Conductance Of Voltage-dependent Anion Channel Research Articles

Voltage dependent anion channel and its interaction with N-acetyl-L-Cysteine (NAC) under oxidative stress on planar lipid bilayer

Mitochondria are the major source of Hydrogen Peroxide (H2O2), a reactive oxygen species, in the cells. The reactive oxygen species generated by the mitochondria oxidize major proteins including Voltage Dependent Anion Channel (VDAC). We were interested to know how the effect of H2O2 is countered by antioxidants present around the mitochondria. N-Acetyl-l-Cysteine (NAC) is a naturally existing antioxidant in the cells. Keeping this in view, the modulatory effect of antioxidant NAC on H2O2 oxidized VDAC has been investigated through in vitro electrophysiological studies. First, the effect of H2O2 and NAC was studied on independently incorporated single-channel VDAC. It was observed that NAC suppresses VDAC conductance with a half-maximal inhibitory concentration (IC50) of ∼1.04 μM. In contrast, H2O2 enhances VDAC conductance. Later, oxidative stress was induced by H2O2 on VDAC increased conductance with half-maximal effective concentration (EC50) of ∼302 nM. An application of 1 μM NAC on H2O2 treated (300 nM) VDAC reversed the effect of oxidation. In the next step, NAC and H2O2 were added in reverse order. When oxidative stress was induced using H2O2, reduction in conductance by NAC was 4.5 ± 0.404 nS. The change in conductance is nearly 6.3%. However, if antioxidant NAC was incubated first followed by H2O2 treatment, the conductance of VDAC was 3.09 ± 0.27 nS. The change in conductance is near 33%. Both H2O2 and NAC also affected various conducting states of VDAC. In-silico studies indicated the binding of NAC at Lysine and Glutamic acid of VDAC. Hence, NAC was found to be effective in protection of VDAC against H2O2-induced oxidative stress due to its strong binding.

Homocysteine-Thiolactone Modulates Gating of Mitochondrial Voltage-Dependent Anion Channel (VDAC) and Protects It from Induced Oxidative Stress.

The gating of the Voltage-Dependent Anion Channel (VDAC) is linked to oxidative stress through increased generation of mitochondrial ROS with increasing mitochondrial membrane potential (ΔΨm). It has been already reported that H2O2 increases the single-channel conductance of VDAC on a bilayer lipid membrane. On the other hand, homocysteine (Hcy) has been reported to induce mitochondria-mediated cell death. It is argued that the thiol-form of homocysteine, HTL could be the plausible molecule responsible for the alteration in the function of proteins, such as VDAC. It is hypothesized that HTL interacts with VDAC that causes functional abnormalities. An investigation was undertaken to study the interaction of HTL with VDAC under H2O2 induced oxidative stress through biophysical and electrophysiological methods. Fluorescence spectroscopic studies indicate that HTL interacts with VDAC, but under induced oxidative stress the effect is prevented partially. Similarly, bilayer electrophysiology studies suggest that HTL shows a reduction in VDAC single-channel conductance, but the effects are partially prevented under an oxidative environment. Gly172 and His181 are predicted through bioinformatics tools to be the most plausible binding residues of HTL in Rat VDAC. The binding of HTL and H2O2 with VDAC appears to be cooperative as per our analysis of experimental data in the light of the Hill-Langmuir equation. The binding energies are estimated to be - 4.7kcalmol-1 and - 2.8kcalmol-1, respectively. The present in vitro studies suggest that when mitochondrial VDAC is under oxidative stress, the effects of amino acid metabolites like HTL are suppressed.

The Role of Voltage-Dependent Anion Channel in Mitochondrial Dysfunction and Human Disease.

The voltage-dependent anion channel (VDAC) is a β-barrel membrane protein located in the outer mitochondrial membrane (OMM). VDAC has two conductance states: an open anion selective state, and a closed and slightly cation-selective state. VDAC conductance states play major roles in regulating permeability of ATP/ADP, regulation of calcium homeostasis, calcium flux within ER-mitochondria contact sites, and apoptotic signaling events. Three reported structures of VDAC provide information on the VDAC open state via X-ray crystallography and nuclear magnetic resonance (NMR). Together, these structures provide insight on how VDAC aids metabolite transport. The interaction partners of VDAC, together with the permeability of the pore, affect the molecular pathology of diseases including Parkinson’s disease (PD), Friedreich’s ataxia (FA), lupus, and cancer. To fully address the molecular role of VDAC in disease pathology, major questions must be answered on the structural conformers of VDAC. For example, further information is needed on the structure of the closed state, how binding partners or membrane potential could lead to the open/closed states, the function and mobility of the N-terminal α-helical domain of VDAC, and the physiological role of VDAC oligomers. This review covers our current understanding of the various states of VDAC, VDAC interaction partners, and the roles they play in mitochondrial regulation pertaining to human diseases.

α-Synuclein emerges as a potent regulator of VDAC-facilitated calcium transport

Voltage-dependent anion channel (VDAC) is the most ubiquitous channel at the mitochondrial outer membrane, and is believed to be the pathway for calcium entering or leaving the mitochondria. Therefore, understanding the molecular mechanisms of how VDAC regulates calcium influx and efflux from the mitochondria is of particular interest for mitochondrial physiology. When the Parkinson’s disease (PD) related neuronal protein, alpha-synuclein (αSyn), is added to the reconstituted VDAC, it reversibly and partially blocks VDAC conductance by its acidic C-terminal tail. Using single-molecule VDAC electrophysiology of reconstituted VDAC we now demonstrate that, at CaCl2 concentrations below 150 mM, αSyn reverses the channel’s selectivity from anionic to cationic. Importantly, we find that the decrease in channel conductance upon its blockage by αSyn is hugely overcompensated by a favorable change in the electrostatic environment for calcium, making the blocked state orders-of-magnitude more selective for calcium and thus increasing its net flux. -Our findings with higher calcium concentrations also demonstrate that the phenomenon of “charge inversion” is taking place at the level of a single polypeptide chain. Measurements of ion selectivity of three VDAC isoforms in CaCl2 gradient show that VDAC3 exhibits the highest calcium permeability among them, followed by VDAC2 and VDAC1, thus pointing to isoform-dependent physiological function. Mutation of the E73 residue – VDAC1 purported calcium binding site – shows that there is no measurable effect of the mutation in either open or αSyn-blocked VDAC1 states. Our results confirm VDACs involvement in calcium signaling and reveal a new regulatory role of αSyn, with clear implications for both normal calcium signaling and PD-associated mitochondrial dysfunction.

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.

Regulation of Single-Channel Conductance of Voltage-Dependent Anion Channel by Mercuric Chloride in a Planar Lipid Bilayer.

The existence of mercury in various forms, e.g., elemental, organic, and inorganic has been known for decades. In any of these forms, it is poisonous to metabolism. In this, an investigation about the effect of the inorganic form of mercury, i.e., mercuric chloride (HgCl2) to the mitochondrial voltage-dependent anion channel (VDAC), has been done after isolation from the cardiac and brain tissues of Wistar rats. In vitro electrophysiology experiments were performed in Cardiolipin planar lipid bilayer membrane (BLM) to study the change in the conductance, selectivity, and gating charge of VDAC post HgCl2 treatment. A reduction in mean conductance of VDAC from 4.3 ± 0.18 to 1.66 ± 0.11 nS was observed. Further, the Gating charge calculated before (± 3.5) and after HgCl2 treatment (± 2.3) showed significant difference. Later, VDAC's behavior was studied at different concentrations of HgCl2 ranging from 0.1μM to 1mM. The Inhibitory concentration (IC50) was calculated from the linear regression plot. The IC50 was found to be 488.1μM. In the asymmetrical HgCl2 (5:1), a permeability ratio of cation to anion was found to be 4.2. It is interpreted that VDAC functioning is affected due to the application of 4mM HgCl2 and a reduction in the conductance, gating charge, and permeability of VDAC was detected. The results provide clues to HgCl2-induced toxicity mediated through VDAC in the Cardiolipin BLM.

VDAC-Tubulin, an Anti-Warburg Pro-Oxidant Switch.

Aerobic enhanced glycolysis characterizes the Warburg phenotype. In cancer cells, suppression of mitochondrial metabolism contributes to maintain a low ATP/ADP ratio that favors glycolysis. We propose that the voltage-dependent anion channel (VDAC) located in the mitochondrial outer membrane is a metabolic link between glycolysis and oxidative phosphorylation in the Warburg phenotype. Most metabolites including respiratory substrates, ADP, and Pi enter mitochondria only through VDAC. Oxidation of respiratory substrates in the Krebs cycle generates NADH that enters the electron transport chain (ETC) to generate a proton motive force utilized to generate ATP and to maintain mitochondrial membrane potential (ΔΨ). The ETC is also the major source of mitochondrial reactive oxygen species (ROS) formation. Dimeric α-β tubulin decreases conductance of VDAC inserted in lipid bilayers, and high free tubulin in cancer cells by closing VDAC, limits the ingress of respiratory substrates and ATP decreasing mitochondrial ΔΨ. VDAC opening regulated by free tubulin operates as a “master key” that “seal–unseal” mitochondria to modulate mitochondrial metabolism, ROS formation, and the intracellular flow of energy. Erastin, a small molecule that binds to VDAC and kills cancer cells, and erastin-like compounds antagonize the inhibitory effect of tubulin on VDAC. Blockage of the VDAC–tubulin switch increases mitochondrial metabolism leading to decreased glycolysis and oxidative stress that promotes mitochondrial dysfunction, bioenergetic failure, and cell death. In summary, VDAC opening-dependent cell death follows a “metabolic double-hit model” characterized by oxidative stress and reversion of the pro-proliferative Warburg phenotype.

Dual mechanism of ion permeation through VDAC revealed with inorganic phosphate ions and phosphate metabolites.

In the exchange of metabolites and ions between the mitochondrion and the cytosol, the voltage-dependent anion channel (VDAC) is a key element, as it forms the major transport pathway for these compounds through the mitochondrial outer membrane. Numerous experimental studies have promoted the idea that VDAC acts as a regulator of essential mitochondrial functions. In this study, using a combination of molecular dynamics simulations, free-energy calculations, and electrophysiological measurements, we investigated the transport of ions through VDAC, with a focus on phosphate ions and metabolites. We showed that selectivity of VDAC towards small anions including monovalent phosphates arises from short-lived interactions with positively charged residues scattered throughout the pore. In dramatic contrast, permeation of divalent phosphate ions and phosphate metabolites (AMP and ATP) involves binding sites along a specific translocation pathway. This permeation mechanism offers an explanation for the decrease in VDAC conductance measured in the presence of ATP or AMP at physiological salt concentration. The binding sites occur at similar locations for the divalent phosphate ions, AMP and ATP, and contain identical basic residues. ATP features a marked affinity for a central region of the pore lined by two lysines and one arginine of the N-terminal helix. This cluster of residues together with a few other basic amino acids forms a “charged brush” which facilitates the passage of the anionic metabolites through the pore. All of this reveals that VDAC controls the transport of the inorganic phosphates and phosphate metabolites studied here through two different mechanisms.

Phosphorylation of voltage-dependent anion channel by c-Jun N-terminal Kinase-3 leads to closure of the channel

Stress activated c-Jun N-terminal Kinase-3 (JNK3) has been reported to act on mitochondrion to promote neuronal cell death. Phosphorylation of mitochondrial Voltage-Dependent Anion Channel (VDAC) plays an important role in mitochondria-mediated cell death. Keeping these in view phosphorylation of rat brain VDAC by JNK3 has been studied in vitro. Pro Q Diamond phospho-protein staining experiment demonstrates VDAC is phosphorylated by JNK3. Bilayer electrophysiological experiments show that single-channel conductance of VDAC phosphorylated by JNK3 is significantly lower than that of the native VDAC at a membrane potential. The opening probability of VDAC undergoes massive reduction due to phosphorylation by JNK3. These indicate closure of VDAC due to phosphorylation by JNK3. Treatment of phosphorylated VDAC with alkaline phosphatase reversed the VDAC functional activity as shown by single-channel current and opening probability. The physiological consequence of closure of VDAC as a result of phosphorylation has been attributed to JNK3 dependent mitochondria-mediated apoptosis.

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.

Antagonists of the Inhibitory Effect of Free Tubulin on VDAC Induce Oxidative Stress and Mitochondrial Dysfunction

Bakground: Mitochondrial membrane potential (ΔΨ) and generation of reactive oxygen species (ROS) requires a respiratory chain fueled by the flux of metabolites into mitochondria through voltage dependent anion channels (VDAC). Free tubulin induces reversible blockage of VDAC both in vitro and in cells. Erastin, a small molecule lethal to cancer cells, antagonizes the inhibitory effect of free tubulin on VDAC and upregulates mitochondrial metabolism. Here, we hypothesized that erastin and erastin-like compounds open VDAC, increase mitochondrial metabolism and ROS formation, and activate JNK, which in turn cause mitochondrial dysfunction. Our AIM was to evaluate the effects of erastin/erastin-like compounds on ΔΨ, NADH, ROS and JNK in intact cells. METHODS: ΔΨ was assessed by confocal microscopy of tetramethylrhodamine methylester (TMRM) fluorescence and ROS by chloromethyldichlorofluorescein (cmDCF) and MitoSOX Red fluorescence. Mitochondrial NADH autofluorescence was assessed by multiphoton microscopy. Total and phosphorylated JNK was determined by Western blotting. RESULTS: In lipid bilayers, erastin antagonized tubulin inhibition on VDAC. In HepG2 human hepatoma cells, erastin increased ΔΨ by 46% and NADH by 30%. Mitochondrial hyperpolarization plateaued after 2 h. Subsequently depolarization occurred (3-4 h), suggesting mitochondrial dysfunction. Erastin-like compounds X1 and X2, identified in a high-throughput screening, similarly caused mitochondrial hyperpolarization/depolarization. Erastin also caused increases of DCF and Mitosox Red fluorescence beginning after ∼30 min and reaching a maximum after 2 h. Additionally, erastin activated JNK (maximum pJNK at 60 min). JNK activation and ROS formation both preceded mitochondrial depolarization. Conclusion: Erastin relieves tubulin-dependent inhibition of VDAC conductance, leading to mitochondrial hyperpolarization that increases ROS production with concomitant activation of the stress kinase JNK. These events, in turn, may induce the mitochondrial permeability transition, mitochondrial dysfunction and ultimately cell death.

Warburg Revisited: Regulation of Mitochondrial Metabolism by Voltage-Dependent Anion Channels in Cancer Cells

The bioenergetics of cancer cells is characterized by a high rate of aerobic glycolysis and suppression of mitochondrial metabolism (Warburg phenomenon). Mitochondrial metabolism requires inward and outward flux of hydrophilic metabolites, including ATP, ADP and respiratory substrates, through voltage-dependent anion channels (VDACs) in the mitochondrial outer membrane. Although VDACs were once considered to be constitutively open, closure of the VDAC is emerging as an adjustable limiter (governator) of mitochondrial metabolism. Studies of VDACs reconstituted into planar lipid bilayers show that tubulin at nanomolar concentrations decreases VDAC conductance. In tumor cell lines, microtubule-destabilizing agents increase cytoplasmic free tubulin and decrease mitochondrial membrane potential (ΔΨ(m)), whereas microtubule stabilization increases ΔΨ(m). Tubulin-dependent suppression of ΔΨ(m) is further potentiated by protein kinase A activation and glycogen synthase kinase-3β inhibition. Knockdown of different VDAC isoforms, especially of the least abundant isoform, VDAC3, also decreases ΔΨ(m), cellular ATP, and NADH/NAD+, suggesting that VDAC1 and VDAC2 are most inhibited by free tubulin. The brake on mitochondrial metabolism imposed by the VDAC governator probably is released when spindles form and free tubulin decreases as cells enter mitosis, which better provides for the high ATP demands of chromosome separation and cytokinesis. In conclusion, tubulin-dependent closure of VDACs represents a new mechanism contributing to the suppression of mitochondrial metabolism in the Warburg phenomenon.

Free Tubulin Modulates Mitochondrial Membrane Potential in Cancer Cells

Formation of the mitochondrial membrane potential (ΔΨ) depends on flux of respiratory substrates, ATP, ADP, and Pi through voltage-dependent anion channels (VDAC). As tubulin promotes single-channel closure of VDAC, we hypothesized that tubulin is a dynamic regulator of ΔΨ, which in cultured cancer cells was assessed by confocal microscopy of the potential-indicating fluorophore tetramethylrhodamine methylester (TMRM). Microtubule destabilizers, rotenone, colchicine, and nocodazole, and the microtubule stabilizer paclitaxel increased and decreased cellular free tubulin, respectively, and in parallel decreased and increased ΔΨ. Protein kinase A (PKA) activation by cAMP analogues and glycogen synthase kinase 3β (GSK-3β) inhibition decreased ΔΨ, whereas PKA inhibition hyperpolarized, consistent with reports that PKA and GSK-3β decrease and increase VDAC conductance, respectively. Plasma membrane potential assessed by DiBAC(4)(3) was not altered by any of the treatments. We propose that inhibition of VDAC by free tubulin limits mitochondrial metabolism in cancer cells.

VDAC1 cysteine residues: topology and function in channel activity and apoptosis

The VDAC (voltage-dependent anion channel) is proposed to control metabolic cross-talk between mitochondria and the cytosol, as well as apoptotic cell death. It has been suggested that apoptosis is modulated by the oxidation state of VDAC. Since cysteine residues are the major target for oxidation/reduction, we verified whether one or both VDAC1 cysteine residues are involved in VDAC1-mediated transport or apoptosis activities. To assess the function of VDAC1 cysteine residues in channel activity and to probe cysteine topology with respect to facing the pore or the bilayer, we used thiol-modifying agents, namely membrane-permeable NEM (N-ethylmaleimide), bulky charged 5-FM (fluorescein-5-maleimide) and the cross-linking reagent BMOE [bis(maleimido)ethane]. Bilayer-reconstituted VDAC conductance was decreased by 5-FM, but not by NEM, whereas 5-FM had no effect on NEM-labelled VDAC conductance. BMOE caused the formation of dimeric VDAC1, suggesting that one of the two VDAC1 cysteine residues is exposed and available for cross-linking. The results thus suggest that one of the VDAC1 cysteine residues faces the VDAC pore, whereas the second is oriented towards the lipid bilayer. Mutated rat VDAC1 in which the two cysteine residues, Cys127 and Cys232, were replaced by alanine residues showed channel activity like native VDAC1 and, when expressed in cells, was localized to mitochondria. Human VDAC1-shRNA (small hairpin RNA)- or -siRNA (small interfering RNA)-treated cells, expressing low levels of endogenous human VDAC1 together with native or cysteine-less rat VDAC1, undergo apoptosis as induced by overexpression of these VDAC1 or upon treatment with reactive oxygen species-producing agents, H2O2, As2O3 or selenite, suggesting that the two cysteine residues are not required for apoptosis or VDAC1 oligomerization.

Free Tubulin and cAMP-Dependent Phosphorylation Modulate Mitochondrial Membrane Potential in Hepg2 Cells: Possible Role of VDAC

BACKGROUND: Conductance of the voltage-dependent anion channel (VDAC) in the mitochondrial outer membrane has been proposed to limit mitochondrial metabolism in cancer cells and contribute to the Warburg effect. Since tubulin binding and phosphorylation promote VDAC closure, we hypothesized that free tubulin and cAMP-dependent phosphorylation by protein kinase A (PKA) modulate ΔΨ in cancer cells by regulating VDAC-dependent flux of substrates into mitochondria. Our AIM was to modulate VDAC closure and opening in intact cells by increasing and decreasing endogenous free tubulin and by promoting and blocking PKA activation. METHODS: HepG2 human hepatoma cells were incubated in Hank's solution with 5% CO2/air, and ΔΨ was assessed by confocal microscopy of TMRM. Free and polymerized tubulin was determined using a commercial kit. RESULTS: Myxothiazol (10 μM), a respiratory inhibitor, caused only a slight decrease of (TMRM fluorescence), but subsequent addition of oligomycin (10 μg/ml), a F1-F0-ATPase inhibitor, collapsed ΔΨ nearly completely, showing that inhibition of both respiration and ATPase are required to collapse ΔΨ. Stabilization of microtubules by paclitaxel (10 μM) increased ΔΨ by 60%, whereas disruption by colchicine (10 μM) or nocodazol (10 μM) decreased ΔΨ by 60-70%. Paclitaxel pretreatment prevented the depolarizing effect of colchicine and nocodazol. Dibutyryl cAMP (1 mM) decreased ΔΨ by 45% whereas H89 (1 μM), a specific inhibitor of PKA, increased ΔΨ by 94% and blocked the effect of dibutyryl cAMP. CONCLUSION: Free tubulin and cAMP/PKA-dependent phosphorylation modulate mitochondrial ΔΨ in HepG2 cells, most likely by regulating VDAC conductance. Up and down regulation of ΔΨ by tubulin polymerization/depolarization and PKA dependent phosphorylation/dephosphorylation is consistent with the hypothesis that VDAC is rate-limiting for mitochondrial metabolism in cancer cells and responsible, at least in part, for the Warburg effect.

Localization of the voltage-dependent anion channel-1 Ca 2+-binding sites

Photoreactive azido ruthenium (AzRu) has been recently shown to specifically interact with Ca 2+-binding proteins and to strongly inhibit their Ca 2+-dependent activities. Upon UV irradiation, AzRu can bind covalently to such proteins. In this study, AzRu was used to localize and characterize Ca 2+-binding sites in the voltage-dependent anion channel (VDAC). AzRu decreased the conductance of VDAC reconstituted into a bilayer while Ca 2+, in the presence of 1 M NaCl, but not Mg 2+, prevented this effect. AzRu had no effect on mutated E72Q- or E202Q-VDAC1 conductance, and [ 103Ru]AzRu labeled native but not E72Q-VDAC1, suggesting that these residues are required for AzRu interaction with the VDAC Ca 2+-binding site(s). AzRu protected against apoptosis induced by over-expression of native but not E72Q- or E202Q- murine VDAC1 in T-REx-293 cells depleted of endogenous hVDAC1. Chymotrypsin and trypsin digestion of AzRu-labeled VDAC followed by MALDI-TOF analysis revealed two AzRu-bound peptides corresponding to E72- and E202-containing sequences. These results suggest that the VDAC Ca 2+-binding site includes E72 and E202, located, according to a proposed VDAC1 topology model, on two distinct cytosolic loops. Furthermore, AzRu protection against apoptosis involves interaction with these residues. Photoreactive AzRu represents an important tool for identifying novel Ca 2+-binding proteins and localizing their Ca 2+-binding sites.

Phosphorylation of rat brain mitochondrial voltage‐dependent anion as a potential tool to control leakage of cytochrome c

Apoptosis is a controlled form of cell death that participates in development, elimination of damaged cells and maintenance of cell homeostasis. Also, it plays a role in neurodegenerative disorders like Alzheimer's disease. Recently, mitochondria have emerged as being pivotal in controlling apoptosis. They house a number of apoptogenic molecules, such as cytochrome c, which are released into the cytoplasm at the onset of apoptosis. When rat brain mitochondrial voltage-dependent anion channel (VDAC), an outer mitochondrial membrane protein, interacts with Bcl-2 family proteins Bax and tBid, its pore size increases, leading to the release of cytochrome c and other apoptogenic molecules into the cytosol and causing cell death. Regulation of this tBid- and Bax-induced increase in pore size of VDAC is a significant step to control cell death induced by cytochrome c. In this work, we have shown, through bilayer electrophysiological experiments, that the increase in VDAC conductance as a result of its interaction with Bax and tBid is reduced because of the action of cyclic AMP-dependent protein kinase A (PKA) in the presence of ATP. This indicates that the increase in the pore size of VDAC after its interaction with Bax and tBid is controlled via phosphorylation of this channel by PKA. This, we believe, could be a mechanism of controlling cytochrome c-mediated cell death in living cells.

Voltage-dependent anion channel (VDAC) as mitochondrial governator—Thinking outside the box

Despite a detailed understanding of their metabolism, mitochondria often behave anomalously. In particular, global suppression of mitochondrial metabolism and metabolite exchange occurs in apoptosis, ischemia and anoxia, cytopathic hypoxia of sepsis and multiple organ failure, alcoholic liver disease, aerobic glycolysis in cancer cells (Warburg effect) and unstimulated pancreatic beta cells. Here, we propose that closure of voltage-dependent anion channels (VDAC) in the mitochondrial outer membrane accounts for global mitochondrial suppression. In anoxia, cytopathic hypoxia and ethanol treatment, reactive oxygen and nitrogen species, cytokines, kinase cascades and increased NADH act to inhibit VDAC conductance and promote selective oxidation of membrane-permeable respiratory substrates like short chain fatty acids and acetaldehyde. In cancer cells, highly expressed hexokinase binds to and inhibits VDAC to suppress mitochondrial function while stimulating glycolysis, but an escape mechanism intervenes when glucose-6-phosphate accumulates and dissociates hexokinase from VDAC. Similarly, glucokinase binds mitochondria of insulin-secreting beta cells, possibly blocking VDAC and suppressing mitochondrial function. We propose that glucose metabolism leads to glucose-6-phosphate-dependent unbinding of glucokinase, relief of VDAC inhibition, release of ATP from mitochondria and ATP-dependent insulin release. In support of the overall proposal, ethanol treatment of isolated rat hepatocytes inhibited mitochondrial respiration and accessibility to adenylate kinase in the intermembrane space, effects that were overcome by digitonin permeabilization of the outer membrane. Overall, these considerations suggest that VDAC is a dynamic regulator, or governator, of global mitochondrial function both in health and disease.

Ion channel transduction in Langmuir-Blodgett lipid membranes

Multilayers of phospholipid films with VDAC (voltage dependent anion channels) or acetylcholine receptors (AChR) have been deposited onto platinum electrodes using the Langmuir-Blodgett technique. Electrical properties of both suspended and platinum electrode-supported phospholipid bilayers containing VDAC were studied. For the VDAC-coated electrodes, the VDAC conductance decreased under appropriate biasing conditions due to the voltage sensitive nature of the protein. The response was asymmetric with respect to the polarity of the offset voltage, possibly the result of steric hindrance of the protein conformational changes by the electrode surface. Physical properties of AChR-lipid films were investigated at the air-water interface as a function of surface pressure. AChR-lipid films were transferred onto electrodes and the conductance of the electrodes was found to increase upon injection of carbamylcholine. Alternative methods for protein transfer onto the electrode have been considered. Stability and sensitivity of these films will be discussed with respect to biosensor development.

Voltage modulation of a gated ion channel admittance in platinum-supported lipid bilayers

Platinum-supported phospholipid bilayers containing voltage-dependent anion channels (VDAC) were used to model physically a chemically sensitive amperometric biosensor which would use gated transmembrane channel proteins. An a.c. electrical technique was used to measure admittance changes caused by channel gating. The magnitude of the responses differed from those observed in bilayer lipid membranes (BLM). However, the bilayers were mechanically stable, and permitted reproducible gating of the VDAC conductance in response to d.c. bias voltages in the range of 0 to −60 mV. The technique may be generally suitable for fabrication of durable biosensors using chemically sensitive protein channels.