Mitochondrial Ca<Superscript>2+</Superscript> cycle mediated by the palmitate-activated cyclosporin a-insensitive pore

Earlier we found that in isolated rat liver mitochondria the reversible opening of the mitochondrial cyclosporin A-insensitive pore induced by low concentrations of palmitic acid (Pal) plus Ca²⁺ results in the brief loss of Δψ [Mironova et al., J Bioenerg Biomembr (2004), 36:171–178]. Now we report that Pal and Ca²⁺, increased to 30 and 70 nmol/mg protein respectively, induce a stable and prolonged (10 min) partial depolarization of the mitochondrial membrane, the release of Ca²⁺ and the swelling of mitochondria. Inhibitors of the Ca²⁺ uniporter, ruthenium red and La³⁺, as well as EGTA added in 10 min after the Pal/Ca²⁺-activated pore opening, prevent the release of Ca²⁺ and repolarize the membrane to initial level. Similar effects can be observed in the absence of exogeneous Pal, upon mitochondria accumulating high [Sr²⁺], which leads to the activation of phospholipase A₂ and appearance of endogenous fatty acids. The paper proposes a new model of the mitochondrial Ca²⁺ cycle, in which Ca²⁺ uptake is mediated by the Ca²⁺ uniporter and Ca²⁺ efflux occurs via a short-living Pal/Ca²⁺-activated pore.     

Bile Acid-Induced Ca<Superscript>2+</Superscript> Efflux from Liver Mitochondria as a Factor Preventing the Formation of Mitochondrial Pores

The effect of bile acids as inducers of Ca²⁺ efflux from the matrix was studied on isolated rat liver mitochondria. Mitochondria in the presence of cyclosporin A (CsA) were energized by succinate, then loaded with Ca²⁺ and after the addition of the calcium uniporter inhibitor ruthenium red were de-energized by malonate. It was shown that under these conditions hydrophobic bile acids lithocholic and chenodeoxycholic at concentrations of 10 and 30 μM respectively and hydrophilic bile acids ursodeoxycholic and cholic at a concentration of 400 μM induce Ca²⁺ efflux from the mitochondrial matrix. It is noted that the efflux of these ions is not associated with damage of the inner mitochondrial membrane by bile acids, since it is accompanied by the generation of Δψ, i.e., the formation of the diffusion potential. It is assumed that along with induction of calcium efflux from the matrix, bile acids are also capable of transporting hydrogen and potassium ions in the opposite direction, i.e., perform H⁺/Ca²⁺ and K⁺/Ca²⁺ exchange. It was found that ruthenium red added to Ca²⁺-loaded energized mitochondria prevents the return of these ions to the matrix and weakens the effect of chenodeoxycholic acid as an inducer of the CsA-sensitive mitochondrial pore and the effect of ursodeoxycholic acid as an inducer of CsA-insensitive permeability of the inner mitochondrial membrane. We conclude that in the conditions of the calcium uniporter activity decrease, Ca²⁺ efflux from the matrix induced by bile acids can be considered as one of the mechanisms reducing their effectiveness as inducers of the Ca²⁺-dependent CsA-sensitive pore and CsA-insensitive permeability transition in mitochondria.     

Effects of Ketamine on the Balance of Ions Ca<Superscript>2+</Superscript>, Mg<Superscript>2+</Superscript>, Cu<Superscript>2+</Superscript> and Zn<Superscript>2+</Superscript> in the Ischemia-reperfusion Affected Spinal Cord Tissues in Rabbits

To test the effects of ketamine on metal ion balance in the spinal cord tissues after ischemic reperfusion (I/R), 24 white adult Japanese rabbits were randomly assigned to sham operation group, I/R group or ketamine-treated I/R group. Spinal cord injuries in I/R group and ketamine-treated I/R group were induced by aortic occlusions. Rabbits in ketamine-treated I/R group were intravenously infused 10 mg/kg ketamine twice: once at 10 min before aortic clamping and once at the onset of reperfusion. Post-operative neurological functions and concentrations of ions Ca²⁺, Mg²⁺, Cu²⁺ and Zn²⁺ in the spinal cord were assessed. Compared with the sham operation group, rabbits in the I/R group showed significantly worsened neurological functions as scored with the modified Tarlov criteria and altered concentrations of ions Ca²⁺, Mg²⁺, Cu²⁺ and Zn²⁺. These unfavorable changes were significantly reversed in the ketamine-treated I/R group, suggesting that the potent protective effects of ketamine against the I/R-induced spinal cord injuries may be due to its ability to maintain ion balance in the I/R affected tissues.     

Towards extracellular Ca<Superscript>2+</Superscript> sensing by MRI: synthesis and calcium-dependent <Superscript>1</Superscript>H and <Superscript>17</Superscript>O relaxation studies of two novel bismacrocyclic Gd<Superscript>3+</Superscript> complexes

Two new bismacrocyclic Gd³⁺ chelates containing a specific Ca²⁺ binding site were synthesized as potential MRI contrast agents for the detection of Ca²⁺ concentration changes at the millimolar level in the extracellular space. In the ligands, the Ca²⁺-sensitive BAPTA-bisamide central part is separated from the DO3A macrocycles either by an ethylene (L¹) or by a propylene (L²) unit [H₄BAPTA is 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid; H₃DO₃A is 1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid]. The sensitivity of the Gd³⁺ complexes towards Ca²⁺ and Mg²⁺ was studied by ¹H relaxometric titrations. A maximum relaxivity increase of 15 and 10% was observed upon Ca²⁺ binding to Gd₂L¹ and Gd₂L², respectively, with a distinct selectivity of Gd₂L¹ towards Ca²⁺ compared with Mg²⁺. For Ca²⁺ binding, association constants of log K = 1.9 (Gd₂L¹) and log K = 2.7 (Gd₂L²) were determined by relaxometry. Luminescence lifetime measurements and UV–vis spectrophotometry on the corresponding Eu³⁺ analogues proved that the complexes exist in the form of monohydrated and nonhydrated species; Ca²⁺ binding in the central part of the ligand induces the formation of the monohydrated state. The increasing hydration number accounts for the relaxivity increase observed on Ca²⁺ addition. A ¹H nuclear magnetic relaxation dispersion and ¹⁷O NMR study on Gd₂L¹ in the absence and in the presence of Ca²⁺ was performed to assess the microscopic parameters influencing relaxivity. On Ca²⁺ binding, the water exchange is slightly accelerated, which is likely related to the increased steric demand of the central part leading to a destabilization of the Ln–water binding interaction. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Synaptotagmin IV Acts as a Multi-Functional Regulator of Ca<Superscript>2+</Superscript>-Dependent Exocytosis

In response to stimuli, secretary cells secrete a variety of signaling molecules packed in vesicles (e.g., neurotransmitters and peptide hormones) into the extracellular space by exocytosis. The vesicle secretion is often triggered by calcium ion (Ca²⁺) entered into secretary cells and achieved by the fusion of secretory vesicles with the plasma membrane. Recent accumulating evidence has indicated that members of the synaptotagmin (Syt) family play a major role in Ca²⁺-dependent exocytosis, and Syt I, in particular, is now widely accepted as the major Ca²⁺-sensor for synchronous neurotransmitter release. Involvement of other Syt isoforms in Ca²⁺-dependent exocytotic events other than neurotransmitter release has also been reported, and the Syt IV isoform is of particular interest, because Syt IV has several unique features not found in Syt I (e.g., immediate early gene product induced by deporalization and postsynaptic localization). In this article, we summarize the literature on the multi-functional role of Syt IV in Ca²⁺-dependent exocytosis.     

Ca<Superscript>2+</Superscript>-ATPase in the symbiosome membrane from broad bean root nodules: further evidence for its functioning as ATP-driven Ca<Superscript>2+</Superscript>/H<Superscript>+</Superscript> exchanger

To date, it has been established that the symbiosome membrane (SM), i.e., plant-derived membrane of symbiosomes, nitrogen-fixing compartments of legume root nodules, is equipped with Ca²⁺-ATPase transporting Ca²⁺ ions through the SM from the cytosol of infected cells into the symbiosome space (SS). Earlier in the experiments on the SM vesicles isolated from broad bean root nodules some data indicating the action of the Ca²⁺-ATPase as ATP-driven Ca²⁺/H⁺ antiporter were obtained. In the present work performed on isolated symbiosomes from the same plant object, further evidence in favor of calcium-proton countertransport mechanism of the pump operation was obtained. These were expressed in vanadate-sensitive alkalinization of the SS coupled with Ca²⁺ uptake by symbiosomes catalyzed by the SM Ca²⁺-ATPase, stimulation of the kinetics of the latter process in the response to artificial acidification of the SS and expectable modulation of ITP-hydrolyzing activity of this enzyme caused by the variation of pH within this compartment. The above findings are discussed in the framework of the model describing the mechanism of Ca²⁺-ATPase operation as an ATP-driven Ca²⁺/H⁺ exchanger and on this base allow us to put forward the hypothesis about the involvement of this enzyme in symbiosome signaling in a Ca²⁺- and pH-dependent manner.     

Change in Intracellular pH Causes the Toxic Ca<Superscript>2+</Superscript> Entry via NCX1 in Neuron- and Glia-Derived Cells

Brain hypoxia or ischemia causes acidosis and the intracellular accumulation of Ca²⁺ in neuron. The aims of the present study were to elucidate the interaction between intracellular pH and Ca²⁺ during transient acidosis and its effects on the viability of neuronal and glial cells. Intracellular Ca²⁺ and pH were measured using the fluorescence of fura-2 and 2′,7′-bis(2-carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl ester in neuroblastoma (IMR-32), glioblastoma (T98G), and astrocytoma (CCF-STTG1) cell lines. The administration of 5 mM propionate caused intracellular acidification in IMR-32 and T98G cells but not in CCF-STTG1 cells. After the removal of propionate, the intracellular pH recovered to the resting level. The intracellular Ca²⁺ transiently increased upon the removal of propionate in IMR-32 and T98G cells but not in CCF-STTG1 cells. The transient Ca²⁺ increase caused by the withdrawal of intracellular acidification was abolished by the removal of external Ca²⁺, diminished by a reduction of external Na⁺, and inhibited by benzamil. Transient acidosis caused cell death, whereas the cells were more viable in the absence of external Ca²⁺. Benzamil alleviated cell death caused by transient acidosis in IMR-32 and T98G cells but not in CCF-STTG1 cells. These results suggest that recovery from intracellular acidosis causes a transient increase in cytosolic Ca²⁺ due to reversal of Ca²⁺ transport via Na⁺/Ca²⁺ exchanger coactivated with Na⁺/H⁺ exchanger, which can cause cell death.     

Ca<Superscript>2+</Superscript> increases the specific coenzyme Q<Subscript>10</Subscript> content in <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>

Of various metal ions (Ca²⁺, Cr³⁺, Cu²⁺, Fe²⁺, Mg²⁺, Mn²⁺, Ni²⁺ and Zn²⁺) added to the culture medium of Agrobacterium tumefaciens at 1 mM, only Ca²⁺ increased Coenzyme Q10 (CoQ₁₀) content in cells without the inhibition of cell growth. In a pH-stat fed-batch culture, supplementation with 40 mM of CaCO₃ increased the specific CoQ₁₀ content and oxidative stress by 22.4 and 48%, respectively. Also, the effect of Ca²⁺ on the increase of CoQ₁₀ content was successfully verified in a pilot-scale (300 L) fermentor. In this study, the increased oxidative stress in A. tumefaciens culture by the supplementation of Ca²⁺ is hypothesized to stimulate the increase of specific CoQ₁₀ content in order to protect the membrane against lipid peroxidation. Our results improve the understanding of Ca²⁺ effect on CoQ₁₀ biosynthesis in A. tumefaciens and should contribute to better industrial production of CoQ₁₀ by biological processes.     

Swelling-Induced Ca<Superscript>2+</Superscript> Influx and K<Superscript>+</Superscript> Efflux in American Alligator Erythrocytes

The American alligator can hibernate during winter, which may lead to osmotic imbalance because of reduced kidney function and lack of food consumption during this period. Accordingly, we hypothesized that their red blood cells would have a well-developed regulatory volume decrease (RVD) to cope with the homeostatic challenges associated with torpor. Osmotic fragility was determined optically, mean cell volume was measured by electronic sizing, and changes in intracellular Ca²⁺ concentration were visualized using fluorescence microscopy and fluo-4-AM. Osmotic fragility increased and the ability to regulate volume was inhibited when extracellular Na⁺ was replaced with K⁺, or when cells were exposed to the K⁺ channel inhibitor quinine, indicating a requirement of K⁺ efflux for RVD. Addition of the ionophore gramicidin to the extracellular medium decreased osmotic fragility and also potentiated volume recovery, even in the presence of quinine. In addition, hypotonic shock (0.5× Ringer) caused an increase in cytosolic Ca²⁺, which resulted from Ca²⁺ influx because it was not observed when extracellular Ca²⁺ was chelated with EGTA (ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid). Furthermore, cells loaded with BAPTA-AM (1,2-bis(2-aminophenoxymethyl)ethane-N,N,N′,N′-tetraacetic acid tetrakis(acetoxymethyl) ester) or exposed to a low Ca²⁺-EGTA hypotonic Ringer had a greater osmotic fragility and also failed to recover from cell swelling, indicating that extracellular Ca²⁺ was needed for RVD. Gramicidin reversed the inhibitory effect of low extracellular Ca²⁺. Finally, and surprisingly, the Ca²⁺ ionophore A23187 increased osmotic fragility and inhibited volume recovery. Taken together, our results show that cell swelling activated a K⁺ permeable pathway via a Ca²⁺-dependent mechanism, and this process mediated K⁺ loss during RVD.     

The voltage-gated Ca2+ channel is the Ca2+ sensor of fast neurotransmitter release

Previously it demonstrated that in the absence of Ca²⁺ entry, evoked secretion occurs neither by membrane depolarization, induction of [Ca²⁺] _i rise, nor by both combined (Ashery, U., Weiss, C., Sela, D., Spira, M. E., and Atlas, D. (1993). Receptors Channels 1:217–220.). These studies designate Ca²⁺ entry as opposed to [Ca²⁺] _i rise, essential for exocytosis. It led us to propose that the channel acts as the Ca²⁺ sensor and modulates secretion through a physical and functional contact with the synaptic proteins. This view was supported by protein–protein interactions reconstituted in the Xenopus oocytes expression system and release experiments in pancreatic cells (Barg, S., Ma, X., Elliasson, L., Galvanovskis, J., Gopel, S. O., Obermuller, S., Platzer, J., Renstrom, E., Trus, M., Atlas, D., Streissnig, G., and Rorsman, P. (2001). Biophys. J.; Wiser, O., Bennett, M. K., and Atlas, D. (1996). EMBO J. 15:4100–4110; Wiser, O., Trus, M., Hernandez, A., Renström, E., Barg, S., Rorsman, P., and Atlas, D. (1999). Proc. Natl. Acad. Sci. U.S.A. 96:248–253). The kinetics of Ca_v1.2 (Lc-type) and Ca_v2.2 (N-type) Ca²⁺ channels were modified in oocytes injected with cRNA encoding syntaxin 1A and SNAP-25. Conserved cysteines (Cys271, Cys272) within the syntaxin 1A transmembrane domain are essential. Synaptotagmin I, a vesicle-associated protein, accelerated the activation kinetics indicating Ca_v2.2 coupling to the vesicle. The unique modifications of Ca_v1.2 and Ca_v2.2 kinetics by syntaxin 1A, SNAP-25, and synaptotagmin combined implied excitosome formation, a primed fusion complex of the channel with synaptic proteins. The Ca_v1.2 cytosolic domain Lc_753–893, acted as a dominant negative modulator, competitively inhibiting insulin release of channel-associated vesicles (CAV), the readily releasable pool of vesicles (RRP) in islet cells. A molecular mechanism is offered to explain fast secretion of vesicles tethered to SNAREs-associated Ca²⁺ channel. The tight arrangement facilitates the propagation of conformational changes induced during depolarization and Ca²⁺-binding at the channel, to the SNAREs to trigger secretion. The results imply a rapid Ca²⁺-dependent CAV (RRP) release, initiated by the binding of Ca²⁺ to the channel, upstream to intracellular Ca²⁺ sensor thus establishing the Ca²⁺ channel as the Ca²⁺ sensor of neurotransmitter release.     

Engineered glucose isomerase from <Emphasis Type="Italic">Streptomyces</Emphasis> sp. SK is resistant to Ca<Superscript>2+</Superscript> inhibition and Co<Superscript>2+</Superscript> independent

The role of two amino acid residues linked to the two catalytic histidines His54 and His220 in kinetics and physicochemical properties of the Streptomyces sp. SK glucose isomerase (SKGI) was investigated by site-directed mutagenesis and molecular modeling. Two single mutations, F53L and G219D, and a double mutation F53L/G219D was introduced into the xylA SKGI gene. The F53L mutation increases the thermostability and the catalytic efficiency and also slightly shifts the optimum pH from 6.5 to 7, but displays a profile being similar to that of the wild-type enzyme concerning the effect of various metal ions. The G219D mutant is resistant to calcium inhibition retaining about 80% of its residual activity in 10 mM Ca²⁺ instead of 10% for the wild-type. This variant is activated by Mn²⁺ ions, but not Co²⁺, as seen for the wild-type enzyme. It does not require the latter for its thermostability, but has its half-life time displaced from 50 to 20 min at 85°C. The double mutation F53L/G219D restores the thermostability as seen for the wild-type enzyme while maintaining the resistance to the calcium inhibition. Molecular modeling suggests that the increase in thermostability is due to new hydrophobic interactions stabilizing α2 helix and that the resistance to calcium inhibition is a result of narrowing the binding site of catalytic ion.     

Pulses of Cell Ca<Superscript>2+</Superscript> and the Dynamics of Tight Junction Opening and Closing

A mathematical modeling of tight junction (TJ) dynamics was elaborated in a previous study (Kassab, F., Marques, R.P., Lacaz-Vieira, F. 2002. Modeling tight junction dynamics and oscillations. J. Gen. Physiol. 120:237–247) to better understand the dynamics of TJ opening and closing, as well as oscillations of TJ permeability that are observed in response to changes of extracellular Ca²⁺ levels. In this model, TJs were assumed to be specifically controlled by the Ca²⁺ concentration levels at the extracellular Ca²⁺ binding sites of zonula adhaerens. Despite the fact that the model predicts all aspects of TJ dynamics, we cannot rule out the likelihood that changes of intracellular Ca²⁺ concentration (Ca²⁺ _cell), which might result from changes \ of extracellular Ca²⁺ concentration (Ca²⁺ _extl), contribute to the observed results. In order to address this aspect of TJ regulation, fast Ca²⁺-switch experiments were performed in which changes of Ca²⁺ _cell were induced using the Ca²⁺ ionophore A23187 or thapsigargin, a specific inhibitor of the sarco-endoplasmic reticulum Ca²⁺-ATPase. The results indicate that the ionophore or thapsigargin per se do not affect basal tissue electrical conductance (G), showing that the sealing of TJs is not affected by a rise in Ca²⁺ _cell. When TJs were kept in a dynamic state, as partially open structures or in oscillation, conditions in which the junctions are very sensitive to disturbances that affect their regulation, a rise of Ca²⁺ _cell never led to a decline of G, indicating that a rise of Ca²⁺ _cell does not trigger per se TJ closure. On the contrary, always the first response to a rise of Ca²⁺ _cell is an increase of G that, in most cases, is a transient response. Despite these observations we cannot assure that a rise of Ca²⁺ _cell is without effect on the TJs, since an increase of Ca²⁺ _cell not only causes a transient increase of G but, in addition, during oscillations a rise of Ca²⁺ _cell induced by the Ca²⁺ ionophore transiently halted the oscillatory pattern of TJs. The main conclusion of this study is that TJ closure that is observed when basolateral Ca²⁺ concentration (Ca²⁺ _bl) is increased after TJs were opened by Ca²⁺ _bl removal cannot be ascribed to a rise of Ca²⁺ _cell and might be a consequence of Ca²⁺ binding to extracellular Ca²⁺ sites.     

Long chain fatty acyl-CoA modulation of H<Subscript>2</Subscript>O<Subscript>2</Subscript> release at mitochondrial complex I

Complex I is responsible for most of the mitochondrial H₂O₂ release, low during the oxidation of the NAD linked substrates and high during succinate oxidation, via reverse electron flow. This H₂O₂ production appear physiological since it occurs at submillimolar concentrations of succinate also in the presence of NAD substrates in heart (present work) and rat brain mitochondria (Zoccarato et al., Biochem J, 406:125–129, 2007). Long chain fatty acyl-CoAs, but not fatty acids, act as strong inhibitors of succinate dependent H₂O₂ release. The inhibitory effect of acyl-CoAs is independent of their oxidation, being relieved by carnitine and unaffected or potentiated by malonyl-CoA. The inhibition appears to depend on the unbound form since the acyl-CoA effect decreases at BSA concentrations higher than 2 mg/ml; it is not dependent on ΔpH or Δp and could depend on the inhibition of reverse electron transfer at complex I, since palmitoyl-CoA inhibits the succinate dependent NAD(P) or acetoacetate reduction.     

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.     

Physiological modulators of cyclosporin A-insensitive nonspecific permeability of the inner membrane of liver mitochondria induced by calcium ions and α,ω-hexadecanedioic acid

Long-chain saturated α,ω-dioic acids can induce nonspecific permeability of the inner membrane (pore opening) of liver mitochondria loaded with Ca²⁺ or Sr²⁺ by the mechanism insensitive to cyclosporin A (CsA). In this work we found that 200 μM Ca²⁺ and 20 μM α,ω-hexadecanedioic acid (HDA) in the presence of 1 μM CsA induced high-amplitude swelling of liver mitochondria (pore opening) only in the presence of succinate as oxidation substrate. Under these conditions protonophore uncoupler of oxidative phosphorylation 2,4-dinitrophenol at the concentration of 75 μM, which is optimal for its uncoupling activity, inhibited mitochondrial swelling induced by Ca²⁺ and HDA, despite the presence of succinate in the incubation medium. Natural uncouplers of oxidative phosphorylation, oleic and linoleic acids, produced a similar effect. These data suggest that energization of organelles, which promotes Ca²⁺ transport into the matrix, is one of the basic requirements of pore opening in liver mitochondria induced by Ca²⁺ and HDA. It is shown that ATP at the physiological concentration of 2 mM inhibits HDA-induced high-amplitude swelling of mitochondria by reducing free Ca²⁺ concentration in the medium. ADP at the same concentration had a similar effect. This modulating effect of nucleotides apparently is attributable to their ability to chelate calcium ions. Polycation spermine, which is known as an inhibitor of the classical CsA-sensitive pore, at the physiological concentration of 1 mM inhibited CsA-insensitive swelling of liver mitochondria induced by sequential addition of Ca²⁺ and HDA. It is assumed that such action of spermine is due to its ability to shield the negative surface charges on the inner membrane of mitochondria. Bovine serum albumin (BSA), which is able to bind free fatty acids and thus prevent the induction of Ca²⁺-dependent pore, inhibited HDA-induced swelling of mitochondria. However, at the same BSA/fatty acid molar ratio inhibitory effect of BSA was much less pronounced if HDA was used as the pore inducer instead of palmitic acid. Apparently, this can be accounted by the fact that BSA binds α,ω-dioic acids weaker than their monocarboxylic analogues.     

Reduced capacity of Ca<Superscript>2+</Superscript> retention in liver as compared to kidney mitochondria. ADP requirement

Ca²⁺ loading in mitochondria promotes the opening of a non-selective transmembrane pathway. Permeability transition is also associated with the interaction of cyclophilin D at the internal surface of the non-specific transmembrane pore. This interaction is circumvented by cyclosporin A and ADP. Our results show that, in the absence of ADP, liver mitochondria were unable to retain Ca²⁺, they underwent a fast and large amplitude swelling, as well as a rapid collapse of the transmembrane potential. In contrast, in the absence of ADP, kidney mitochondria retained Ca²⁺, swelling did not occur, and the collapse of the membrane potential was delayed. Ca²⁺ efflux was reversed by the addition of ADP and cyclosporin A. Our findings indicate that the differences between liver and kidney mitochondria are due to the low association of cyclophilin D to the ADP/ATP carrier found in kidney mitochondria as compared to liver mitochondria.     

Anoxia-induced elevation of cytosolic Ca<Superscript>2+</Superscript> concentration depends on different Ca<Superscript>2+</Superscript> sources in rice and wheat protoplasts

The anoxia-dependent elevation of cytosolic Ca²⁺ concentration, [Ca²⁺]_cyt, was investigated in plants differing in tolerance to hypoxia. The [Ca²⁺]_cyt was measured by fluorescence microscopy in single protoplasts loaded with the calcium-fluoroprobe Fura 2-AM. Imposition of anoxia led to a fast (within 3 min) significant elevation of [Ca²⁺]_cyt in rice leaf protoplasts. A tenfold drop in the external Ca²⁺ concentration (to 0.1 mM) resulted in considerable decrease of the [Ca²⁺]_cyt shift. Rice root protoplasts reacted upon anoxia with higher amplitude. Addition of plasma membrane (verapamil, La³⁺ and EGTA) and intracellular membrane Ca²⁺-channel antagonists (Li⁺, ruthenium red and cyclosporine A) reduced the anoxic Ca²⁺-accumulation in rice. Wheat protoplasts responded to anoxia by smaller changes of [Ca²⁺]_cyt. In wheat leaf protoplasts, the amplitude of the Ca²⁺-shift little depended on the external level of Ca²⁺. Wheat root protoplasts were characterized by a small shift of [Ca²⁺]_cyt under anoxia. Plasmalemma Ca²⁺-channel blockers had little effect on the elevation of cytosolic Ca²⁺ in wheat protoplasts. Intact rice seedlings absorbed Ca²⁺ from the external medium under anoxic treatment. On the contrary, wheat seedlings were characterized by leakage of Ca²⁺. Verapamil abolished the Ca²⁺ influx in rice roots and Ca²⁺ efflux from wheat roots. Anoxia-induced [Ca²⁺]_cyt elevation was high particularly in rice, a hypoxia-tolerant species. In conclusion, both external and internal Ca²⁺ stores are important for anoxic [Ca²⁺]_cyt elevation in rice, whereas the hypoxia-intolerant wheat does not require external sources for [Ca²⁺]_cyt rise. Leaf and root protoplasts similarly responded to anoxia, independent of their organ origin.     

Effect of Mg<Superscript>2+</Superscript> versus Ca<Superscript>2+</Superscript> on the behavior of Annexin A5 in a membrane-bound state

Annexin A5 (AnxA5) binds to negatively charged phospholipid membranes in a Ca²⁺ dependent manner. Several studies already demonstrate that Mg²⁺ ions cannot induce the binding. In this paper, quartz crystal microbalance with dissipation monitoring (QCM-D), Brewster angle microscopy (BAM), polarization modulation infrared reflection absorption spectroscopy (PMIRRAS) and molecular dynamics (MD) were performed to elucidate the high specificity of Ca²⁺ versus Mg²⁺ on AnxA5 binding to membrane models. In the presence of Ca²⁺, AnxA5 showed a strong interaction with lipids, the protein is adsorbed mainly in α-helix under the DMPS monolayer, with an orientation of the α-helices axes slightly tilted with respect to the normal of the phospholipid monolayer as revealed by PMIRRAS. The Ca²⁺ ions interact strongly with the phosphate group of the phospholipid monolayer. In the presence of Mg²⁺, instead of Ca²⁺, no interaction of AnxA5 with lipids was detected. Molecular dynamics simulations allow us to explain the high specificity of calcium. Ca²⁺ ions are well exposed and surrounded by labile water molecules at the surface of the protein, which then favour their binding to the phosphate group of the membrane, explaining their specificity. To the contrary, Mg²⁺ ions are embedded in the protein structure, with a smaller number of water molecules strongly bound. We conclude that the embedded Mg²⁺ ions inside the AnxA5 structure are not able to link the protein to the phosphate group of the phospholipids for this reason.     

Damage-induced cell–cell communication in different cochlear cell types via two distinct ATP-dependent Ca<Superscript>2+</Superscript> waves

Intercellular Ca²⁺ waves can coordinate the action of large numbers of cells over significant distances. Recent work in many different systems has indicated that the release of ATP is fundamental for the propagation of most Ca²⁺ waves. In the organ of hearing, the cochlea, ATP release is involved in critical signalling events during tissue maturation. ATP-dependent signalling is also implicated in the normal hearing process and in sensing cochlear damage. Here, we show that two distinct Ca²⁺ waves are triggered during damage to cochlear explants. Both Ca²⁺ waves are elicited by extracellular ATP acting on P2 receptors, but they differ in their source of Ca²⁺, their velocity, their extent of spread and the cell type through which they propagate. A slower Ca²⁺ wave (14 μm/s) communicates between Deiters’ cells and is mediated by P2Y receptors and Ca²⁺ release from IP₃-sensitive stores. In contrast, a faster Ca²⁺ wave (41 μm/s) propagates through sensory hair cells and is mediated by Ca²⁺ influx from the external environment. Using inhibitors and selective agonists of P2 receptors, we suggest that the faster Ca²⁺ wave is mediated by P2X₄ receptors. Thus, in complex tissues, the expression of different receptors determines the propagation of distinct intercellular communication signals.     

Mitochondrial Ca<Superscript>2+</Superscript> and regulation of the permeability transition pore

The mitochondrial permeability transition pore was originally described in the 1970’s as a Ca²⁺ activated pore and has since been attributed to the pathogenesis of many diseases. Here we evaluate how each of the current models of the pore complex fit to what is known about how Ca²⁺ regulates the pore, and any insight that provides into the molecular identity of the pore complex. We also discuss the central role of Ca²⁺ in modulating the pore’s open probability by directly regulating processes, such as ATP/ADP balance through the tricarboxylic acid cycle, electron transport chain, and mitochondrial membrane potential. We review how Ca²⁺ influences second messengers such as reactive oxygen/nitrogen species production and polyphosphate formation. We discuss the evidence for how Ca²⁺ regulates post-translational modification of cyclophilin D including phosphorylation by glycogen synthase kinase 3 beta, deacetylation by sirtuins, and oxidation/ nitrosylation of key residues. Lastly we introduce a novel view into how Ca²⁺ activated proteolysis through calpains in the mitochondria may be a driver of sustained pore opening during pathologies such as ischemia reperfusion injury.