Mitochondrial glycosidic residues contribute to the interaction between ruthenium amine complexes and the calcium uniporter

The role of glycosidic residues in the inhibitory properties of ruthenium complexes on mitochondrial calcium uptake was determined in mitoplasts.Our results showed that the binding and inhibitory properties of ruthenium amine complexes were modified when mitoplasts were exposed to N-glycosidase F action, but calcium uptake was not altered. N-linked proteins of the mitochondrial inner membrane were identified. We detected an 18-kDa protein that binds labeled Ru₃₆₀ under control conditions, but failed to bind the inhibitor after deglycosilation. A relationship between this protein and the action of ruthenium amine inhibitors of the mitochondrial uniporter is proposed.     

Outstanding questions regarding the permeation,selectivity, and regulation of the mitochondrial calcium uniporter

The recent discovery of genes encoding the mitochondrial calcium (Ca²⁺) uniporter has revealed new opportunities for studying how abnormal Ca²⁺ signals cause disease. Ca²⁺ transport across the mitochondrial inner membrane is highly regulated, and the uniporter is the channel that acts as a major portal for Ca²⁺ influx. Low amounts of mitochondrial Ca²⁺ can boost ATP synthesis, but excess amounts, such as following cytoplasmic Ca²⁺ overload in heart failure, triggers mitochondrial failure and cell death. In fact, precisely because mitochondrial Ca²⁺ transport is so tightly regulated, a fundamental understanding of how the uniporter functions is necessary. Two key uniporter features allow Ca²⁺ influx without mitochondrial damage during normal physiology. First, the channel is significantly more selective than other known Ca²⁺ channels. This prevents the permeation of other ions and uncoupling of the electrochemical gradient. Second, the uniporter becomes active at only high Ca²⁺ concentrations, preventing a resting leak of cytoplasmic Ca²⁺ itself. Now possessing the identities of the various proteins forming the uniporter, we can proceed with efforts to define the molecular determinants of permeation, selectivity and Ca²⁺-regulation.     

Cholera-toxin-dependent ADP-ribosylation of the adenylate cyclase regulatory protein in turkey erythrocyte membranes

Incubation of turkey erythrocyte membranes with cholera toxin and [³²P]NAD caused toxin-dependent incorporation of ³²P into a 42,000 M_r peptide which could be distinguished from toxin-independent ³²P incorporation into other membrane proteins. The radiolabeled 42,000 M_r peptide could be extracted from the membranes using Lubrol PX. When toxin-treated membranes were incubated with isoproterenol and GMP before detergent solubilization, the 42,000 M_r labeled peptide was adsorbed by GTP-γ-agarose which, with the same conditions, adsorbed the adenylate cyclase guanine nucleotide regulatory protein. The labeled peptide and guanine nucleotide regulatory protein activity were coeluted from the affinity matrix by guanylyl-β,γ-imidodiphosphate, GDP, and GMP. Guanosine 5′-O-(2-thiodiphosphate), an analog of GDP which blocks guanine nucleotide- and fluoride-stimulated adenylate cyclase activity, caused elution of labeled peptide which exhibited no regulatory protein activity. Our data support the view that the 42,000 M_r peptide is part of the adenylate cyclase guanine nucleotide regulatory protein. The labeled peptide allows identification of both active and inactive regulatory protein and should be useful in monitoring the purification of the regulatory protein from turkey erythrocytes.     

Calcium-promoted resonance energy transfer between fluorescently labeled proteins during aggregation of chromaffin granule membranes

Proteins of the chromaffin granule membrane were covalently labeled in situ with sulfhydryl-specific fluorophores. Using MIANS (maleimide iodoaminonaphthyl sulfonate) as the donor and fluorescein mercury acetate or fluorescein-5-maleimide as the acceptor, Förster fluorescence resonance energy transfer (FRET) could be employed to measure the degree of inter-membrane and intra-membrane protein-protein contact upon Ca²⁺-induced aggregation of the membranes. The four major findings were: (1) Raising the Ca²⁺ concentration to approx. 500 μM causes the proteins to aggregate in the plane of the membrane. This is demonstrated by Ca²⁺-induced increases in the fluorescence resonance energy transfer in double labeled membranes. This effect is not protein-concentration dependent and occurs at calcium concentrations too low for granule aggregation, implying intra-membrane protein clustering or patching. To our knowledge this is the first direct demonstration of the fluid mosaic nature of subcellular organelles. (2) If two sets of granules are labeled separately, Ca²⁺-induced aggregation brings at least 74% of the labeled proteins into close transmembrane proximity. This effect is also observed at 10–100-fold slower rates in the absence of calcium and can be greatly reduced by depleting the granule membrane of labeled peripheral proteins. It is enhanced if the granules are aggregated by Ca²⁺ or K⁺. We conclude that (some) peripheral proteins can transfer from one membrane surface to another. (3) Aggregation of separately labeled sets of membranes by Ca²⁺ also produces transmembrane energy transfer since: (a) the K_m for Ca²⁺-induced quantum transfer is in the same range as the K_m for aggregation; (b) the reaction is protein-concentration dependent; (c) reversal of aggregation also (partially) reverses donor quenching. (4) A kinetic analysis of the transmembrane effect shows it to be 5–10-fold slower than aggregation itself, supporting earlier suggestions (Haynes, D.H., Kolber, M. and Morris, S.J., (1979) J. Theor. Biol. 81, 713–743) that lipid and protein rearrangements are secondary to granule membrane aggregation.     

Redox regulation of ER and mitochondrial Ca2+ signaling in cell survival and death

Physiological signaling by reactive oxygen species (ROS) and their pathophysiological role in cell death are well recognized. This review focuses on two ROS targets that are key to local Ca²⁺ signaling at the ER/mitochondrial interface – notably, inositol trisphosphate receptors (IP₃Rs) and the mitochondrial calcium uniporter (MCU). Both transport systems are central to molecular mechanisms in cell survival and death. Methods for the measurement of the redox state of these proteins and for the detection of ROS nanodomains are described. Recent results on the redox regulation of these proteins are reviewed.     

Inhibitory Properties of Ruthenium Amine Complexes on Mitochondrial Calcium Uptake

The recent finding that the inhibition of Ca²⁺-stimulated respiration by ruthenium red is mainlydue to a binuclear ruthenium complex (Ru₃₆₀) present in the commercial samples of the classicalinhibitor ruthenium red (Ying et. al., 1991), showed that this complex is the more potent andspecific inhibitor of the mitochondrial calcium uniporter. This work was aimed to provideinsights into the mechanism by which Ru₃₆₀ and other ruthenium-related compounds inhibitscalcium uptake. Ruthenium red and a synthesized analog (Rrphen) were compared with Ru₃₆₀.The inhibition by this binuclear complex was noncompetitive, with a K _i of 9.89 nM. Thenumber of specific binding sites for Ru₃₆₀ was 6.2 pmol/mg protein. Ruthenium red and Ru₃₆₀were mutually exclusive inhibitors. Bound La³⁺ was not displaced by Ru₃₆₀. Rrphen was theleast effective for inhibiting calcium uptake. The results support the notion of a specific bindingsite in the uniporter for the polycationic complexes and a negative charged region from thephospholipids in the membrane, closely associated with the uniporter inhibitor-binding site.     

Essential sulfhydryl groups in the catalytic center of the tonoplast H+-ATPase from coleoptiles ofZea mays L. as demonstrated by the biotin-streptavidin-peroxidase system

Functional properties and the localization of essential SH-groups of the tonoplast H⁺-ATPase fromZea mays L. were studied. In contrast to the pyrophosphate-dependent H⁺-translocation activity of the tonoplast, the H⁺-ATPase activity was inhibited by SH-blocking agents, such as N-ethylmaleimide and iodoacetic acid. In the case ofp-hydroxymercuribenzoate, HgCl₂ and oxidized glutathione, the inhibition could be reversed by adding reduced glutathione or dithiothreitol. Incubation of tonoplast vesicles with oxidized glutathione or N-ethylmaleimide in the presence of Mg·ADP—a competitive inhibitor of the ATP-dependent H⁺ pump—avoided the inhibition of the H⁺-pumping activity. This effect is an indication for the occurrence of essential SH-groups at the catalytic site of the H⁺-ATPase. In order to characterize the active center these thiols were specifically labeled with maleimidobutyrylbiocytin. Subsequently, the membrane proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to an immobilizing membrane. The maleimidobutyrylbiocytin-labeled active-center protein was detected by a biotin-streptavidin-peroxidase staining system and was shown to be a 70-kDa subunit of the tonoplast H⁺-ATPase. It is suggested that the oxidation state of the critical sulfhydryl groups within the active center of the enzyme and their reversible blocking by endogenous compounds might be of great importance for the regulation of the enzyme activity in vivo.     

Dictyostelium discoideum as Expression Host: Isotopic Labeling of a Recombinant Glycoprotein for NMR Studies

The advantages of the organism Dictyostelium discoideum as an expression host for recombinant glycoproteins have been exploited for the production of an isotopically labeled cell surface protein for NMR structure studies. Growth medium containing [¹⁵N]NH₄Cl and [¹³C]glycerol was used to generate isotopically labeled Escherichia coli, which was subsequently introduced to D. discoideum cells in simple Mes buffer. A variety of growth conditions were screened to establish minimal amounts of nitrogen and carbon metabolites for a cost-effective protocol. Following single-step purification by anion-exchange chromatography, 8 mg of uniformly ¹³C,¹⁵N-labeled protein secreted by approximately 10¹⁰D. discoideum cells was isolated from 3.3 liters of supernatant. Mass spectrometry showed the recombinant protein of 16 kDa to have incorporated greater than 99.9% isotopic label. The two-dimensional ¹H-¹³C HSQC spectrum confirms ¹³C labeling of both glycan and amino acid residues of the glycoprotein. All heteronuclear NMR spectra showed a good dispersion of cross-peaks essential for high-quality structure determination.     

Effects of polyamines on mitochondrial Ca transport

Mammalian mitochondria are able to enhance Ca²⁺ accumulation in the presence of polyamines by activating the saturable systems of Ca²⁺ inward transport and buffering extramitochondrial Ca²⁺ concentrations to levels similar to those in the cytosol of resting cells. This effect renders them responsive to regulate free Ca²⁺ concentrations in the physioloical range. The mechanism involved is due to a rise in the affinity of the Ca²⁺ transport system, induced by polyamines, most probably exhibiting allosteric behaviour. The regulatory site of this mechanism is the so-called S₁ binding site of polyamines, which operates in physiological conditions and is located in the energy well between the two peaks present in the energy profile of mitochondrial spermine transport. Spermine is bidirectionally transported across teh inner membrane by cycling, in which influx and efflux are driven by electrical and pH gradients, respectively. Most probably, polyamine affects the Ca²⁺ transport system when it acts from the outside-that is, in the direction of its uniporter channel, in order to reach the S₁ site. Important physiological functions are related to activation of Ca²⁺ transport systems by polyamines and their interactions with the S₁ site. These functions include a rise in the metabolic rate for energy supply and modulation of mitochondrial permeability transition induction, with consequent effects on the triggering of the apoptotic pathway.     

Comparison of In Vitro and In Vivo Labeling of Virus-Induced L-Cell Interferon

Preparations of NDV_uv-induced L-cell interferon were labeled in vitro with ¹²⁵I and ³H gas, or in vivo through incorporation of amino acids-³H during synthesis. Prior to purification, more than 90% of the interferon titer was lost during in vitro labeling by either procedure, whereas 34% of the initial activity of in vivo-labeled material was preserved during preparatory handling. Purification by carboxymethyl-Sephadex chromatography and electrophoresis in polyacrylamide gels was about 100-fold, and electrophoretic profiles revealed close concordance between isotopes and interferon titers in all instances. Noninterferon proteins from control cells, although less extensively labeled with tritium during synthesis than proteins from interferon-producing cells and released in lesser amounts, also contained components of identical electrophoretic mobility and distribution in acrylamide gels as interferon. The highest specific activity (6 x 10⁶ U/mg protein) but lowest cpm per interferon unit ratio (0.3) were exhibited by in vivo-labeled interferon. The advantage of better isotope incorporation through in vitro labeling techniques was largely offset by extensive losses in interferon activity.     

Inhibition of the mitochondrial calcium uniporter by antibodies against a 40-kDa glycorproteinT

Polyclonal rabbit antibodies against a Ca²⁺-binding mitochondrial glycoprotein were found to inhibit the uniporter-mediated transport of Ca²⁺ in mitoplasts prepared from rat liver mitochondria. Spermine, a modulator of the uniporter, decreased the inhibition. This glycoprotein ofM _r 40,000, isolated from beef heart mitochondria and earlier shown to form Ca²⁺-conducting channels in black-lipid membranes, thus is a good candidate for being a component of the uniporter. Antibody-IgG was found to specifically bind to mitochondria in human fibroblasts.     

Vitamin E up-regulates arachidonic acid release and phospholipase A2 in megakaryocytes

The alteration in calcium transport in the liver nuclei of rats orally administered carbon tetrachloride (CCl4) was investigated. Rats received a single oral administration of CCl₄(5, 10, and 25%, 1.0ml/100 g body weight), and 5, 24 and 48 h later the animals were sacrificed. The administration of CCl₄ (25%) caused a remarkable elevetion of calcium content in the liver tissues and the nuclei of rats. Liver nuclear Ca2+-ATPase activity was markedly decreased by CCl₄ (25%) administration. The presence of dibutyryl cyclic AMP(10^-4 and 10^-3 M) or inositol 1,4,5-trisphosphate (10^-6 and 10^-5 M) in the enzyme reaction mixture caused a significant decrease in Ca²⁺-ATPase activity in the liver nuclei obtained from normal rat, while the enzyme activity was significantly increased by calmodulin (1.0 and 2.0 g/ml). These signaling factor's effects were completely impaired in the liver nuclei obtained from CCl₄ (25%)-administered rats. DNA fragmentation in the liver nuclei obtained from CCl₄ -administered rats was significantly decreased by the presence of EGTA (2 mM) in the reaction mixture, suggesting that the endogenous calcium activates nuclear DNA fragmentation. The present study demonstrates that calcium transport system in the liver nuclei is impaired by liver injury with CCl₄ administration in rats.     

Reconstitution of cAMP-Dependent protein kinase regulated renal Na+−H+ exchanger

Summary Studies were performed to determine if the Na⁺–H⁺ exchanger, solubilized from renal brush border membranes from the rabbit and assayed in reconstituted artificial proteoliposomes, could be regulated by cAMP-dependent protein kinase. Octyl glucoside solubilized renal apical membrane proteins from the rabbit kidney were phosphorylated by incubation with ATP and highly purified catalytic subunit of cAMP-dependent kinase.²²Na⁺ uptake was determined subsequently after reconstitution of the proteins into proteoliposomes. cAMP-dependent protein kinase resulted in sustained protein phosphorylation and a concentration-dependent decrease in the amiloride-sensitive component of pH gradient-stimulated sodium uptake. The inhibitory effect of cAMP-dependent protein kinase demonstrated an absolute requirement for ATP and was blocked by the specific protein inhibitor of this kinase. cAMP-dependent protein kinase also inhibited²²Na⁺ uptake in the absence of a pH gradient (pH_in 6.0. pH_out 6.0) and the inhibitory effect was blocked by the specific inhibitor of the kinase. Solubilized membrane proteins exhibited little endogenous protein kinase or protein phosphatase activity.These studies indicate that Na⁺–H⁺ exchange activity of proteoliposomes reconstituted with proteins from renal brush border membranes is inhibited by phosphorylation of selected proteins by cAMP-dependent protein kinase. These findings also indicate that the regulatory components of the Na⁺–H⁺ exchanger remain active during the process of solubilization and reconstitution of renal apical membrane proteins.     

Disruption of energy transduction in sarcoplasmic reticulum by trypsin cleavage of (Ca2++Mg2+)-ATPase

Summary Proteolytic digestion of sarcoplasmic reticulum vesicles with trypsin has been used as a structural modification with which to examine the interaction between the ATP hydrolysis site and calcium transport sites of the (Ca²⁺+Mg²⁺)-ATPase. The kinetics of trypsin fragmentation were examined and the time course of fragment production compared with ATP hydrolytic and calcium uptake activities of the digested vesicles. The initial cleavage (TD 1) of the native ATPase to A and B peptides has no effect on the functional integrity of the enzyme, hydrolytic and transport activities remaining at the levels of the undigested control. Concomitant with the second tryptic cleavage (TD 2) of the A peptide to A₁ and A₂ fragments, calcium transport is inhibited. Kinetic analysis demonstrates that the rate constant for inhibition of calcium uptake is correlated with the rate constant of a fragment disappearance. Both Ca²⁺-dependent and total ATPase activities are unaffected by this second cleavage. Passive loading of vesicles with calcium and subsequent efflux measurements show that transport inhibition is not due to increased permeability of the membrane to calcium even at substantial extents of digestion. Steady-state levels of acidstable phosphoenzyme are unaffected by either TD 1 or TD 2, indicating that uncoupling of the hydrolytic and transport functions does not increase the turnover rate of the enzyme and that TD 2 does not change the essential characteristics of the ATP hydrolysis site. Sarcoplasmic reticulum (SR) vesicles were examined for the presence of tightly bound nucleotides and are shown to contain 2.8–3.0 nmol ATP and 2.6–2.7 nmol ADP per mg SR protein. The ADP content of SR remains essentially unchanged with TD 1 cleavage of the ATPase enzyme to A and B peptides, but declines upon TD 2 in parallel with the digestion of the A fragment and the loss of calcium uptake activity of the vesicles. The ATP content is essentially constant throughout the course of trypsin digestion. The results are discussed in terms of current models of the SR calcium pump and the molecular mechanism of energy transduction.     

Cationic lipids involved in gene transfer mobilize intracellular calcium

Cationic lipids are efficient tools to introduce nucleic acids and proteins into cells. Elucidation of the mechanism and cellular pathways associated with such transport has been relatively tedious, even though significant progress has been made in the characterization of the intracellular trafficking of lipid/DNA complexes. Surprisingly little is known about the effects of these delivery vectors on cell functioning. In this report, we show that both cationic lipids and cationic lipid/DNA complexes mobilize the intracellular calcium. Removal of extracellular calcium did not significantly abolish this effect and preincubating cells with thapsigargin led to a decrease in [Ca²⁺]_i, indicating that calcium was released mainly from internal calcium stores sensitive to thapsigargin. Pretreatment of the cells with the phospholipase C inhibitor U73122, blocked the [Ca²⁺]_i rise, suggesting an inositol dependent mechanism.     

Molecularly Distinct Routes of Mitochondrial Ca2+ Uptake Are Activated Depending on the Activity of the Sarco/Endoplasmic Reticulum Ca2+ ATPase (SERCA)

The transfer of Ca²⁺ across the inner mitochondrial membrane is an important physiological process linked to the regulation of metabolism, signal transduction, and cell death. While the definite molecular composition of mitochondrial Ca²⁺ uptake sites remains unknown, several proteins of the inner mitochondrial membrane, that are likely to accomplish mitochondrial Ca²⁺ fluxes, have been described: the novel uncoupling proteins 2 and 3, the leucine zipper-EF-hand containing transmembrane protein 1 and the mitochondrial calcium uniporter. It is unclear whether these proteins contribute to one unique mitochondrial Ca²⁺ uptake pathway or establish distinct routes for mitochondrial Ca²⁺ sequestration. In this study, we show that a modulation of Ca²⁺ release from the endoplasmic reticulum by inhibition of the sarco/endoplasmatic reticulum ATPase modifies cytosolic Ca²⁺ signals and consequently switches mitochondrial Ca²⁺ uptake from an uncoupling protein 3- and mitochondrial calcium uniporter-dependent, but leucine zipper-EF-hand containing transmembrane protein 1-independent to a leucine zipper-EF-hand containing transmembrane protein 1- and mitochondrial calcium uniporter-mediated, but uncoupling protein 3-independent pathway. Thus, the activity of sarco/endoplasmatic reticulum ATPase is significant for the mode of mitochondrial Ca²⁺ sequestration and determines which mitochondrial proteins might actually accomplish the transfer of Ca²⁺ across the inner mitochondrial membrane. Moreover, our findings herein support the existence of distinct mitochondrial Ca²⁺ uptake routes that might be essential to ensure an efficient ion transfer into mitochondria despite heterogeneous cytosolic Ca²⁺ rises.     

Model of active transport of ions in cardiac cell

A model of the active transport of ions in a cardiac muscle cell, which takes into account the active transport of Na⁺, K⁺, Ca²⁺, Mg²⁺, HCO₃⁻ and Cl⁻ ions, has been constructed. The model allows independent calculations of the resting potential at the biomembrane and concentrations of basic ions (sodium, potassium, chlorine, magnesium and calcium) in a cell. For the analysis of transport processes in cardiac cell hierarchical algorithm “one ion-one transport system” was offered. The dependence of the resting potential on concentrations of the ions outside a cell has been established. It was shown, that ions of calcium and magnesium, despite their rather small concentration, play an essential role in maintenance of resting potential in cardiac cell. The calculated internal concentrations of ions are in good agreement with the corresponding experimental values.