Voltage-dependent Anion Channel Closure Research Articles

Small molecules targeting the NADH-binding pocket of VDAC modulate mitochondrial metabolism in hepatocarcinoma cells.

Voltage dependent anion channels (VDAC) control the flux of most anionic respiratory substrates, ATP, ADP, and small cations, crossing the outer mitochondrial membrane. VDAC closure contributes to the partial suppression of mitochondrial metabolism that favors the Warburg phenotype of cancer cells. Recently, it has been shown that NADH binds to a specific pocket in the inner surface of VDAC1, also conserved in VDAC2 and 3, closing the channel. We hypothesized that binding of small molecules to the NADH pocket, maintain VDAC in an open configuration by preventing closure induced by NADH and possible other endogenous regulators. We screened in silico, the South Carolina Compound Collection SC3 (~ 100,000 proprietary molecules), using shape-based queries of the NADH binding region of VDAC. After molecular docking of selected compounds, we physically screened candidates using mitochondrial membrane potential (ΔΨm), as an overall readout of mitochondrial metabolism. We identified SC18, as the most potent compound. SC18 bound to VDAC1, as assessed by a thermal shift assay. Short-term treatment with SC18 decreased ΔΨm in SNU-449 and HepG2 human hepatocarcinoma cells. Mitochondrial depolarization was similar in wild type, VDAC1/2, 1/3, and 2/3 double KO HepG2 cells indicating that the effect of SC18 was not VDAC isoform-dependent. In addition, SC18 decreased mitochondrial NADH and cellular ATP production; and increased basal respiration. Long-term exposure to SC18, decreased cell proliferation as determined by wound-healing and cell viability assays. In summary, SC18 is a novel VDAC-targeting small molecule that induces mitochondrial dysfunction and inhibits cell proliferation.

A Rheostat of Ceramide and Sphingosine-1-Phosphate as a Determinant of Oxidative Stress-Mediated Kidney Injury.

Reactive oxygen species (ROS) modulate sphingolipid metabolism, including enzymes that generate ceramide and sphingosine-1-phosphate (S1P), and a ROS-antioxidant rheostat determines the metabolism of ceramide-S1P. ROS induce ceramide production by activating ceramide-producing enzymes, leading to apoptosis, while they inhibit S1P production, which promotes survival by suppressing sphingosine kinases (SphKs). A ceramide-S1P rheostat regulates ROS-induced mitochondrial dysfunction, apoptotic/anti-apoptotic Bcl-2 family proteins and signaling pathways, leading to apoptosis, survival, cell proliferation, inflammation and fibrosis in the kidney. Ceramide inhibits the mitochondrial respiration chain and induces ceramide channel formation and the closure of voltage-dependent anion channels, leading to mitochondrial dysfunction, altered Bcl-2 family protein expression, ROS generation and disturbed calcium homeostasis. This activates ceramide-induced signaling pathways, leading to apoptosis. These events are mitigated by S1P/S1P receptors (S1PRs) that restore mitochondrial function and activate signaling pathways. SphK1 promotes survival and cell proliferation and inhibits inflammation, while SphK2 has the opposite effect. However, both SphK1 and SphK2 promote fibrosis. Thus, a ceramide-SphKs/S1P rheostat modulates oxidant-induced kidney injury by affecting mitochondrial function, ROS production, Bcl-2 family proteins, calcium homeostasis and their downstream signaling pathways. This review will summarize the current evidence for a role of interaction between ROS-antioxidants and ceramide-SphKs/S1P and of a ceramide-SphKs/S1P rheostat in the regulation of oxidative stress-mediated kidney diseases.

Possible new mitochondrial mechanism of vitamin C anticancer activity

The main feature of fast-growing malignant tumors is the Warburg-type metabolism, which is directly related to an extremely high quantity of hexokinase (HK) bound to the voltage-dependent anion channels (VDACs) in the mitochondrial outer membrane. Previously, we explained the Warburg type metabolism as a result of the electrical suppression of mitochondria due to the closure of free VDACs that are not bound to HK. Here, we propose a possible new mechanism of high-dose vitamin C (ascorbate) anticancer activity estimated with a simplified computational model. According to the proposed hypothesis and the model, ascorbate oxidation in mitochondria leads to the generation of the negative outer membrane potential (OMP) of opposite sign to the positive OMP generated by the VDAC-HK complexes in cancer cells. The model demonstrates that negative OMP generated by any mechanism, even of relatively low magnitudes, leads tothe reopening of the electrically closed VDACs, thus reprogramming cell energy metabolism.According to the hypothesis, redox mediators, which increase the rate of ascorbate oxidation in mitochondria, should synergistically increase anticancer effects of high-dose ascorbate in accordance with experimental data recorded in the literature. The model shows that even small changes in the VDAC-voltage sensitivity and/or quantity of the VDAC-HK complexes, known to be caused by various physiological factors, might strongly influence the proposed mitochondrial mechanism of ascorbate anticancer activity.

Solving the Gating Mechanism of the Mitochondrial β-Barrel Metabolite Channel VDAC

The Voltage-Dependent Anion Channel (VDAC) is a β-barrel protein at the mitochondrial outer membrane (MOM) serving as the major pathway for ions and respiratory metabolites between mitochondria and the cytosol. Under applied voltage, VDAC transitions from an anionic “open” state, allowing the passive diffusion of neutral and anionic metabolites, to multiple low-conducting “closed” states with reduced anion selectivity. Importantly, VDAC closed states are virtually impermeable to ATP. A potential implication of these closed states in regulating metabolite traffic across the MOM has triggered vigorous investigations on their structural nature. Still, the mechanism behind VDAC closure remains enigmatic. Here, we study VDAC1 voltage-gating by using ion-channel electrophysiology guided by ANTON2 multi-microsecond Molecular Dynamics (MD) simulations. MD simulations highlight the particular role played by conformational dynamics of a “trigger” region, which is especially impacted by voltage polarity or solution acidification. We found that a basic residue in this region is key for initiating gating transitions at neutral pH, and its mutation to the oppositely charged residue hinders channel closure and reverses selectivity. We further explore VDAC voltage-gating by combining selectivity and pH-titration experiments with Markov State Model analysis on the motions of charged residues obtained from MD simulations. We propose a VDAC gating mechanism in which all charged residues are part of an ensemble where the motion of the single basic residue at the trigger region acts as a seed coordinating distinct dynamic rearrangements of the salt-bridge complex network. Thus, VDAC closure does not imply significant conformational changes of the β-barrel. This is strikingly different from the gating mechanism of most highly ion-selective channels and thus establishes a new paradigm of β-barrel channel gating that does not involve the translocation of a voltage sensor or a major change in pore geometry.

A Unifying Hypothesis Linking Hepatic Adaptations for Ethanol Metabolism to the Proinflammatory and Profibrotic Events of Alcoholic Liver Disease.

The pathogenesis of alcoholic liver disease (ALD) remains poorly understood but is likely a multihit pathophysiological process. Here, we propose a hypothesis of how early mitochondrial adaptations for alcohol metabolism lead to ALD pathogenesis. Acutely, ethanol (EtOH) feeding causes a near doubling of hepatic EtOH metabolism and oxygen consumption within 2 to 3hours. This swift increase in alcohol metabolism (SIAM) is an adaptive response to hasten metabolic elimination of both EtOH and its more toxic metabolite, acetaldehyde (AcAld). In association with SIAM, EtOH causes widespread hepatic mitochondrial depolarization (mtDepo), which stimulates oxygen consumption. In parallel, voltage-dependent anion channels (VDAC) in the mitochondrial outer membrane close. Together, VDAC closure and respiratory stimulation promote selective and more rapid oxidation of EtOH first to AcAld in the cytosol and then to nontoxic acetate in mitochondria, since membrane-permeant AcAld does not require VDAC to enter mitochondria. VDAC closure also inhibits mitochondrial fatty acid oxidation and ATP release, promoting steatosis and a decrease in cytosolic ATP. After acute EtOH, these changes revert as EtOH is eliminated with little hepatocellular cytolethality. mtDepo also stimulates mitochondrial autophagy (mitophagy). After chronic high EtOH exposure, the capacity to process depolarized mitochondria by mitophagy becomes compromised, leading to intra- and extracellular release of damaged mitochondria, mitophagosomes, and/or autolysosomes containing mitochondrial damage-associated molecular pattern (mtDAMP) molecules. mtDAMPs cause inflammasome activation and promote inflammatory and profibrogenic responses, causing hepatitis and fibrosis. We propose that persistence of mitochondrial responses to EtOH metabolism becomes a tipping point, which links initial adaptive EtOH metabolism to maladaptive changes initiating onset and progression of ALD.

Erastin-Like Anti-Warburg Agents Prevent Mitochondrial Depolarization Induced by Free Tubulin and Decrease Lactate Formation in Cancer Cells

In Warburg metabolism, suppression of mitochondrial metabolism contributes to a low cytosolic ATP/ADP ratio favoring enhanced aerobic glycolysis. Flux of metabolites across the mitochondrial outer membrane occurs through voltage-dependent anion channels (VDAC). In cancer cells, free dimeric tubulin induces VDAC closure and dynamically regulates mitochondrial membrane potential (ΔΨ). Erastin, a small molecule that binds to VDAC, antagonizes the inhibitory effect of tubulin on VDAC and hyperpolarizes mitochondria in intact cells. Here, our aim was to identify novel compounds from the ChemBridge DIVERSet library that block the inhibitory effect of tubulin on ΔΨ using cell-based screening. HCC4006 cells were treated with nocodazole (NCZ) to increase free tubulin and decrease ΔΨ in the presence or absence of library compounds. Tetramethylrhodamine methylester (TMRM) fluorescence was assessed by high-content imaging to determine changes in ΔΨ. Compounds were considered positive if ΔΨ increased in the presence of NCZ. Using confocal microscopy, we identified and validated six lead molecules that antagonized the depolarizing effect of NCZ. Lead compounds and erastin did not promote microtubule stabilization, so changes in ΔΨ were independent of tubulin dynamics. The most potent lead compound also decreased lactate formation. These novel small molecules represent a potential new class of anti-Warburg drugs.

Evolution of Voltage-Dependent Anion Channel Function: From Molecular Sieve to Governator to Actuator of Ferroptosis.

The voltage-dependent anion channel (VDAC) is well known as the pathway for passive diffusion of anionic hydrophilic mitochondrial metabolites across the outer membrane, but a more complex functionality of the three isoforms of VDAC has emerged, as addressed in the Frontiers in Oncology Research Topic on “Uncovering the Function of the Mitochondrial Protein VDAC in Health and Disease: from Structure-Function to Novel Therapeutic Strategies.” VDAC as the single most abundant protein in mitochondrial outer membranes is typically involved in isoform-specific interactions of the mitochondrion with its surroundings as, for example, during mitochondria-dependent pathways of cell death. VDAC closure can also act as an adjustable limiter (governator) of global mitochondrial metabolism, as during hepatic ethanol metabolism to promote selective oxidation of membrane-permeant acetaldehyde. In cancer cells, high free tubulin inhibits VDAC1 and VDAC2, contributing to suppression of mitochondrial function in the Warburg phenomenon. Erastin, the canonical inducer of ferroptosis, opens VDAC in the presence of tubulin and hyperpolarizes mitochondria, leading to mitochondrial production of reactive oxygen species, mitochondrial dysfunction, and cell death. Our understanding of VDAC function continues to evolve.

Number of Open Mitochondrial Voltage-Dependent Anion Channels and Intracellular Diffusion Coefficient in Heart Muscle

In cardiomyocytes, there are two diffusion obstacles that have shown to interfere with energy transfer from mitochondria to ATPases: mitochondrial outer membrane (MOM) and cytosolic diffusion barriers grouping ATP-producers and -consumers. While the mechanism responsible for restricting diffusion through MOM involves interaction between voltage-dependent anion channel (VDAC) and tubulin leading to the closure of VDAC for ATP, the number of open and closed VDACs in vivo remains unknown. In addition, there is no method developed so far that can clearly distinguish contributions of cytosolic barriers and MOM to the overall diffusion restriction in cardiomyocyte. The aim of this work is to establish the partitioning of intracellular diffusion obstacles in cardiomyocyte. For that, we studied the response of mitochondrial oxidative phosphorylation of permeabilized rat cardiomyocytes to changes in extracellular ADP by recording 3D image stacks of NADH autofluorescence. 3D image stacks were used as an input to finite element model specifically constructed for each studied cell. Using reaction-diffusion model, optical properties of fluorescence microscope, and comparing the model solution to the measured data, we are able to find permeability of MOM and cytosolic barriers. Our results demonstrate that MOM and cytosolic barriers have similar contribution to overall diffusion restriction. In our conditions, most of VDAC are in closed conformation with only ∼10% VDACs open per single mitochondria and cytosolic diffusion barriers reducing overall diffusion coefficient by 6-10x. Such profound diffusion restrictions are expected to play a major role in energy transfer, signaling, distribution of apoptotic factors and ROS.

Tubulin Tail Sequences and Post-translational Modifications Regulate Closure of Mitochondrial Voltage-dependent Anion Channel (VDAC)

It was previously shown that tubulin dimer interaction with the mitochondrial outer membrane protein voltage-dependent anion channel (VDAC) blocks traffic through the channel and reduces oxidative metabolism and that this requires the unstructured anionic C-terminal tail peptides found on both α- and β-tubulin subunits. It was unclear whether the α- and β-tubulin tails contribute equally to VDAC blockade and what effects might be due to sequence variations in these tail peptides or to tubulin post-translational modifications, which mostly occur on the tails. The nature of the contribution of the tubulin body beyond acting as an anchor for the tails had not been clarified either. Here we present peptide-protein chimeras to address these questions. These constructs allow us to easily combine a tail peptide with different proteins or combine different tail peptides with a particular protein. The results show that a single tail grafted to an inert protein is sufficient to produce channel closure similar to that observed with tubulin. We show that the β-tail is more than an order of magnitude more potent than the α-tail and that the lower α-tail activity is largely due to the presence of a terminal tyrosine. Detyrosination activates the α-tail, and activation is reversed by the removal of the glutamic acid penultimate to the tyrosine. Nitration of tyrosine reverses the tyrosine inhibition of binding and even induces prolonged VDAC closures. Our results demonstrate that small changes in sequence or post-translational modification of the unstructured tails of tubulin result in substantial changes in VDAC closure.

Abstract 3044: The ability of tubulin to close mitochondrial VDAC pores depends on beta tubulin isotype

Abstract Tubulin heterodimers can contain any of a number of beta-tubulin isotypes, and differential beta isotype expression has been found to have prognostic significance in human cancer and in chemotherapy response. We have shown that tubulin dimers can bind to and close the pore of the mitochondrial outer membrane protein VDAC (Voltage Dependent Anion Channel). VDAC acts as the “Gatekeeper” of metabolite flux into or out of the mitochondria, including all movement of ADP and ATP (and hence is a regulator of oxidative phosphorylation), and is also involved in early steps of apoptosis. We wished to test if all beta isotypes are equivalent in their ability to close VDAC, or if there are differences which may correlate to isotype expression in patient tumors. We previously showed that tubulin closure of VDAC blocks channel traffic and reduces oxidative metabolism, and that this requires the unstructured anionic tail peptides found on both the alpha- and beta-tubulin subunits. These intrinsically disordered tails contain only ∼3% of the mass but ∼40% of the net charge on the tubulin heterodimer. While all of the tails share some features, there are significant differences in length and sequence between alpha- and beta-tubulin tails, and between beta tubulin isotypes. Our earlier studies left unclear if the alpha- and beta-tails contribute equally to VDAC blockade, whether tails from different beta isotypes are all equivalent, and the role, if any, of tubulin posttranslational modifications (PTM), which mostly occur on the tails. Here we use single molecule binding studies to show that channel closure is due to the tails alone, and not the tubulin body, since grafting the tails to albumin gives the same VDAC closure extent as tubulin. The method involves electrophysiological recording from single VDAC molecules inserted in lipid bilayers and exposed to controlled concentrations of tubulin or BSA-tail constructs. We show that the beta-I tail is ∼100-fold more potent than the alpha-tail, and that this difference is largely due to the presence or absence of a terminal tyrosine on alpha. The activation of the alpha-tail by detyrosination is reversed by subsequent removal of the next residue. We also show that BSA-tail constructs from different beta isotypes differ widely in their VDAC closure activity. In particular, beta-I tails are the most potent of those tested, while beta-III is at least 100-fold less active, although the sequence differences between the two isotypes are not great. Other beta isotypes show intermediate levels of closure activity. Our results demonstrate that small changes in sequence or PTM of the disordered tails of tubulin result in substantial changes in VDAC binding and closure. Sequence differences between beta tubulin isotypes have major consequences on VDAC closure activity. In particular switching from the common epithelial beta-I to the beta-III isotype results in at least 100-fold reduction of VDAC closure. Citation Format: Kely L. Sheldon, Dan L. Sackett. The ability of tubulin to close mitochondrial VDAC pores depends on beta tubulin isotype. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3044. doi:10.1158/1538-7445.AM2015-3044

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.

Voltage Dependent Anion Channels (VDAC) and Regulation of Mitochondrial Metabolism

Voltage dependent anion channels (VDAC) are responsible for permeability of mitochondrial outer membranes to hydrophilic metabolites like ATP, ADP and respiratory substrates. Although previously assumed to remain open, VDAC closure is emerging as an important mechanism for regulation of global mitochondrial function. Acetaldehyde formation from hepatic ethanol metabolism leads to VDAC closure, which suppresses exchange of mitochondrial metabolites and inhibits ureagenesis. In vivo, VDAC closure after ethanol occurs coordinately with mitochondrial depolarization. Together, VDAC closure and uncoupling foster selective and more rapid mitochondrial oxidation of toxic acetaldehyde formed by ethanol metabolism. Glycolysis and suppression of mitochondrial metabolism are metabolic characteristics of cancer cells that promote cell proliferation (Warburg phenomenon). High free αβ-tubulin dimers in cancer cells block VDAC and suppress mitochondrial function, and reversal of tubulin inhibition of VDAC with erastin and erastin-like small molecules has an anti-Warburg effect that hyperpolarizes mitochondria, enhances oxidative phosphorylation and decreases glycolysis. Erastin-like compounds also enhance mitochondria formation of reactive oxygen species in cancer cells, leading eventually to mitochondrial dysfunction and cell death. Thus, VDAC is a key regulator of mitochondrial function (DK073336, DK037034 and 14.Z50.31.0028).

Channeling of Mitochondrial Energy in Cardiac and Cancer Cells by the Metabolically-Dependent Outer Membrane Potential

Mitochondria are the main source of energy in eukaryotic non-proliferating aerobic cells. In cancer cells, significant part of the energy metabolism is supported by the aerobic glycolysis. Oxidative phosphorylation metabolites cross the mitochondrial outer membrane through the voltage dependent anion channel (VDAC). The possibility of the voltage-gating functioning of VDAC under physiological conditions and proposed mechanisms of generation of the outer membrane potential (OMP) require further study. In this work, we present a computational thermodynamic analysis of a possible generation of OMP by the creatine kinase(CK)-VDAC and VDAC-hexokinase(HK) complexes, as well as by their intermembrane complexes with the adenine nucleotide translocator (ANT). The CK-VDAC and VDAC-HK complexes function as direct steady-state voltage generators, using the free energy of kinase reactions, while the ANT-CK-VDAC and ANT-VDAC-HK complexes allow the application of a part of the sum of the corresponding “kinase voltage” and the inner membrane potential to the outer membrane. The developed computational models demonstrate a high probability of generation of the metabolically-dependent OMP with magnitudes high enough to close free VDACs. We suggest that in case of cardiac cells, the CK-VDAC-mediated channeling of mitochondrial energy, earlier postulated by Saks and Wallimann, leads to the metabolically-dependent generation of a high OMP during systole, thus causing electrical closure of free VDACs and essentially avoiding the mitochondrial ATP usage by non-contraction system consumers. In case of cancer cells, that have a very high percentage of hexokinase attached to mitochondria, the electrical closure of free VDACs by OMP allows the preferential VDAC-HK-mediated channeling of the mitochondrial ATP to initiate the aerobic glycolysis by an anti-turbo mode. (Research grants #111852128625 and #520154531565, Colciencias).

ATP/ADP ratio, the missed connection between mitochondria and the Warburg effect

Non-proliferating cells generate the bulk of cellular ATP by fully oxidizing respiratory substrates in mitochondria. Respiratory substrates cross the mitochondrial outer membrane through only one channel, the voltage dependent anion channel (VDAC). Once in the matrix, respiratory substrates are oxidized in the tricarboxylic acid cycle to generate mostly NADH that is further oxidized in the respiratory chain to generate a proton motive force comprised mainly of membrane potential (ΔΨ) to synthesize ATP. Mitochondrial ΔΨ then drives the release of ATP4− from the matrix in exchange for ADP3− in the cytosol via the adenine nucleotide translocator (ANT) located in the mitochondrial inner membrane. Thus, mitochondrial function in non-proliferating cells drives a high cytosolic ATP/ADP ratio, essential to inhibit glycolysis. By contrast, the bioenergetics of the Warburg phenotype of proliferating cells is characterized by enhanced aerobic glycolysis and the suppression of mitochondrial metabolism. Suppressed mitochondrial function leads to lower production of mitochondrial ATP and hence lower cytosolic ATP/ADP ratios that favor enhanced glycolysis. Thus, the cytosolic ATP/ADP ratio is a key feature that determines if cell metabolism is predominantly oxidative or glycolytic. Here, we describe two novel mechanisms to explain the suppression of mitochondrial metabolism in cancer cells: the relative closure of VDAC by free tubulin and the inactivation of ANT. Both mechanisms contribute to low ATP/ADP ratios that activate glycolysis.

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. The model showed that the inhibition of the conductance and/or an increase in the voltage sensitivity of a relatively small fraction of VDACs by factors like tubulin potentiate the electrical closure of the remaining free VDACs. The extrusion of calcium ions from the mitochondrial intermembrane space by the generated OMP, positive inside, might increase cancer cell resistance to death. Within the VGC model, the known effect of induction of ATP release from mitochondria by accumulated glucose-6-phosphate in pancreatic beta cells might result not only of the known effect of GK dissociation from the VDAC-GK complex, but also of a decrease in the free energy of glucokinase reaction, leading to the OMP decrease and VDAC opening. We suggest that the VDAC-mediated electrical control of the mitochondrial outer membrane permeability, dependent on metabolic conditions, is a fundamental physiological mechanism of global regulation of mitochondrial functions and of cell death.

Mitochondrial Dysfunction in Gliomas

Mitochondrial (mt) dysfunction in gliomas has been linked to abnormalities of mt energy metabolism, marked by a metabolic shift from oxidative phosphorylation to glycolysis ("Warburg effect"), disturbances in mt membrane potential regulation and apoptotic signaling, as well as to somatic mutations involving the Krebs cycle enzyme isocitrate dehydrogenase. Evolving biological concepts with potential therapeutic implications include interaction between microtubule proteins and mitochondria (mt) in the control of closure of voltage-dependent anion channels and in the regulation of mt dynamics and the mt-endoplasmic reticulum network. The cytoskeletal protein βIII-tubulin, which is overexpressed in malignant gliomas, has emerged as a prosurvival factor associated in part with mt and also as a marker of chemoresistance. Mt-targeted therapeutic strategies that are discussed include the following: (1) metabolic modulation with emphasis on dichloroacetate, a pyruvate dehydrogenase kinase inhibitor; (2) tumor cell death via apoptosis induced by tricyclic antidepressants, microtubule-modulating drugs, and small molecules or compounds capable of inflicting reactive oxygen species-dependent tumor cell death; and (3) pretreatment mt priming and mt-targeted prodrug cancer therapy.

β-Barrel Mobility Underlies Closure of the Voltage-Dependent Anion Channel

The voltage-dependent anion channel (VDAC) is themajor protein in the outer mitochondrial membrane, where it mediates transport of ATP and ADP. Changes in its permeability, induced by voltage or apoptosis-related proteins, have been implicated in apoptotic pathways. The three-dimensional structure of VDAC has recently been determined asa 19-stranded β-barrel with an in-lying N-terminal helix. However, its gating mechanism is still unclear. Using solid-state NMR spectroscopy, molecular dynamics simulations, and electrophysiology, we show that deletion of the rigid N-terminal helix sharply increases overall motion in VDAC's β-barrel, resulting in elliptic, semicollapsed barrel shapes. These states quantitatively reproduce conductance and selectivity of the closed VDAC conformation. Mutation of the N-terminal helix leads to a phenotype intermediate to the open and closed states. These data suggest that the N-terminal helix controls entry into elliptic β-barrel states which underlie VDAC closure. Our results also indicate that β-barrel channels are intrinsically flexible.

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.

Ethanol Suppresses Ureagenesis in Rat Hepatocytes: ROLE OF ACETALDEHYDE

We proposed previously that closure of voltage-dependent anion channels (VDAC) in the mitochondrial outer membrane after ethanol exposure leads to suppression of mitochondrial metabolite exchange. Because ureagenesis requires extensive mitochondrial metabolite exchange, we characterized the effect of ethanol and its metabolite, acetaldehyde (AcAld), on total and ureagenic respiration in cultured rat hepatocytes. Ureagenic substrates increased cellular respiration from 15.8 ± 0.9 nmol O(2)/min/10(6) cells (base line) to 29.4 ± 1.7 nmol O(2)/min/10(6) cells in about 30 min. Ethanol (0-200 mM) suppressed extra respiration after ureagenic substrates (ureagenic respiration) by up to 51% but not base line respiration. Urea formation also declined proportionately. Inhibition of alcohol dehydrogenase, cytochrome P450 2E1, and catalase with 4-methylpyrazole, trans-1,2-dichloroethylene, and 3-amino-1,2,3-triazole restored ethanol-suppressed ureagenic respiration by 46, 37, and 66%, respectively. By contrast, inhibition of aldehyde dehydrogenase with phenethyl isothiocyanate increased the inhibitory effect of ethanol on ureagenic respiration by an additional 60%. AcAld, an intermediate product of ethanol oxidation, suppressed ureagenic respiration with an apparent IC(50) of 125 μM. AcAld also inhibited entry of 3-kDa rhodamine-conjugated dextran in the mitochondrial intermembrane space of digitonin-permeabilized hepatocytes, indicative of VDAC closure. In conclusion, AcAld, derived from ethanol metabolism, suppresses ureagenesis in hepatocytes mediated by closure of VDAC.

Knockdown of VDAC Isoforms Decreases Mitochondrial Membrane Potential and Modulates the Effects of Free Tubulin in HepG2 Hepatoma Cells

BACKGROUND: Mitochondrial membrane potential (ΔΨ) depends on flux of metabolites through voltage dependent anion channels (VDAC) in the mitochondrial outer membrane. In vitro, tubulin binding induces VDAC closure. In intact cells, nocodazole (Ncz) increases free tubulin and decreases ΔΨ, whereas paclitaxel (Ptx) promotes tubulin polymerization and increases ΔΨ. These effects are attributed to closing and opening of VDAC. Here, we hypothesize that VDAC knockdown will decrease ΔΨ and block the inhibitory effect of free tubulin on ΔΨ. METHODS: HepG2 cells were transfected with individual siRNAs for VDAC1/2/3 (5 nM, Ambion) for 48 h. To assess ΔΨ, fluorescence of tetramethylrhodamine methylester (TMRM) was measured by confocal microscopy. RESULTS: VDAC1/2/3 relative mRNA abundance by qPCR was ∼39, 50 and 10%, respectively. Each siRNA decreased its corresponding VDAC mRNA by ∼90% without affecting other isoforms. After VDAC1/2/3 knockdown, TMRM fluorescence was 58, 41 and 21%, respectively, compared to 100% after non-target siRNA. In control cells, Ncz decreased TMRM fluorescence by 70%, and Ptx increased fluorescence by 51%. After knockdown of VDAC1/2/3, Ncz decreased TMRM fluorescence by 25, 22 and 9%, respectively, whereas Ptx increased fluorescence by 26, 28, and 27% . CONCLUSION: Each of the three VDAC isoforms contributes to maintenance of ΔΨ in HepG2 cells. After knockdown, the relative response to changes of free tubulin was attenuated. These results are consistent with the conclusion that free tubulin dynamically regulates ΔΨ in HepG2 cells by interacting with VDAC.