Membrane Potential Generation Research Articles

Toxicity and Biotransformation of Carbon-Based Nanomaterials in Marine Microalgae Heterosigma akashiwo.

This work is related to the environmental toxicology risk assessment and evaluation of the possible transformation of carbon-based nanomaterials (CNMs) after contact with marine microalgae. The materials used in the study represent common and widely applied multi-walled carbon nanotubes (CNTs), fullerene (C60), graphene (Gr), and graphene oxide (GrO). The toxicity was evaluated as growth rate inhibition, esterase activity, membrane potential, and reactive oxygen species generation changes. The measurement was performed with flow cytometry after 3, 24, 96 h, and 7 days. The biotransformation of nanomaterials was evaluated after 7 days of microalgae cultivation with CNMs by FTIR and Raman spectroscopy. The calculated toxic level (EC50 in mg/L, 96 h) of used CNMs reduced in the following order: CNTs (18.98) > GrO (76.77) > Gr (159.40) > C60 (414.0). Oxidative stress and membrane depolarization were the main toxic action of CNTs and GrO. At the same time, Gr and C60 decreased the toxic action with time and had no negative impact on microalgae after 7 days of exposure even at the concentration of 125 mg/L. Moreover, C60 and Gr after 7 days of contact with microalgae cells obtained structural deformations.

The Comparative Toxic Impact Assessment of Carbon Nanotubes, Fullerene, Graphene, and Graphene Oxide on Marine Microalgae Porphyridium purpureum.

The growing production and application of carbon-based nanomaterials (CNMs) represent possible risks for aquatic systems. However, the variety of CNMs with different physical and chemical properties and different morphology complicate the understanding of their potential toxicity. This paper aims to evaluate and compare the toxic impact of the four most common CNMs, namely multiwalled carbon nanotubes (CNTs), fullerene (C60), graphene (Gr), and graphene oxide (GrO) on the marine microalgae Porphyridium purpureum. The microalgae cells were exposed to the CNMs for 96 h and measured by flow cytometry. Based on the obtained results, we determined no observed effect level (NOEL), and calculated EC10 and EC50 concentrations for growth rate inhibition, esterase activity, membrane potential, and reactive oxygen species (ROS) generation changes for each tested CNM. According to the sensitivity (growth rate inhibition) of P. purpureum, the used CNMs can be listed in the following order (EC50 in mg/L, 96 h): CNTs (2.08) > GrO (23.37) > Gr (94.88) > C60 (>131.0). The toxicity of CNTs was significantly higher than the toxic effect of the other used CNMs, and only this sample caused an increase in ROS generation in microalgae cells. This effect was apparently caused by the high affinity between particles and microalgae associated with the presence of exopolysaccharide coverage on P. purpureum cells.

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). In turn, VDACs interact with some enzymes, other proteins and molecules, including drugs. This work aimed to analyze various literature experimental data related to targeting mitochondrial VDACs and VDAC-kinase complexes on the basis of the hypothesis of generation of the outer membrane potential (OMP) and OMP-dependent reprogramming of cell energy metabolism. Our previous model of the VDAC-hexokinase-linked generation of OMP was further complemented in this study with an additional regulation of the MOM permeability by the OMP-dependent docking of cytosolic proteins like tubulin to VDACs. Computational analysis of the model suggests that OMP changes might be involved in the mechanisms of apoptosis promotion through the so-called transient hyperpolarization of mitochondria. The high concordance of the performed computational estimations with many published experimental data allows concluding that OMP generation under physiological conditions is highly probable and VDAC might function as an OMP-dependent gatekeeper of mitochondria, controlling cell life and death. The proposed model of OMP generation allows understanding in more detail the mechanisms of cancer death resistance and anticancer action of various drugs and treatments influencing VDAC voltage-gating properties, VDAC content, mitochondrial hexokinase activity and VDAC-kinase interactions in MOM.

α-Mangostin Exhibits a Therapeutic Effect on Spinal Cystic Echinococcosis by Affecting Glutamine Metabolism.

Spinal cystic echinococcosis, a severely neglected, rare disease, is characterized by high morbidity, disability, and mortality in prevalent regions. Due to the high-risk nature of surgical treatment and the ineffectiveness of conventional drugs, there is an unmet need for novel safe and effective drugs for the treatment of this disease. In this study, we examined the therapeutic effects of α-mangostin for spinal cystic echinococcosis, and explored its potential pharmacological mechanism. The repurposed drug exhibited a potent in vitro protoscolicidal effect and significantly inhibited the evolution of larval encystation. Moreover, it demonstrated a remarkable anti-spinal cystic echinococcosis effect in gerbil models. Mechanistically, we found that α-mangostin intervention led to intracellular depolarization of mitochondrial membrane potential and reactive oxygen species generation. In addition, we observed elevated expression of autophagic proteins, aggregation of autophagic lysosomes, activated autophagic flux, and disrupted larval microstructure in protoscoleces. Further metabolite profiling showed that glutamine was imperative for autophagic activation and anti-echinococcal effects mediated by α-mangostin. These results suggest that α-mangostin is a potentially valuable therapeutic option against spinal cystic echinococcosis through its effect on glutamine metabolism.

Small molecule II-6s synergises with fluconazole against Candida albicans

BackgroundCandida albicans (C. albicans) is the most common opportunistic fungal species in the oral cavity. The emergence of drug resistance of C. albicans has necessitated the development of novel antifungal agents. ObjectivesThis study evaluated the antifungal activity of a previously developed antimicrobial small molecule, namely II-6s, and explored its potential synergism with fluconazole against C. albicans and the underlying mechanisms. MethodsThe antifungal effects of II-6s against C. albicans were evaluated with microdilution and time-killing assays. Synergism of II-6s with fluconazole was determined by calculating the fractional inhibitory concentration index (FICI). The effects of II-6s on efflux pump, mitochondrial function and energy metabolism were examined to investigate the underlying mechanism of synergism. The antifungal mechanism of II-6s against C. albicans was further explored with RNA-seq and validated with specific mutant strains. ResultsII-6s exhibited a fungicidal effect against C. albicans with a minimum fungicidal concentration of 31.25 µg/mL. II-6s also inhibited C. albicans biofilm with a sessile minimum inhibitory concentration at 500 µg/mL. More importantly, II-6s showed a synergistic effect with fluconazole against a fluconazole-resistant strain of C. albicans, which expressed elevated levels of CDR1 (FICI < 0.5). II-6s inhibited the efflux pump activity of C. albicans. Consistently, II-6s inhibited energy metabolism of C. albicans by reducing the mitochondrial membrane potential and ATP generation, and inhibited utilisation of the non-fermentable carbon source. II-6s also inhibited the mitogen-activated protein kinase signal pathway, particularly HOG1, which may explain its antifungal activity against C. albicans. ConclusionThe small molecule II-6s inhibits the growth of C. albicans by targeting HOG1. II-6s also synergises with fluconazole by inhibiting the drug efflux pump, representing a promising antifungal agent for the control of candidiasis.

Oriented Insertion of ESR-Containing Hybrid Proteins in Proteoliposomes.

Microbial rhodopsins comprise a diverse family of retinal-containing membrane proteins that convert absorbed light energy to transmembrane ion transport or sensory signals. Incorporation of these proteins in proteoliposomes allows their properties to be studied in a native-like environment; however, unidirectional protein orientation in the artificial membranes is rarely observed. We aimed to obtain proteoliposomes with unidirectional orientation using a proton-pumping retinal protein from Exiguobacterium sibiricum, ESR, as a model. Three ESR hybrids with soluble protein domains (mCherry or thioredoxin at the C-terminus and Caf1M chaperone at the N-terminus) were obtained and characterized. The photocycle of the hybrid proteins incorporated in proteoliposomes demonstrated a higher pKa of the M state accumulation compared to that of the wild-type ESR. Large negative electrogenic phases and an increase in the relative amplitude of kinetic components in the microsecond time range in the kinetics of membrane potential generation of ESR-Cherry and ESR-Trx indicate a decrease in the efficiency of transmembrane proton transport. On the contrary, Caf-ESR demonstrates a native-like kinetics of membrane potential generation and the corresponding electrogenic stages. Our experiments show that the hybrid with Caf1M promotes the unidirectional orientation of ESR in proteoliposomes.

Synthetic transmembrane channel molecules formed by acyclic cucurbiturils and pillararene: Tuning cation selectivity and generating membrane potential

Biomimetic transmembrane channel molecules developed through chemical synthesis strategies were used for treat genetic diseases which due to the alterations in the channel structure of native membrane proteins and ion transport is deregulated. In this paper, we designed and constructed a novel synthetic transmembrane channel molecules (STCMs), which based on acyclic cucurbiturils (ACBs) and pillar[5]arene (PA[5]). STCMs could imitate the function of natural channel protein molecules and reveal the possible existence structure, there is a hope for new treatment options for genetic diseases. The structure of STCMs have been identified by NMR and FT-IR, the results of fluorescence experiments showed that it could effectively insert into the phospholipid bilayer, specifically recognize and transmit K+. Whole-cell patch-clamp experiments showed that STCMs could insert into the cell membrane of HEK293T cells, and could form channels on the cell membrane to generate current. These works will help to understand the relationship between the structures and properties of STCMs and the selectivity and efficiency of ions or polar molecules. This study presents the molecular structure and function of STCMs which may form a basis for explore the scientific laws of transmembrane transporter.

Isofraxidin enhances hyperthermia‑induced apoptosis via redox modification in acute monocytic leukemia U937 cells.

The cell‑killing potential of most chemotherapeutic agents is enhanced by a temperature elevation. Isofraxidin (IF) is a coumarin compound widely found in plants, such as the Umbelliferae or Chloranthaceae families. IF induces anticancer effects in lung and colorectal cancer. To the best of our knowledge, the combined effects of hyperthermia (HT) and IF on heat‑induced apoptosis have not been reported. Acute monocytic leukemia U937 cells were exposed to HT with or without IF pre‑treatment. Apoptosis was measured by Annexin V‑FITC/PI double staining assay using flow cytometry and cell viability was observed by cell counting kit assay, DNA fragmentation. The mechanism involved in the combination was explored by measuring changes in the mitochondrial membrane potential, (MMP), intracellular ROS generation, expression of apoptosis related protein, and intracellular calcium ion level. It was demonstrated that IF enhanced HT‑induced apoptosis in U937 cells. The results demonstrated that combined treatment enhanced mitochondrial membrane potential loss and transient superoxide generation increased protein expression levels of caspase‑3, caspase‑8 and phosphorylated‑JNK and intracellular calcium levels. Moreover, the role of caspases and JNK was confirmed using a pan caspase inhibitor (zVAD‑FMK) and JNK inhibitor (SP600125) in U937 cells. Collectively, the data demonstrated that IF enhanced HT‑induced apoptosis via a reactive oxygen species mediated mitochondria/caspase‑dependent pathway in U937 cells.

Lactate protects against oxidative stress-induced retinal degeneration by activating autophagy.

Age-related macular degeneration is a common cause of blindless among the aged, which can mainly be attributed to oxidative stress and dysregulated autophagy in retinal pigment epithelium cells. Lactate was reported to act as a signaling molecule and exerted beneficial effect against oxidative stress. This study aims to investigate the protective effect of lactate against oxidative stress-induced retinal degeneration. Here, H2O2-induced oxidative stress cell model and sodium iodate-induced mice retinal degeneration model were established. It was found that H2O2 inhibited cell viability in ARPE-19cells and sodium iodate induced deterioration of retinal pigment epithelium as well as apoptosis in retina. Pretreatment with lactate alleviated oxidative stress-induced cell death and retinal degeneration. Molecularly, lactate activated autophagy by up-regulating the ratio of LC3II/I, increased formation of LC3 puncta and autophagic vacuole. Further, lactate prevented H2O2-induced mitochondrial fission and maintained mitochondrial function by alleviating H2O2-induced mitochondrial membrane potential disruption and intracellular ROS generation. In contrast, application of 3-methyladenine, an inhibitor of autophagy, effectively weakened the protective effect of lactate against oxidative stress in vivo and in vitro. Taken together, all data in this study indicate that lactate protects against oxidative stress-induced retinal degeneration and preserves mitochondrial function by activating autophagy.

Влияние оксопроизводных азотсодержащих гетероциклов CBR-384 и CBR-386 на дыхательную активность и уровень внеклеточных серосодержащих соединений у бактерий Escherichia coli

The biological activity of two representatives of oxo-derivative nitrogen-containing heterocycles CBR-384 and CBR-386, planned for use as drugs, on growing aerobically gram-negative bacteria Escherichia coli was studied. Compound CBR-384 completely inhibited growth rate and biomass accumulation as measured by optical density (OD600). Continuous recording of dissolved oxygen (dO2) with a Clark electrode directly in the growing culture showed that CBR-384 caused a rapid and irreversible increase in oxygen levels in the medium, which indicated a decrease in the respiratory activity of cells. In time, the phase of rapid decline in respiration coincided with the phase of decreased growth rate. In aerobic cultures of E. coli, respiratory activity is closely related to the generation of membrane potential. However, only a small, but statistically significant, decrease in membrane potential, measured using the fluorescent dye DiBAC4(3), was found with CBR-384. It is known that in aerobic E. coli cultures growing on sulfate as a sulfur source, stress-induced growth inhibition is accompanied by an increase in extracellular glutathione (GSH) and H2S export. The use of a sulfide-specific electrode re-vealed that when E. coli growth is stopped by CBR-384, sulfide is also exported into the medium. Under these conditions, an increase in extracellular GSH was also noted. The effect of CBR-386 on E. coli, assessed by these four parameters, was less pronounced. The differences in the biological activities of CBR-384 and CBR-386 may be due to differences in their structures.

Unraveling the physicochemical and toxicological properties of food product isolated E341/E171

AbstractNanostructured materials have been extensively exploited in the food sector for nutrient delivery, sensing, packaging, and food additives. It has exhibited size, shape, chemical nature, and surface area‐dependent physicochemical and biological properties. Currently, nanoscale ingredients are identified in approved food additives. Still, food‐grade nanostructured material's impact on biological systems is unexplored well. Thus, in this study, we have identified and analyzed the physicochemical properties of nanoscale ingredients in commercial food product isolate E341 and E171 combinations. Also, we have evaluated the E341/E171 combination cytotoxic potential on human mesenchymal stem cells. The food isolated E341/E171 exhibits spherical nature with a 60–200 nm diameter. The E341 and E171 combination exposure increased the cell viability loss to ~36% at a high dose in human mesenchymal stem cells (hMSCs). The E341/E171 treatment‐induced cellular and nuclear damage at a moderate dose. The mitochondrial membrane potential loss and ROS generation were observed in E341/E171 treated cells. The E341/E171 alters the gene expression pattern in hMSCs dose‐dependently. The GSR and POR gene expression is upregulated significantly in E341/E171 treatment. Our study results revealed that E341/E171 induced toxicity in hMSCs at high concentrations. Thus, E341/E171 potential impact on the biological system should be reexamined for food industrial implications.

Lupane Triterpene Derivatives Improve Antiproliferative Effect on Leukemia Cells through Apoptosis Induction

Leukemia is one of the most frequent types of cancer. No effective treatment currently exists, driving a search for new compounds. Simple structural modifications were made to novel triterpenes isolated from Phoradendron wattii. Of the three resulting derivatives, 3α-methoxy-24-hydroxylup-20(29)-en-28-oic acid (T1m) caused a decrease in the median inhibitory concentration (IC50) on the K562 cell line. Its mode of action was apparently apoptosis, ROS generation, and loss of mitochondrial membrane potential (MMP). Molecular docking analysis showed T1m to produce lower binding energies than its precursor for the Bcl-2 and EGFR proteins. Small, simple, and viable modifications to triterpenes can improve their activity against leukemia cell lines. T1m is a potentially promising element for future research. Clarifying the targets in its mode of action will improve its applicability.

Mitoregulin controls mitochondrial function and stress-adaptation response during early phase of endoplasmic reticulum stress in breast cancer cells

The proper regulation of mitochondrial function is important for cellular homeostasis. Especially, in cancer cells, dysregulation of mitochondria is associated with diverse cellular events such as metabolism, redox status, and stress responses. Mitoregulin (MTLN), a micro protein encoded by LINC00116, recently has been reported to control mitochondrial functions in skeletal muscle cells and adipocytes. However, the role of MTLN in cancer cells remains unclear. In the present study, we found that MTLN regulates membrane potential and reactive oxygen species (ROS) generation of mitochondria in breast cancer cells. Moreover, MTLN deficiency resulted in abnormal mitochondria-associated ER membranes (MAMs) formation, which is crucial for stress adaptation. Indeed, the MTLN-deficient breast cancer cells failed to successfully resolve ER (endoplasmic reticulum) stress, and cell vulnerability to ER-stress inducers was significantly enhanced by the downregulation of MTLN. In conclusion, MTLN controls stress-adaptation responses in breast cancer cells as a key regulator of mitochondria-ER harmonization, and thereby its expression level may serve as an indicator of the responsiveness of cancer cells to proteasome inhibitors.

Combination treatment with cyclosporin A and arsenic trioxide induce synergistic cell death via non-apoptotic pathway in uterine cervical cancer cells

Cyclosporin A is an immunosuppressive drug with anti-cancer effect. Arsenic trioxide (As2O3), a well-known cancer-inhibiting drug, induced cytotoxicity via apoptosis and autophagy. The aim of this study is to evaluate the effect of combinational treatment with cyclosporin A and arsenic trioxide on cell viability inhibition in cervical cancer cells. Using MTT assay and combination index, combinational treatment with cyclosporin A and arsenic trioxide induced a synergistic cytotoxic effect in Caski and SiHa cells. Cyclosporin A and arsenic trioxide triggered cell death via non-apoptotic pathway by using annexin V/propidium iodide (PI) assay. Cyclosporin A and arsenic trioxide combined treatment decreased mitochondrial membrane potential and increase reactive oxygen species (ROS) generation. This co-treatment increased LC3B-II expression and autophagosome formation in cervical cancer cells. This study first demonstrated that combinational treatment with cyclosporin A and As2O3 trigger synergistic cytotoxic effect via autophagy in cervical cancer cells.

MFN2 mediates ER-mitochondrial coupling during ER stress through specialized stable contact sites.

Endoplasmic reticulum (ER) functions critically depend on a suitable ATP supply to fuel ER chaperons and protein trafficking. A disruption of the ability of the ER to traffic and fold proteins leads to ER stress and the unfolded protein response (UPR). Using structured illumination super-resolution microscopy, we revealed increased stability and lifetime of mitochondrial associated ER membranes (MAM) during ER stress. The consequent increase of basal mitochondrial Ca2+ leads to increased TCA cycle activity and enhanced mitochondrial membrane potential, OXPHOS, and ATP generation during ER stress. Subsequently, OXPHOS derived ATP trafficking towards the ER was increased. We found that the increased lifetime and stability of MAMs during ER stress depended on the mitochondrial fusion protein Mitofusin2 (MFN2). Knockdown of MFN2 blunted mitochondrial Ca2+ effect during ER stress, switched mitochondrial F1FO-ATPase activity into reverse mode, and strongly reduced the ATP supply for the ER during ER stress. These findings suggest a critical role of MFN2-dependent MAM stability and lifetime during ER stress to compensate UPR by strengthening ER ATP supply by the mitochondria.

Catalpol induces apoptosis in breast cancer in vitro and in vivo: Involvement of mitochondria apoptosis pathway and post-translational modifications

Breast cancer is a fatal cancer with the highest mortality in female. New strategies for anti-breast cancer are still urgently needed. Catalpol, an iridoid glycoside extracted from the traditional Chinese medicinal plant Rehmannia glutinosa, has shown anticancer efficacy in various cancer cells. However, its effect on breast cancer remains unclear. In this study, we aim to investigate the anti-breast cancer activity of catalpol and elucidate its underlying mechanism. Cell counting kit-8 (CCK-8) and morphology change showed that catalpol could inhibit the proliferation and viability of MCF-7 cells. Catalpol administration reduced the tumor volume in xenograft model. Catalpol induced apoptosis in MCF-7 cells confirmed by Hoechst 33342 staining and Annexin V-FITC/PI double staining. In vivo, catalpol also induced apoptosis as seen from the increased level of terminal-deoxynucleoitidyl transferase mediated nick end labeling (TUNEL) in tumor. According to JC-1 and Dichlorodi-hydrofluorescein Diacetate (DCFH-DA) staining, loss of mitochondrial membrane potential (MMP) and reactive oxygen species (ROS) generation was found in MCF-7 cells treated with catalpol. Furthermore, catalpol also increased the level of cytoplasmic cytochrome c and activity of caspase-3 in MCF-7 cells. Likewise, histopathological and immunohistochemical (IHC) assay also found that catalpol enhanced the levels of cytochrome c and caspase-3 in breast cancer tissues. Ultimately, acetylation, 2-hydroxyisobutyrylation and lactylation were dramatically increased, whereas succinylation, malonylation and phosphorylation were markedly decreased in the breast cancer tumor treated with catalpol. Taken together, catalpol inhibited breast cancer in vitro and in vivo through induction of apoptosis via mitochondria apoptosis pathway and regulation of protein post-translational modifications (PTMs). Thus, it can be considered as an excellent candidate compound for treatment of breast cancer.

Enhancing autophagy and energy metabolism in the meniscus can delay the occurrence of PTOA in ACLT rat.

Osteoarthritis (OA) is a progressive degenerative joint disease characterized by the destruction of the articular cartilage, meniscus and the like. Autophagy and cellular energy metabolism are the mechanisms by which cells maintain homeostasis. However, little is known about the effects of autophagy and cellular energy metabolism on meniscus degeneration, and the pathogenesis of posttraumatic osteoarthritis (PTOA) after the meniscal injury is rarely reported. Therefore, this study aimed to investigate the relationship between changes in autophagy and cellular energy metabolism in the meniscus following anterior cruciate ligament transection (ACLT) and PTOA induced by subsequent articular cartilage injury. In this study, we use a combination of cell experiments in vitro and animal experiments in vivo. On the one hand, cell experiment results show that inhibiting the mTORC1 signaling pathway by inhibiting the phosphorylation of S6K and AKT proteins in meniscal cells will lead to the increase of Beclin1, LC-3B, ATG12, ULK1, P62, and activate autophagy-related signaling pathways, which in turn protects the extracellular matrix component COL1 of meniscal cells from degradation by catabolic factor MMP13. In addition, it increased the generation of mitochondrial membrane potential in meniscal cells, increased the expression of anti-apoptotic factor BCL-XL, decreased the expression of pro-apoptotic factors BAD and BAX, and reduced the apoptosis of meniscal cells. More importantly, under the stimulation of inflammatory factor IL-1β, the secretion of meniscus cells can reduce the elevated levels of MMP13 and Adamts5 caused by chondrocytes affected by IL-1β. On the other hand, the results of animal experiments in vivo further proved the validity of the results of the cell experiments, and also proved that the meniscus injury did prior to the articular cartilage degeneration after ACLT. In conclusion, this study suggests that the meniscus prior to articular cartilage damage during the development of PTOA after ACLT, and that promoting autophagy and energy metabolism of meniscal cells may be a potential therapeutic target for delaying PTOA.

Positively charged mineral surfaces promoted the accumulation of organic intermediates at the origin of metabolism.

Identifying plausible mechanisms for compartmentalization and accumulation of the organic intermediates of early metabolic cycles in primitive cells has been a major challenge in theories of life's origins. Here, we propose a mechanism, where positive membrane potentials elevate the concentration of the organic intermediates. Positive membrane potentials are generated by positively charged surfaces of protocell membranes due to accumulation of transition metals. We find that (i) positive membrane potentials comparable in magnitude to those of modern cells can increase the concentration of the organic intermediates by several orders of magnitude; (ii) generation of large membrane potentials destabilize ion distributions; (iii) violation of electroneutrality is necessary to induce nonzero membrane potentials; and (iv) violation of electroneutrality enhances osmotic pressure and diminishes reaction efficiency, resulting in an evolutionary driving force for the formation of lipid membranes, specialized ion channels, and active transport systems.

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. However, computational analysis performed in the presented work suggests that the kinase-linked generation of the positive OMP might take place under the described conditions, because the measured potential includes the algebraic sum of both IMP and OMP. Alternatively to the suggested mild depolarization of mitochondria, the reported experimental data might reflect mainly a change of the positive OMP generated by the VDAC-kinase complexes. We also demonstrate that the reported in the literature mitochondrial hyperpolarization induced by erastin (known to prevent VDAC-tubulin interactions) and the depolarization caused by the mitochondrial VDAC knockdowns in the cancer cells might actually represent a decrease or increase, respectively, of the magnitude of the kinase-linked positive OMP. This is consistent with our hypothesis that VDAC voltage gating by the kinase-linked metabolically-dependent OMP plays a very important physiological role in regulating the cell energy metabolism under normal and pathological conditions, in the maintenance of the cell death resistance and even in the genetic aging program.

Interaction of Venturicidin and Fo·F1-ATPase/ATP Synthase of Tightly Coupled Subbacterial Particles of Paracoccus denitrificans in Energized Membranes.

Proton-translocating Fo×F1-ATPase/synthase that catalyzes synthesis and hydrolysis of ATP is commonly considered to be a reversibly functioning complex. We have previously shown that venturicidin, a specific Fo-directed inhibitor, blocks the synthesis and hydrolysis of ATP with a significant difference in the affinity [Zharova, T. V. and Vinogradov, A. D. (2017) Biochim. Biophys. Acta, 1858, 939-944]. In this paper, we have studied in detail inhibition of Fo×F1-ATPase/synthase by venturicidin in tightly coupled membranes of Paracoccus denitrificans under conditions of membrane potential generation. ATP hydrolysis was followed by the ATP-dependent succinate-supported NAD+ reduction (potential-dependent reverse electron transfer) catalyzed by the respiratory chain complex I. It has been demonstrated that membrane energization did not affect the affinity of Fo×F1-ATPase/synthase for venturicidin. The dependence of the residual ATP synthase activity on the concentration of venturicidin approximated a linear function, whereas the dependence of ATP hydrolysis was sigmoidal: at low inhibitor concentrations venturicidin strongly inhibited ATP synthesis without decrease in the rate of ATP hydrolysis. A model is proposed suggesting that ATP synthesis and ATP hydrolysis are catalyzed by two different forms of Fo×F1.