Voltage-dependent Anion Channel Activity Research Articles

Modulation of the mitochondrial voltage-dependent anion channel (VDAC) by hydrogen peroxide and its recovery by curcumin.

The Voltage-Dependent Anion Channel (VDAC) plays a vital role in mitochondria-mediated transport of ions and metabolites. It is well established that mitochondria are a site for production of hydrogen peroxide (H2O2). Excess production of H2O2 is toxic to the cell and causes oxidative stress. Therefore, the effect of H2O2 on the single-channel conductance of VDAC was investigated. In vitro bilayer electrophysiology experiments were performed on VDAC isolated from rat brain mitochondria, which consists predominately of the isoform VDAC1. VDAC was treated with H2O2 on a planar bilayer membrane (BLM). The conductance of VDAC increased upon H2O2 treatment, whereas the same concentration of H2O2 was unable to affect the BLM (without protein) over a long period of time. Subsequently, the sequential addition of curcumin to H2O2-treated VDAC reduced the conductance. Experimental results (bilayer electrophysiology) demonstrate the role of curcumin in the restoration of the activity of VDAC affected by H2O2. In silico docking studies enables identification of the probable binding site of H2O2 on VDAC. We further find that the oligomerization of VDAC that results in its increased conductance is an effect of lipid oxidation by H2O2.

Penehyclidine hydrochloride prevents anoxia/reoxygenation injury and induces H9c2 cardiomyocyte apoptosis via a mitochondrial pathway

Penehyclidine hydrochloride (PHC) is an anticholinergic drug that has been widely used in the clinic for years, evaluating for anesthetic premedication, anti-muscarinic, or improving microcirculation. However, very little is known about its protective effects against anoxia/reoxygenation (A/R) injury in myocardiocytes. The aim of our study was to investigate the protective effects of PHC pretreatment on A/R injury, as well as the underlying mechanisms involved. To investigate apoptosis, we used the cell viability assay, Annexin-V/PI assay, and lactate dehydrogenase (LDH) and creatine kinase (CK) activity measurements. Intracellular Ca2+ concentrations, reactive oxygen species (ROS), malonaldehyde (MDA), superoxide dismutase (SOD), and mitochondrial membrane potential were also measured. To analyze the molecular mechanisms, mitochondrial permeability transition pore (MPTP) activity and mRNA expressions of Bcl-2, Bax, cytochrome C (Cyt-C), caspase-3, and voltage-dependent anion channel (VDAC) were assessed. Pretreatment with PHC significantly increased cell viability and decreased the percentage of apoptotic cells, as well as LDH and CK activity, which was accompanied by a reduction in intracellular Ca2+ concentration, ROS, and MDA, as well as an increase in SOD levels. PHC pretreatment also restored mitochondrial membrane potential. Moreover, pre-incubation with PHC significantly attenuated MPTP activity and mRNA expressions of Bax, Cyt-C, caspase-3, and VDAC. Our results showed that PHC pretreatment protected H9c2 cells against A/R injury by modulating the mitochondrial apoptosis pathway.

Markov chain Monte Carlo based analysis of post-translationally modified VDAC gating kinetics.

The voltage-dependent anion channel (VDAC) is the main conduit for permeation of solutes (including nucleotides and metabolites) of up to 5 kDa across the mitochondrial outer membrane (MOM). Recent studies suggest that VDAC activity is regulated via post-translational modifications (PTMs). Yet the nature and effect of these modifications is not understood. Herein, single channel currents of wild-type, nitrosated, and phosphorylated VDAC are analyzed using a generalized continuous-time Markov chain Monte Carlo (MCMC) method. This developed method describes three distinct conducting states (open, half-open, and closed) of VDAC activity. Lipid bilayer experiments are also performed to record single VDAC activity under un-phosphorylated and phosphorylated conditions, and are analyzed using the developed stochastic search method. Experimental data show significant alteration in VDAC gating kinetics and conductance as a result of PTMs. The effect of PTMs on VDAC kinetics is captured in the parameters associated with the identified Markov model. Stationary distributions of the Markov model suggest that nitrosation of VDAC not only decreased its conductance but also significantly locked VDAC in a closed state. On the other hand, stationary distributions of the model associated with un-phosphorylated and phosphorylated VDAC suggest a reversal in channel conformation from relatively closed state to an open state. Model analyses of the nitrosated data suggest that faster reaction of nitric oxide with Cys-127 thiol group might be responsible for the biphasic effect of nitric oxide on basal VDAC conductance.

Expression and purification of OsVDAC4.

The voltage-dependent anion channel (VDAC), a major component of the mitochondrial outer membrane, has emerged as an important player in cell function, survival, and death signaling. VDAC function is modulated by its interaction with proteins such as hexokinase, adenine nucleotide translocator, and apoptotic proteins like Bax. Monitoring the activity of VDAC and its modulation in the complex cellular milieu is fraught with complications. Minimizing the number of components in the study is one approach to teasing apart various aspects of its function. In this chapter, we have described detailed protocols for the purification of a rice VDAC isoform, OsVDAC4 after overexpression in a bacterial system. The protein is solubilized with LDAO and then reconstituted into liposomes or planar bilayers to verify its competence to fold into a functionally active form.

VDAC phosphorylation impairment causes mitochondrial membrane permeability during lipid accumulation

Non‐alcoholic steatosis is a chronic disease characterized by lipid accumulation in the cytoplasm of hepatocytes involving early mitochondrial impairment and late hepatocyte cell death. The voltage‐dependent anion channel (VDAC) is a mitochondrial membrane channel mediating the flux of metabolites, calcium and water across the mitochondrial outer membrane. Upon lethal stress, VDAC can mediate mitochondrial membrane permeabilization (MMP) and cell death. Combination of four experimental models of steatosis (human biopsies, isolated mitochondria from ob/ob obese mice, high fat diet‐fed mice or immortalized cell lines) displayed, in steatotic livers, favored calcium‐induced MMP induction and permeability of VDAC. Thus, lipid accumulation increased the influx of water and calcium into mitochondria and sensitized the organelle to matrix swelling, depolarization and cytochrome c release. Moreover, NADH oxidase activity and channel properties of VDAC are sensitized to calcium regulation in steatosis models. These findings are associated with the hypophosphorylation of VDAC on a threonine residue and the loss of its interaction with the anti‐apoptotic Bcl‐XL and GSK3 kinase. In conclusion, VDAC acts as an early sensor of lipid toxicity and its GSK3‐mediated phosphorylation status controls outer mitochondrial membrane permeabilization in steatosis.

The Voltage-dependent Anion Channel (VDAC) Binds Tissue-type Plasminogen Activator and Promotes Activation of Plasminogen on the Cell Surface

The voltage-dependent anion channel (VDAC), a major pore-forming protein in the outer membrane of mitochondria, is also found in the plasma membrane of a large number of cells where in addition to its role in regulating cellular ATP release and volume control it is important for maintaining redox homeostasis. Cell surface VDAC is a receptor for plasminogen kringle 5, which promotes partial closure of the channel. In this study, we demonstrate that VDAC binds tissue-type plasminogen activator (t-PA) on human neuroblastoma SK-N-SH cells. Binding of t-PA to VDAC induced a decrease in K(m) and an increase in the V(max) for activation of its substrate, plasminogen (Pg). This resulted in accelerated Pg activation when VDAC, t-PA, and Pg were bound together. VDAC is also a substrate for plasmin; hence, it mimics fibrin activity. Binding of t-PA to VDAC occurs between a t-PA fibronectin type I finger domain located between amino acids Ile(5) and Asn(37) and a VDAC region including amino acids (20)GYGFG(24). These VDAC residues correspond to a GXXXG repeat motif commonly found in amyloid β peptides that is necessary for aggregation when these peptides form fibrillar deposits on the cell surface. Furthermore, we also show that Pg kringle 5 is a substrate for the NADH-dependent reductase activity of VDAC. This ternary complex is an efficient proteolytic complex that may facilitate removal of amyloid β peptide deposits from the normal brain and cell debris from injured brain tissue.

Mitochondria and calcium flux as targets of neuroprotection caused by minocycline in cerebellar granule cells

Minocycline, an antibiotic of the tetracycline family, has attracted considerable interest for its theoretical therapeutic applications in neurodegenerative diseases. However, the mechanism of action underlying its effect remains elusive. Here we have studied the effect of minocycline under excitotoxic conditions. Fluorescence and bioluminescence imaging studies in rat cerebellar granular neuron cultures using fura2/AM and mitochondria-targeted aequorin revealed that minocycline, at concentrations higher than those shown to block inflammation and inflammation-induced neuronal death, inhibited NMDA-induced cytosolic and mitochondrial rises in Ca2+ concentrations in a reversible manner. Moreover, minocycline added in the course of NMDA stimulation decreased Ca2+ intracellular levels, but not when induced by depolarization with a high K+ medium. We also found that minocycline, at the same concentrations, partially depolarized mitochondria by about 5–30mV, prevented mitochondrial Ca2+ uptake under conditions of environmental stress, and abrogated NMDA-induced reactive oxygen species (ROS) formation. Consistently, minocycline also abrogates the rise in ROS induced by 75μM Ca2+ in isolated brain mitochondria. In search for the mechanism of mitochondrial depolarization, we found that minocycline markedly inhibited state 3 respiration of rat brain mitochondria, although distinctly increased oxygen uptake in state 4. Minocycline inhibited NADH–cytochrome c reductase and cytochrome c oxidase activities, whereas the activity of succinate–cytochrome c reductase was not modified, suggesting selective inhibition of complexes I and IV. Finally, minocycline affected activity of voltage-dependent anion channel (VDAC) as determined in the reconstituted system. Taken together, our results indicate that mitochondria are a critical factor in minocycline-mediated neuroprotection.

Abstract 5420: Dephosphorylation of the Cardiac Mitochondrial Voltage-Dependent Anion Channel Prevents Inhibition by Hexokinase

The mitochondrial permeability transition pore (mPTP) has been implicated as the end effector in ischemic and pharmacological preconditioning. Though the molecular composition of the mPTP is thought to consist of cyclophilin D located in the mitochondrial matrix, adenine nucleotide translocase on the inner mitochondrial membrane, and the voltage-dependent anion channel (VDAC) on the outer mitochondrial membrane, recent studies have raised the possibility that VDAC may be a regulatory, rather than a major, component of mPTP. Nevertheless, VDAC is likely to be a critical component of the preconditioning signaling pathway since it is the main conduit for metabolite diffusion across the mitochondrial outer membrane. Yet, the direct measurements of cardiac VDAC activity and modulation have been limited. In the present study, we purified VDAC from rat hearts using standard procedure and investigated its modulation by phosphatase and hexokinase. VDAC was incorporated into planar lipid bilayer for measurements of channel activities. The channel exhibited the reported voltage-dependent gating. Several conductance states were identified, with the most prevalent between 1.5 to 2 nS in 0.5 M NaCl. Koenig’s polyanion, a VDAC blocker, triggered channel flickering and decreased the mean current by 78±6%. In the presence of phosphatase (1 unit/ml), the mean conductance significantly increased from 1.81±0.03 to 3.68±0.61 nS (n=9; mean±SEM). However, the addition of a recombinant hexokinase (5 units/ml; GenWay Biotech) had no significant effect on the phosphatase-enhanced VDAC current (n=4). In contrast, recombinant hexokinase alone significantly decreased the mean conductance from 1.75±0.05 to 0.79±0.19 nS (n=4). The addition of phosphatase reversed the inhibitory effect of hexokinase and further enhanced VDAC activity, increasing the mean conductance to 2.69±0.19 nS (n=4). Our results suggest that the dephosphorylation of VDAC prevents the inhibitory effects of hexokinase. Furthermore, VDAC activity suppressed by hexokinase can be reversed by dephosphorylation of the channel. In conclusion, we have reported on a novel observation at the functional level that basal phosphorylation of the cardiac VDAC may be required for its modulation by hexokinase.

Nucleotide-binding Sites in the Voltage-dependent Anion Channel: CHARACTERIZATION AND LOCALIZATION

In this study, we addressed the presence and location of nucleotide-binding sites in the voltage-dependent anion channel (VDAC). VDAC bound to reactive red 120-agarose, from which it was eluted by ATP, less effectively by ADP and AMP, but not by NADH. The photoreactive ATP analog, benzoyl-benzoyl-ATP (BzATP), was used to identify and characterize the ATP-binding sites in VDAC. [alpha-(32)P]BzATP bound to purified VDAC at two or more binding sites with apparent high and low binding affinities. Matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) analysis of BzATP-labeled VDAC confirmed the binding of at least two BzATP molecules to VDAC. The VDAC BzATP-binding sites showed higher specificity for purine than for pyrimidine nucleotides and higher affinity for negatively charged nucleotide species. VDAC treatment with the lysyl residue modifying reagent, fluorescein 5'-isothiocyanate, markedly inhibited VDAC labeling with BzATP. The VDAC nucleotide-binding sites were localized using chemical and enzymatic cleavage. Digestion of [alpha-(32)P]BzATP-labeled VDAC with CNBr or V8 protease resulted in the appearance of approximately 17- and approximately 14-kDa labeled fragments. Further digestion, high performance liquid chromatography separation, and sequencing of the selected V8 peptides suggested that the labeled fragments originated from two different regions of the VDAC molecule. MALDI-TOF analysis of BzATP-labeled, tryptic VDAC fragments indicated and localized three nucleotide binding sites, two of which were at the N and C termini of VDAC. Thus, the presence of two or more nucleotide-binding sites in VDAC is suggested, and their possible function in the control of VDAC activity, and, thereby, of outer mitochondrial membrane permeability is discussed.

The Voltage-dependent Anion Channel in Endoplasmic/Sarcoplasmic Reticulum: Characterization, Modulation and Possible Function

In recent years, it has been recognized that there is a metabolic coupling between the cytosol, ER/SR and mitochondria. In this cross-talk, mitochondrial Ca(2+) homeostasis and ATP production and supply play a major role. The primary transporter of adenine nucleotides, Ca(2+)and other metabolites into and out of mitochondria is the voltage-dependent anion channel (VDAC) located at the outer mitochondrial membrane, at a crucial position in the cell. VDAC has been established as a key player in mitochondrial metabolite and ion signaling and it has also been proposed that VDAC is present in extramitochondrial membranes. Thus, regulation of VDAC, as the main interface between mitochondrial and cellular metabolism, by other molecules is of utmost importance. This article reviews localization and function of VDAC, and focuses on VDAC as a skeletal muscle sarcoplasmic reticulum channel. The regulation of VDAC activity by associated proteins and by inhibitors is also presented. Several aspects of the physiological relevance of VDAC to Ca(2+) homeostasis and mitochondria-mediated apoptosis will be discussed.

In self-defence: hexokinase promotes voltage-dependent anion channel closure and prevents mitochondria-mediated apoptotic cell death.

In tumour cells, elevated levels of mitochondria-bound isoforms of hexokinase (HK-I and HK-II) result in the evasion of apoptosis, thereby allowing the cells to continue proliferating. The molecular mechanisms by which bound HK promotes cell survival are not yet fully understood. Our studies relying on the purified mitochondrial outer membrane protein VDAC (voltage-dependent anion channel), isolated mitochondria or cells in culture suggested that the anti-apoptotic activity of HK-I occurs via modulation of the mitochondrial phase of apoptosis. In the present paper, a direct interaction of HK-I with bilayer-reconstituted purified VDAC, inducing channel closure, is demonstrated for the first time. Moreover, HK-I prevented the Ca(2+)-dependent opening of the mitochondrial PTP (permeability transition pore) and release of the pro-apoptotic protein cytochrome c. The effects of HK-I on VDAC activity and PTP opening were prevented by the HK reaction product glucose 6-phosphate, a metabolic intermediate in most biosynthetic pathways. Furthermore, glucose 6-phosphate re-opened both the VDAC and the PTP closed by HK-I. The HK-I-mediated effects on VDAC and PTP were not observed using either yeast HK or HK-I lacking the N-terminal hydrophobic peptide responsible for binding to mitochondria, or in the presence of an antibody specific for the N-terminus of HK-I. Finally, HK-I overexpression in leukaemia-derived U-937 or vascular smooth muscle cells protected against staurosporine-induced apoptosis, with a decrease of up to 70% in cell death. These results offer insight into the mechanisms by which bound HK promotes tumour cell survival, and suggests that its overexpression not only ensures supplies of energy and phosphometabolites, but also reflects an anti-apoptotic defence mechanism.

Activation of mitochondrial voltage-dependent anion channel by apro-apoptotic BH3-only protein Bim

Bcl-2 family of proteins regulates apoptosis by controlling mitochondrial membrane permeability. We have previously shown that the voltage-dependent anion channel (VDAC) plays a crucial role in apoptotic changes of the mitochondria and its activity is directly regulated by some Bcl-2 family members, including Bcl-2/Bcl-x(L) and Bax/Bak but not Bid. Here, we showed that in isolated mitochondria, Bim induced loss of membrane potential and cytochrome c release like Bax/Bak, with these changes being inhibited by an anti-VDAC antibody. In addition, microinjection of the anti-VDAC antibody significantly reduced Bim-induced apoptosis. Study using purified proteins indicated that Bim directly interacts with the VDAC. Immunoprecipitation analysis revealed that Bim interacts with the VDAC and the interaction is remarkably enhanced during apoptosis. An experiment using liposomes indicated that Bim enhanced VDAC activity, as did Bax/Bak. Furthermore, Bim (but not tBid) was able to induce apoptotic changes of yeast mitochondria in a VDAC-dependent manner, and also induced the lysis of red blood cells, with this effect being inhibited by the anti-VDAC antibody. These results indicate that Bim has an ability to activate directly the VDAC, which plays an important role in apoptosis of mammalian cells.

Essential role of voltage-dependent anion channel in various forms of apoptosis in mammalian cells.

Through direct interaction with the voltage-dependent anion channel (VDAC), proapoptotic members of the Bcl-2 family such as Bax and Bak induce apoptogenic cytochrome c release in isolated mitochondria, whereas BH3-only proteins such as Bid and Bik do not directly target the VDAC to induce cytochrome c release. To investigate the biological significance of the VDAC for apoptosis in mammalian cells, we produced two kinds of anti-VDAC antibodies that inhibited VDAC activity. In isolated mitochondria, these antibodies prevented Bax-induced cytochrome c release and loss of the mitochondrial membrane potential (Deltapsi), but not Bid-induced cytochrome c release. When microinjected into cells, these anti-VDAC antibodies, but not control antibodies, also prevented Bax-induced cytochrome c release and apoptosis, whereas the antibodies did not prevent Bid-induced apoptosis, indicating that the VDAC is essential for Bax-induced, but not Bid-induced, apoptogenic mitochondrial changes and apoptotic cell death. In addition, microinjection of these anti-VDAC antibodies significantly inhibited etoposide-, paclitaxel-, and staurosporine-induced apoptosis. Furthermore, we used these antibodies to show that Bax- and Bak-induced lysis of red blood cells was also mediated by the VDAC on plasma membrane. Taken together, our data provide evidence that the VDAC plays an essential role in apoptogenic cytochrome c release and apoptosis in mammalian cells.

Human gelsolin prevents apoptosis by inhibiting apoptotic mitochondrial changes via closing VDAC.

Gelsolin is a Ca2+-dependent actin-regulatory protein that modulates actin assembly and disassembly, and is believed to regulate cell motility through modulation of the actin network. Gelsolin was also recently suggested to be involved in the regulation of apoptosis: human gelsolin (hGsn) has anti-apoptotic activity, whereas mouse gelsolin (mGsn) exerts either proapoptotic or anti-apoptotic activity depending on different cell types. Here, we studied the basis of anti-apoptotic activity of hGsn. We showed that both endogenous and overexpressed hGsn has anti-apoptotic activity, that depends on its C-terminal half. We also found that hGsn and its C-terminal half but not mGsn could prevent apoptotic mitochondrial changes such as Apsi loss and cytochrome c release in isolated mitochondria to a similar extent as Bcl-xL, indicating that hGsn targets the mitochondria to prevent apoptosis via its C-terminal half. In the same way as anti-apoptotic Bcl-xL, which we recently found to prevent apoptotic mitochondrial changes by binding and closing the voltage-dependent anion channel (VDAC), hGsn and its C-terminal half inhibited the activity of VDAC on liposomes through direct binding in a Ca2+-dependent manner. These results suggest that hGsn inhibits apoptosis by blocking mitochondrial VDAC activity.

Proapoptotic BH3-only Bcl-2 family members induce cytochromecrelease, but not mitochondrial membrane potential loss, and do not directly modulate voltage-dependent anion channel activity

Through direct interaction with the voltage-dependent anion channel (VDAC), proapoptotic Bcl-2 family members such as Bax and Bak induce apoptogenic mitochondrial cytochrome c release and membrane potential (Deltapsi) loss in isolated mitochondria. Using isolated mitochondria, we showed that Bid and Bik, BH3-only proteins from the Bcl-2 family, induced cytochrome c release but not Deltapsi loss. Unlike Bax/Bak, the cytochrome c release induced by Bid/Bik was Ca(2+)-independent, cyclosporin A-insensitive, and respiration-independent. Furthermore, in contrast to Bax/Bak, Bid/Bik neither interacted with VDAC nor directly affected the VDAC activity in liposomes. Consistently, Bid/Bik induced apoptosis without Deltapsi loss, whereas Bax induced apoptosis with Deltapsi loss. These findings indicated the involvement of a different mechanism in BH3-only, protein-induced apoptogenic cytochrome c release.