Is the mitochondrial ATP synthesis solely H-driven, K,H symport-driven or K/H antiport-driven?

Some recent literature experimental data indicate that the mitochondrial ATP synthesis might be not solely H-driven, but K,H symport-driven membrane potential-dependent, considered as a further development of Mitchell's chemiosmotic theory, in contrast to the anti-Mitchell's hypothesis of K/H antiport-driven mechanism. In this short communication, the attention was pointed to a possible influence of the ionic strength of the used KCl incubation medium, versus of only K ions, and of the Mg-induced mitochondrial aggregation in the sucrose medium on the reported rates of the mitochondrial respiration and ATP synthesis. These observations were based on the own author's experimental works published earlier.

VDAC as a voltage-dependent mitochondrial gatekeeper under physiological conditions.

Mitochondria, composed of two membranes, play a key role in energy production in eukaryotic cells. The main function of the inner membrane is oxidative phosphorylation, while the mitochondrial outer membrane (MOM) seems to control the energy flux and exchange of various charged metabolites between mitochondria and the cytosol. Metabolites cross MOM via the various isoforms of voltage-dependent anion channel (VDAC).

Apparent "mild depolarization of the inner mitochondrial membrane" as a result of a possible generation of the outer membrane potential.

Recently reported kinase-linked mild depolarization of mitochondria, which prevents the generation of the reactive oxygen species (ROS) and disappears in various organs of the old mice, has been assumed to represent a crucial component of the mitochondrial anti-aging program. To measure mitochondrial inner membrane potential (IMP), the authors used fluorescent probe safranin O. It is widely accepted that the accumulation of such cationic probes in the mitochondrial matrix depends exclusively on IMP, thus completely ignoring the possibility of the outer membrane potential (OMP) generation.

Cytolytic activity of peptides derived from the Cry11Bb insecticidal toxin of B. thuringiensis subsp. medellin.

A few Bacillus thuringiensis Cry proteins, known as parasporins, have demonstrated cell proliferation inhibition of human cancer cells in vitro after protease activation. In this work, eight peptides derived from the Cry11Bb protoxin produced by B. thuringiensis subsp.

Electrical control of the cell energy metabolism at the level of mitochondrial outer membrane.

Energy, generated by the mitochondrial oxidative phosphorylation system, is transferred to the cytosol across the mitochondrial outer membrane (MOM), through the voltage-dependent anion channels (VDACs). The role of the VDAC's voltage-gating process to control the transfer of ATP, creatine phosphate and other negatively charged metabolites across MOM might be crucial for the cell energy metabolism regulation. However, it depends on the probability of the outer membrane potential (OMP) generation by a currently undefined mechanism that has usually been considered doubtful, based on the assumption that VDACs always stay in the electrically open state.

VDAC electronics: 5. Mechanism and computational model of hexokinase-dependent generation of the outer membrane potential in brain mitochondria.

Glycolysis plays a key role in brain energy metabolism. The initial and rate-limiting step of brain glycolysis is catalyzed mainly by hexokinase I (HKI), the majority of which is bound to the mitochondrial outer membrane (MOM), mostly through the mitochondrial inter-membrane contact sites formed by the voltage-dependent anion channel (VDAC, outer membrane) and the adenine nucleotide translocator (ANT, inner membrane). Earlier, we proposed a mechanism for the generation of the mitochondrial outer membrane potential (OMP) as a result of partial application of the inner membrane potential (IMP) to MOM through the electrogenic ANT-VDAC-HK inter-membrane contact sites.

VDAC electronics: 4. Novel electrical mechanism and thermodynamic estimations of glucose repression of yeast respiration.

Inhibition of cell respiration by high concentrations of glucose (glucose repression), known as "Crabtree effect", has been demonstrated for various cancerous strains, highly proliferating cells and yeast lines. Although significant progress in understanding metabolic events associated with the glucose repression of cell respiration has been achieved, it is not yet clear whether the Crabtree effect is the result of a limited activity of the respiratory chain, or of some glucose-mediated regulation of mitochondrial metabolic state. In this work we propose an electrical mechanism of glucose repression of the yeast S.

VDAC electronics: 3. VDAC-Creatine kinase-dependent generation of the outer membrane potential in respiring mitochondria.

Mitochondrial energy in cardiac cells has been reported to be channeled into the cytosol through the intermembrane contact sites formed by the adenine nucleotide translocator, creatine kinase and VDAC. Computational analysis performed in this study showed a high probability of the outer membrane potential (OMP) generation coupled to such a mechanism of energy channeling in respiring mitochondria. OMPs, positive inside, calculated at elevated concentrations of creatine are high enough to restrict ATP release from mitochondria, to significantly decrease the apparent K(m,ADP) for state 3 respiration and to maintain low concentrations of Ca(2+) in the mitochondrial intermembrane space.

Computational models for monitoring the trans-membrane potential with fluorescent probes: the DiSC(5) case.

Fluorescent permeant charged probes are commonly used for monitoring the trans-membrane potential in lipid vesicles and biological membranes, which has been earlier described by various mathematical models. In the present study, we developed a more complex model based on the computational step-by-step analysis of the influence of various factors, such as the membrane surface potential, ionic strength, and the aggregation properties of cationic cyanine probe DiSC(5) in the membrane and aqueous phases, in addition to the Nernstian distribution of the probe across the membrane and the hydrophobic interaction with the lipid bilayer. The final full model allows prediction of the optimal experimental conditions for monitoring the trans-membrane potential, such as the probe/lipid ratio and the concentration of liposomes, with a given percentage of negatively charged phospholipids in the membrane, the ionic strength of the aqueous media, the "membrane-water" partition coefficient and the aggregation properties of the probe, as well as the most adequate mode of fluorescence measurement.

VDAC electronics: 2. A new, anaerobic mechanism of generation of the membrane potentials in mitochondria.

Mitochondrial hexokinase (HK) and creatine kinase (CK) known to form complexes with a voltage dependent anion channel (VDAC) have been reported to increase cell death resistance under hypoxia/anoxia. In this work we propose a new, non-Mitchell mechanism of generation of the inner and outer membrane potentials at anaerobic conditions. The driving force is provided by the Gibbs free energy of the HK and CK reactions associated with the VDAC-HK and the ANT (adenine nucleotide translocator)-CK-VDAC complexes, respectively, both functioning as voltage generators.

Competitive interactions of amphipathic polycationic peptides and cationic fluorescent probes with lipid membrane: experimental approaches and computational model.

The electrostatic interaction of polycationic peptides with negatively charged biomembranes has been recognized as the first and very important step of their selective binding to many bacteria and transformed cells. In this work we demonstrated the phenomenon of competition of some earlier designed polycationic peptides and fluorescent probes for their binding to the negatively charged inner membrane of mitochondria and to the PC/PG (9:1) liposomes. Rat liver mitochondria swelling induced by the antimicrobial polycationic peptide BTM-P1 (VAPIAKYLATALAKWALKQGFAKLKS) and by the retro-BTM-P1 was significantly diminished in the presence of 10μM fluorescent probe safranin O.

VDAC electronics: 1. VDAC-hexo(gluco)kinase generator of the mitochondrial outer membrane potential.

The simplest mechanism of the generation of the mitochondrial outer membrane potential (OMP) by the VDAC (voltage-dependent anion channel)-hexokinase complex (VHC), suggested earlier, and by the VDAC-glucokinase complex (VGC), was computationally analyzed. Even at less than 4% of VDACs bound to hexokinase, the calculated OMP is high enough to trigger the electrical closure of VDACs beyond the complexes at threshold concentrations of glucose. These results confirmed our previous hypothesis that the Warburg effect is caused by the electrical closure of VDACs, leading to global restriction of the outer membrane permeability coupled to aerobic glycolysis.

Electrical potentiation of the membrane permeabilization by new peptides with anticancer properties.

New polycationic peptides were designed on the basis of 16-mer and 14-mer fragments of the peptide BTM-P1, derived from the Cry11Bb protoxin. The peptides caused mitochondrial, but not red blood cell membrane permeabilization. Conjugation of the cell penetrating hepta-arginine vector to their N- or C-termini through two glycine residues resulted in more active peptides, which also permeabilized the red blood cells with a relatively high plasma membrane potential generated in the presence of valinomycin.

Electrical hypothesis of toxicity of the Cry toxins for mosquito larvae.

Many electrical properties of insect larval guts have been studied, but their importance for toxicity of the Cry-type toxins has never been reported in the literature. In the present work, we observed potential-dependent permeabilization of plasma membrane by several polycationic peptides derived from the Cry11Bb protoxin. The peptide BTM-P1d, all D-type amino acid analogue of the earlier reported peptide BTM-P1, demonstrated high membrane-permeabilizing activity in experiments with isolated rat liver mitochondria, RBC (red blood cells) and mitochondria in homogenates of Aedes aegypti larval guts.

Permeabilization of mitochondria and red blood cells by polycationic peptides BTM-P1 and retro-BTM-P1.

Mitochondrial and plasma membrane permeabilization by polycationic peptides BTM-P1 and retro-BTM-P1 were studied. BTM-P1 was more active than its retro-analog. In the sucrose medium, the capacity of BTM-P1 to permeabilize mitochondria was lower than in salt media.

Redox state-dependent aggregation of mitochondria induced by cytochrome c.

Cytochrome c is known to play central role in apoptosis. Here, it is shown that ferricytochrome c, but not ferrocytochrome c is able to directly induce the aggregation of rat liver mitochondria, similar to the effect caused by magnesium ions at high concentrations. The aggregation was revealed by a decrease in light dispersion of mitochondrial suspension and it was confirmed by the optical microscopy.

Red blood cell permeabilization by hypotonic treatments, saponin, and anticancer avicins.

Plasma membrane permeabilization by saponin and anticancer avicins was studied using light dispersion measurements, since high correlation between light dispersion changes and hemolysis has been demonstrated. Nevertheless, we observed that rat red blood cell swelling in moderately hypotonic media was accompanied by up to 20% decrease of light dispersion, when hemolysis was not yet detectable. Avicin G and avicin D were significantly more efficient than saponin in inducing cytotoxicity in PC3 human prostate cancer cells.

Potential-dependent membrane permeabilization and mitochondrial aggregation caused by anticancer polyarginine-KLA peptides.

The anticancer activity of the polycationic peptide (KLAKLAK)(2), as a possible mitochondria-damaging agent, named KLA (l-form) or kla (d-form), has been increased by the fusion with hepta-arginine cell delivery vectors r7 and R7 (peptides r7-kla and R7-KLA, respectively), as shown in the literature. We demonstrated that 3.6muM r7-kla or R7-KLA, but not kla, caused significant permeabilization of the inner and the outer membranes of energized rat liver mitochondria.

Potential-dependent permeabilization of plasma membrane by the peptide BTM-P1 derived from the Cry11Bb1 protoxin.

The peptide BTM-P1, which is derived from the amino acid sequence of the Cry11Bb1 protoxin, is able to permeabilize mitochondrial membranes and reveals antimicrobial activity. In this work we demonstrated that the permeabilizing activity of BTM-P1 for the plasma membrane of rat red blood cells increased in a dose-dependent manner for the concentration range of 1-4 microg/ml. Using osmotic protectants, the radius of pores formed at 4 microg/ml BTM-P1 was determined as 0.

Hemoglobin precipitation by polyethylene glycols leads to underestimation of membrane pore sizes.

The size of pores formed in the plasma membrane by various substances is frequently determined using polyethylene glycols as osmotic protectants. In this work, we have found that the size of pores formed by saponin in the red blood cell membrane determined by hemolysis versus molecular weight of polyethylene glycol was different to that estimated by light dispersion of cell suspensions. After complete swelling of cells induced by saponin in semiisotonic salt media containing 150 mOsm PEG-4000 or PEG-3000, a significant increase in the light absorbance at 640 nm was developed resulting from the formation of hemoglobin precipitates.

Synergistic inhibition of mitochondrial respiration by anticancer agent erucylphosphohomocholine and cyclosporin A.

Alkylphosphocholines are a new class of anticancer agents. The mechanisms by which these drugs display their antitumor activities are not known. In this work, we show that erucylphosphohomocholine, a new antineoplastic compound, significantly decreased ATP synthesis in isolated rat liver mitochondria at a concentration of 50 microm or higher via permeabilization of the inner membrane.

Theoretical evaluation of a possible nature of the outer membrane potential of mitochondria.

A possibility of generation of the outer membrane potential in mitochondria has been suggested earlier in the literature, but the potential has not been directly measured yet. Even its nature, metabolic impact and a possible range of magnitudes are not clear, and require further theoretical and experimental analysis. Here, using simple mathematical model, we evaluated a possible contribution of the Donnan and metabolically derived potentials to the outer membrane potential, concluding that the superposition of both is most probable; exclusively Donnan origin of the potential is doubtful because unrealistically high concentrations of charged macromolecules are needed for maintaining its relatively high levels.

Avicins, natural anticancer saponins, permeabilize mitochondrial membranes.

Avicins are a class of natural saponins with selective pro-apoptotic activity in cancer cells. In this work, we studied the influence of avicins on metabolic state of rat liver mitochondria. Avicin-treated mitochondria underwent a significant decrease in oxygen consumption rate that was completely restored by addition of exogenous cytochrome c.

A possible restriction of ferro- and ferricyanide oxidoreductase activities of rat liver mitochondria by the outer membrane.

In this work, various ferro-ferricyanide oxidoreductase activities of rat liver mitochondria were studied to find conditions under which the outer membrane might restrict the flux of these highly charged non-biological anions. When the isotonic low ionic strength medium was supplemented with 25mM KCl, a several-fold increase in the succinate-ferricyanide reductase activity of mitochondria and in the rate of external NADH oxidation in the presence of ferrocyanide was observed. Mitochondrial respiration with 5mM ferrocyanide was almost completely inhibited after consumption of 3.

Mitochondria permeabilization by a novel polycation peptide BTM-P1.

Bacillus thuringiensis subsp. medellin is known to produce the Cry11Bb protein of 94 kDa, which is toxic for mosquito larvae due to permeabilization of the plasma membrane of midgut epithelial cells. Earlier we found that a 2.