a Cyclosporine A‐Insensitive Permeability Transition in Trypanosoma cruzi Mitochondria

Previous results provided evidence that Cratylia mollis seed lectin (Cramoll 1,4) promotes Trypanosoma cruzi epimastigotes death by necrosis via a mechanism involving plasma membrane permeabilization to Ca²⁺ and mitochondrial dysfunction due to matrix Ca²⁺ overload. In order to investigate the mechanism of Ca²⁺‐induced mitochondrial impairment, experiments were performed analyzing the effects of this lectin on T. cruzi mitochondrial fraction and in isolated rat liver mitochondria (RLM), as a control. Confocal microscopy of T. cruzi whole cell revealed that Cramoll 1,4 binding to the plasma membrane glycoconjugates is followed by its internalization and binding to the mitochondrion. Electrical membrane potential (?Ψ_m) of T. cruzi mitochondrial fraction suspended in a reaction medium containing 10 μM Ca²⁺ was significantly decreased by 50 μg/ml Cramoll 1,4 via a mechanism insensitive to cyclosporine A (CsA, membrane permeability transition (MPT) inhibitor), but sensitive to catalase or 125 mM glucose. In RLM suspended in a medium containing 10 μM Ca²⁺ this lectin, at 50 μg/ml, induced increase in the rate of hydrogen peroxide release, mitochondrial swelling, and ?Ψ_m disruption. All these mitochondrial alterations were sensitive to CsA, catalase, and EGTA. These results indicate that Cramoll 1, 4 leads to inner mitochondrial membrane permeabilization through Ca²⁺ dependent mechanisms in both mitochondria. The sensitivity to CsA in RLM characterizes this lectin as a MPT inducer and the lack of CsA effect identifies a CsA‐insensitive MPT in T. cruzi mitochondria.     

Salt stress-induced programmed cell death via Ca<Superscript>2+</Superscript>-mediated mitochondrial permeability transition in tobacco protoplasts

The change in cytosolic free concentration of calcium ([Ca²⁺]_cyt) plays a key role in regulating apoptosis in animal cells. In our experiment, we tried to investigate the function of Ca²⁺ in programmed cell death (PCD) in tobacco (Nicotiana tobacum, cultivar BY-2) protoplasts induced by salt stress. An obvious increase in [Ca²⁺]_cyt was observed a few minutes after treatment and the onset of a decrease in mitochondrial membrane potential (ΔΨ_m) was also observed before the appearance of PCD, pre-treatment of protoplasts with EGTA or LaCl₃ effectively retarded the increase in [Ca²⁺]_cyt, which was concomitant with the decrease in the percentage of cell death and higher ΔΨ_m, pre-treatment with cyclosporine A (CsA) also effectively retarded the increase in [Ca²⁺]_cyt, the decrease in ΔΨ_m and the onset of PCD. All these results suggest that Ca²⁺ is a necessary element in regulating PCD and the increase in [Ca²⁺]_cyt and the opening of mitochondrial permeability transition pore (MPTP) could promote each other in regulating PCD in tobacco protoplasts induced by salt stress.Jiusheng Lin and Yuan Wang-These authors contributed equally for this work.