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1.  Two-color STED microscopy reveals different degrees of colocalization between hexokinase-I and the three human VDAC isoforms 
PMC Biophysics  2010;3:4.
The voltage-dependent anion channel (VDAC, also known as mitochondrial porin) is the major transport channel mediating the transport of metabolites, including ATP, across the mitochondrial outer membrane. Biochemical data demonstrate the binding of the cytosolic protein hexokinase-I to VDAC, facilitating the direct access of hexokinase-I to the transported ATP. In human cells, three hVDAC isoforms have been identified. However, little is known on the distribution of these isoforms within the outer membrane of mitochondria and to what extent they colocalize with hexokinase-I. In this study we show that whereas hVDAC1 and hVDAC2 are localized predominantly within the same distinct domains in the outer membrane, hVDAC3 is mostly uniformly distributed over the surface of the mitochondrion. We used two-color stimulated emission depletion (STED) microscopy enabling a lateral resolution of ~40 nm to determine the detailed sub-mitochondrial distribution of the three hVDAC isoforms and hexokinase-I. Individual hVDAC and hexokinase-I clusters could thus be resolved which were concealed in the confocal images. Quantitative colocalization analysis of two-color STED images demonstrates that within the attained resolution, hexokinase-I and hVDAC3 exhibit a higher degree of colocalization than hexokinase-I with either hVDAC1 or hVDAC2. Furthermore, a substantial fraction of the mitochondria-bound hexokinase-I pool does not colocalize with any of the three hVDAC isoforms, suggesting a more complex interplay of these proteins than previously anticipated. This study demonstrates that two-color STED microscopy in conjunction with quantitative colocalization analysis is a powerful tool to study the complex distribution of membrane proteins in organelles such as mitochondria.
PACS: 87.16.Tb, 87.85.Rs
doi:10.1186/1757-5036-3-4
PMCID: PMC2838807  PMID: 20205711
2.  Charged Residues Distribution Modulates Selectivity of the Open State of Human Isoforms of the Voltage Dependent Anion-Selective Channel 
PLoS ONE  2014;9(8):e103879.
Voltage Dependent Anion-selective Channels (VDACs) are pore-forming proteins located in the outer mitochondrial membrane. They are responsible for the access of ions and energetic metabolites into the inner membrane transport systems. Three VDAC isoforms exist in mammalian, but their specific role is unknown. In this work we have performed extensive (overall ∼5 µs) Molecular Dynamics (MD) simulations of the human VDAC isoforms to detect structural and conformational variations among them, possibly related to specific functional roles of these proteins. Secondary structure analysis of the N-terminal domain shows a high similarity among the three human isoforms of VDAC but with a different plasticity. In particular, the N-terminal domain of the hVDAC1 is characterized by a higher plasticity, with a ∼20% occurrence for the ‘unstructured’ conformation throughout the folded segment, while hVDAC2, containing a peculiar extension of 11 amino acids at the N-terminal end, presents an additional 310-helical folded portion comprising residues 10′ to 3, adhering to the barrel wall. The N-terminal sequences of hVDAC isoforms are predicted to have a low flexibility, with possible consequences in the dynamics of the human VDACs. Clear differences were found between hVDAC1 and hVDAC3 against hVDAC2: a significantly modified dynamics with possible important consequence on the voltage-gating mechanism. Charge distribution inside and at the mouth of the pore is responsible for a different preferential localization of ions with opposite charge and provide a valuable rationale for hVDAC1 and hVDAC3 having a Cl−/K+ selectivity ratio of 1.8, whereas hVDAC2 of 1.4. Our conclusion is that hVDAC isoforms, despite sharing a similar scaffold, have modified working features and a biological work is now requested to give evidence to the described dissimilarities.
doi:10.1371/journal.pone.0103879
PMCID: PMC4146382  PMID: 25084457
3.  Influence of Protein – Micelle Ratios and Cysteine Residues on the Kinetic Stability and Unfolding Rates of Human Mitochondrial VDAC-2 
PLoS ONE  2014;9(1):e87701.
Delineating the kinetic and thermodynamic factors which contribute to the stability of transmembrane β-barrels is critical to gain an in-depth understanding of membrane protein behavior. Human mitochondrial voltage-dependent anion channel isoform 2 (hVDAC-2), one of the key anti-apoptotic eukaryotic β-barrel proteins, is of paramount importance, owing to its indispensable role in cell survival. We demonstrate here that the stability of hVDAC-2 bears a strong kinetic contribution that is dependent on the absolute micellar concentration used for barrel folding. The refolding efficiency and ensuing stability is sensitive to the lipid-to-protein (LPR) ratio, and displays a non-linear relationship, with both low and high micellar amounts being detrimental to hVDAC-2 structure. Unfolding and aggregation process are sequential events and show strong temperature dependence. We demonstrate that an optimal lipid-to-protein ratio of 2600∶1 – 13000∶1 offers the highest protection against thermal denaturation. Activation energies derived only for lower LPRs are ∼17 kcal mol−1 for full-length hVDAC-2 and ∼23 kcal mol−1 for the Cys-less mutant, suggesting that the nine cysteine residues of hVDAC-2 impart additional malleability to the barrel scaffold. Our studies reveal that cysteine residues play a key role in the kinetic stability of the protein, determine barrel rigidity and thereby give rise to strong micellar association of hVDAC-2. Non-linearity of the Arrhenius plot at high LPRs coupled with observation of protein aggregation upon thermal denaturation indicates that contributions from both kinetic and thermodynamic components stabilize the 19-stranded β-barrel. Lipid-protein interaction and the linked kinetic contribution to free energy of the folded protein are together expected to play a key role in hVDAC-2 recycling and the functional switch at the onset of apoptosis.
doi:10.1371/journal.pone.0087701
PMCID: PMC3907894  PMID: 24494036
4.  Solution NMR Spectroscopic Characterization of Human VDAC-2 in Detergent Micelles and Lipid Bilayer Nanodiscs 
Biochimica et Biophysica Acta  2011;1818(6):1562-1569.
Three isoforms of the human voltage-dependent anion channel (VDAC), located in the outer mitochondrial membrane, are crucial regulators of mitochondrial function. Numerous studies have been carried out to elucidate biochemical properties, as well as the three-dimensional structure of VDAC-1. However, functional and structural studies of VDAC-2 and VDAC-3 at atomic resolution are still scarce. VDAC-2 is highly similar to VDAC-1 in amino acid sequence, but has substantially different biochemical functions and expression profiles. Here, we report the reconstitution of functional VDAC-2 in lauryldimethylamine-oxide (LDAO) detergent micelles and 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) lipid bilayer nanodiscs. We find that VDAC-2 is probably folded in both membrane-mimicking systems and that structural and functional characterization by solution NMR spectroscopy is feasible.
doi:10.1016/j.bbamem.2011.11.012
PMCID: PMC3321126  PMID: 22119777
5.  NMR Structural Investigation of the Mitochondrial Outer Membrane Protein VDAC and its Interaction with Anti-apoptotic Bcl-xL† 
Biochemistry  2007;46(2):514-525.
Bcl-2 family proteins are essential regulators of cell death and exert their primary pro- or anti-apoptotic roles at the mitochondrial outer membrane. Previously, pro- and anti-apoptotic Bcl-2 proteins have been shown to interact with the voltage dependent anion channel (VDAC) of the outer mitochondrial membrane. VDAC is a 283-residue integral membrane protein that forms an aqueous pore in the outer mitochondrial membrane, through which metabolites and other small molecules pass between the cytosol and intermembrane space. The essential life-sustaining function of VDAC in metabolite trafficking is believed to be regulated by proteins of the Bcl-2 family. The protective role of anti-apoptotic Bcl-xL may be through its interaction with VDAC. Here, VDAC has been expressed, purified, and refolded into a functional form amenable to NMR studies. Various biophysical experiments indicate that micelle-bound VDAC is in intermediate exchange between monomer and trimer. Using NMR spectroscopy, gel filtration, and chemical cross-linking we obtained direct evidence for binding of Bcl-xL to VDAC in a detergent micelle system. The VDAC-interacting region of Bcl-xL was characterized by NMR with chemical shift perturbation and transferred cross saturation. The interaction region was mapped to a putative helical hairpin motif of Bcl-xL that was found to insert into detergent micelles. Our results suggest that Bcl-xL can bind to 1 or 2 VDAC molecules forming heterodimers and heterotrimers. Our characterization of the VDAC/Bcl-xL complex offer initial structural insight into the role of anti-apoptotic Bcl-xL in regulating apoptotic events in the mitochondrial outer membrane.
doi:10.1021/bi061577h
PMCID: PMC2579276  PMID: 17209561
VDAC; Bcl-xL; NMR; membrane protein; apoptosis
6.  Multicopy suppressors of phenotypes resulting from the absence of yeast VDAC encode a VDAC-like protein. 
Molecular and Cellular Biology  1997;17(10):5727-5738.
The permeability of the outer mitochondrial membrane to most metabolites is believed to be based in an outer membrane, channel-forming protein known as VDAC (voltage-dependent anion channel). Although multiple isoforms of VDAC have been identified in multicellular organisms, the yeast Saccharomyces cerevisiae has been thought to contain a single VDAC gene, designated POR1. However, cells missing the POR1 gene (delta por1) were able to grow on yeast media containing a nonfermentable carbon source (glycerol) but not on such media at elevated temperature (37 degrees C). If VDAC normally provides the pathway for metabolites to pass through the outer membrane, some other protein(s) must be able to partially substitute for that function. To identify proteins that could functionally substitute for POR1, we have screened a yeast genomic library for genes which, when overexpressed, can correct the growth defect of delta por1 yeast grown on glycerol at 37 degrees C. This screen identified a second yeast VDAC gene, POR2, encoding a protein (YVDAC2) with 49% amino acid sequence identity to the previously identified yeast VDAC protein (YVDAC1). YVDAC2 can functionally complement defects present in delta por1 strains only when it is overexpressed. Deletion of the POR2 gene alone had no detectable phenotype, while yeasts with deletions of both the POR1 and POR2 genes were viable and able to grow on glycerol at 30 degrees C, albeit more slowly than delta por1 single mutants. Like delta por1 single mutants, they could not grow on glycerol at 37 degrees C. Subcellular fractionation studies with antibodies which distinguish YVDAC1 and YVDAC2 indicate that YVDAC2 is normally present in the outer mitochondrial membrane. However, no YVDAC2 channels were detected electrophysiologically in reconstituted systems. Therefore, mitochondrial membranes made from wild-type cells, delta por1 cells, delta por1 delta por2 cells, and delta por1 cells overexpressing YVDAC2 were incorporated into liposomes and the permeability of resulting liposomes to nonelectrolytes of different sizes was determined. The results indicate that YVDAC2 does not confer any additional permeability to these liposomes, suggesting that it may not normally form a channel. In contrast, when the VDAC gene from Drosophila melanogaster was expressed in delta por1 yeast cells, VDAC-like channels could be detected in the mitochondria by both bilayer and liposome techniques, yet the cells failed to grow on glycerol at 37 degrees C. Thus, channel-forming activity does not seem to be either necessary or sufficient to restore growth on nonfermentable carbon sources, indicating that VDAC mediates cellular functions that do not depend on the ability to form channels.
PMCID: PMC232421  PMID: 9315631
7.  Functional Model of Metabolite Gating by Human Voltage-Dependent Anion Channel 2 
Biochemistry  2011;50(17):3408-3410.
Voltage-dependent anion channels (VDACs) are critical regulators of outer mitochondrial membrane permeability in eukaryotic cells. VDACs have also been postulated to regulate cell death mechanisms. Erastin, a small molecule quinazolinone that is selectively lethal to tumor cells expressing mutant RAS, has previously been reported as a ligand for hVDAC2. While significant efforts have been made to elucidate the structure and function of hVDAC1, structural and functional characterization of hVDAC2 remains lacking. Here, we present an in vitro system that provides a platform for both functional and structural investigation of hVDAC2 and its small molecule modulator, erastin. Using this system, we found that erastin increases permeability of VDAC2 liposomes to NADH in a manner that requires the amino-terminal region of VDAC2. Furthermore, we confirmed that this VDAC2-lipsome sample is folded using solid-state NMR.
doi:10.1021/bi2003247
PMCID: PMC3082971  PMID: 21425834
8.  Cysteine Residues Impact the Stability and Micelle Interaction Dynamics of the Human Mitochondrial β-Barrel Anion Channel hVDAC-2 
PLoS ONE  2014;9(3):e92183.
The anti-apoptotic 19-stranded transmembrane human voltage dependent anion channel isoform 2 (hVDAC-2) β-barrel stability is crucial for anion transport in mitochondria. The role of the unusually high number of cysteine residues in this isoform is poorly understood. Using a Cys-less construct of hVDAC-2, we haveinvestigated the contribution of cysteines to channel function, barrel stability and its influence on the strength of protein-micelle interactions. We observe that despite the overall preservation in barrel structure upon cysteine mutation, subtle local variations in the mode of interaction of the barrel with its refolded micellar environment arise, which may manifest itself in the channel activity of both the proteins.Fluorescence measurements of the Trp residues in hVDAC-2 point to possible differences in the association of the barrel with lauryldimethylamine oxide (LDAO) micelles. Upon replacement of cysteines in hVDAC-2, our data suggests greater barrel rigidity by way of intra-protein interactions. This, in turn, lowers the equilibrium barrel thermodynamic parameters in LDAOby perturbingthe stability of the protein-micelle complex. In addition to this, we also find a difference in the cooperativity of unfolding upon increasing the LDAO concentration, implying the importance of micelle concentration and micelle-protein ratios on the stability of this barrel. Our results indicate that the nine cysteine residues of hVDAC-2 are the key in establishing strong(er) barrel interactions with its environment and also impart additional malleability to the barrel scaffold.
doi:10.1371/journal.pone.0092183
PMCID: PMC3967697  PMID: 24642864
9.  VDAC regulation: role of cytosolic proteins and mitochondrial lipids 
It was recently asserted that the voltage-dependent anion channel (VDAC) serves as a global regulator, or governor, of mitochondrial function (Lemasters and Holmuhamedov, Biochim Biophys Acta 1762:181–190, 2006). Indeed, VDAC, positioned on the interface between mitochondria and the cytosol (Colombini, Mol Cell Biochem 256:107–115, 2004), is at the control point of mitochondria life and death. This large channel plays the role of a “switch” that defines in which direction mitochondria will go: to normal respiration or to suppression of mitochondria metabolism that leads to apoptosis and cell death. As the most abundant protein in the mitochondrial outer membrane (MOM), VDAC is known to be responsible for ATP/ADP exchange and for the fluxes of other metabolites across MOM. It controls them by switching between the open and “closed” states that are virtually impermeable to ATP and ADP. This control has dual importance: in maintaining normal mitochondria respiration and in triggering apoptosis when cytochrome c and other apoptogenic factors are released from the intermembrane space into the cytosol. Emerging evidence indicates that VDAC closure promotes apoptotic signals without direct involvement of VDAC in the permeability transition pore or hypothetical Bax-containing cytochrome c permeable pores. VDAC gating has been studied extensively for the last 30 years on reconstituted VDAC channels. In this review we focus exclusively on physiologically relevant regulators of VDAC gating such as endogenous cytosolic proteins and mitochondrial lipids. Closure of VDAC induced by such dissimilar cytosolic proteins as pro-apoptotic tBid and dimeric tubulin is compared to show that the involved mechanisms are rather distinct. While tBid mostly modulates VDAC voltage gating, tubulin blocks the channel with the efficiency of blockage controlled by voltage. We also discuss how characteristic mitochondrial lipids, phospatidylethanolamine and cardiolipin, could regulate VDAC gating. Overall, we demonstrate that VDAC gating is not just an observation made under artificial conditions of channel reconstitution but is a major mechanism of MOM permeability control.
doi:10.1007/s10863-008-9145-y
PMCID: PMC2671000  PMID: 18654841
Apoptosis; Mitochondria; Mitochondria outer membrane; Voltage dependent anion channel; VDAC; Channel gating; Tubulin; tBid; Cardiolipin; Lipid packing stress
10.  Concentration Dependent Ion Selectivity in VDAC: A Molecular Dynamics Simulation Study 
PLoS ONE  2011;6(12):e27994.
The voltage-dependent anion channel (VDAC) forms the major pore in the outer mitochondrial membrane. Its high conducting open state features a moderate anion selectivity. There is some evidence indicating that the electrophysiological properties of VDAC vary with the salt concentration. Using a theoretical approach the molecular basis for this concentration dependence was investigated. Molecular dynamics simulations and continuum electrostatic calculations performed on the mouse VDAC1 isoform clearly demonstrate that the distribution of fixed charges in the channel creates an electric field, which determines the anion preference of VDAC at low salt concentration. Increasing the salt concentration in the bulk results in a higher concentration of ions in the VDAC wide pore. This event induces a large electrostatic screening of the charged residues promoting a less anion selective channel. Residues that are responsible for the electrostatic pattern of the channel were identified using the molecular dynamics trajectories. Some of these residues are found to be conserved suggesting that ion permeation between different VDAC species occurs through a common mechanism. This inference is buttressed by electrophysiological experiments performed on bean VDAC32 protein akin to mouse VDAC.
doi:10.1371/journal.pone.0027994
PMCID: PMC3229507  PMID: 22164223
11.  Crystal packing analysis of murine VDAC1 crystals in a lipidic environment reveals novel insights on oligomerization and orientation 
Channels (Austin, Tex.)  2009;3(3):167-170.
All eukaryotic cells require efficient trafficking of metabolites between the mitochondria and the rest of the cell. This exchange is carried out by the dominant protein in the outer mitochondrial membrane (OMM), the Voltage Dependent Anion Channel (VDAC), which serves as the primary pathway for the exchange of ions and metabolites between the cytoplasm and the intermembrane space of the mitochondria. Additionally, VDAC provides a scaffold for the binding of modulator proteins to the mitochondria and has been implicated in mitochondria-dependent cell death. We recently determined the structure of the murine VDAC1 (mVDAC1) at 2.3Å resolution crystallized in a native-like bilayer environment. The high-resolution structure provided concise structural details about the voltage-sensing N-terminal domain and catalyzed new hypotheses regarding the gating mechanisms for metabolites and ions that transit the OMM. In this study, the crystal packing of mVDAC1 is analyzed revealing a strong antiparallel dimer that further assemble as hexamers mimicking the native oligomeric packing observed in EM and AFM images of the OMM. Oligomerization has been shown to be important for VDAC regulation and function, and mVDAC1 crystal packing in a lipidic medium reveals insights on how oligomerization is accomplished using protein-protein and protein-lipid interactions. Furthermore, orientation of VDAC in the OMM remains uncertain due to inconsistencies in antibody labeling studies. The physiological implications of a novel antiparallel arrangement are addressed that may clarify these conflicting biochemical data.
PMCID: PMC3719987  PMID: 19574737
VDAC; crystal structure; oligomerization; orientation
12.  Expression Profiling of Mitochondrial Voltage-Dependent Anion Channel-1 Associated Genes Predicts Recurrence-Free Survival in Human Carcinomas 
PLoS ONE  2014;9(10):e110094.
Background
Mitochondrial voltage-dependent anion channels (VDACs) play a key role in mitochondria-mediated apoptosis. Both in vivo and in vitro evidences indicate that VDACs are actively involved in tumor progression. Specifically, VDAC-1, one member of the VDAC family, was thought to be a potential anti-cancer therapeutic target. Our previous study demonstrated that the human gene VDAC1 (encoding the VDAC-1 isoform) was significantly up-regulated in lung tumor tissue compared with normal tissue. Also, we found a significant positive correlation between the gene expression of VDAC1 and histological grade in breast cancer. However, the prognostic power of VDAC1 and its associated genes in human cancers is largely unknown.
Methods
We systematically analyzed the expression pattern of VDAC1 and its interacting genes in breast, colon, liver, lung, pancreatic, and thyroid cancers. The genes differentially expressed between normal and tumor tissues in human carcinomas were identified.
Results
The expression level of VDAC1 was uniformly up-regulated in tumor tissue compared with normal tissue in breast, colon, liver, lung, pancreatic, and thyroid cancers. Forty-four VDAC1 interacting genes were identified as being commonly differentially expressed between normal and tumor tissues in human carcinomas. We designated VDAC1 and the 44 dysregulated interacting genes as the VDAC1 associated gene signature (VAG). We demonstrate that the VAG signature is a robust prognostic biomarker to predict recurrence-free survival in breast, colon, and lung cancers, and is independent of standard clinical and pathological prognostic factors.
Conclusions
VAG represents a promising prognostic biomarker in human cancers, which may enhance prediction accuracy in identifying patients at higher risk for recurrence. Future therapies aimed specifically at VDAC1 associated genes may lead to novel agents in the treatment of cancer.
doi:10.1371/journal.pone.0110094
PMCID: PMC4198298  PMID: 25333947
13.  Voltage-dependent Anion Channel-1 (VDAC-1) Contributes to ATP Release and Cell Volume Regulation in Murine Cells 
The Journal of General Physiology  2004;124(5):513-526.
Extracellular ATP regulates several elements of the mucus clearance process important for pulmonary host defense. However, the mechanisms mediating ATP release onto airway surfaces remain unknown. Mitochondrial voltage-dependent anion channels (mt-VDACs) translocate a variety of metabolites, including ATP and ADP, across the mitochondrial outer membrane, and a plasmalemmal splice variant (pl-VDAC-1) has been proposed to mediate ATP translocation across the plasma membrane. We tested the involvement of VDAC-1 in ATP release in a series of studies in murine cells. First, the full-length coding sequence was cloned from a mouse airway epithelial cell line (MTE7b−) and transfected into NIH 3T3 cells, and pl-VDAC-1-transfected cells exhibited higher rates of ATP release in response to medium change compared with mock-transfected cells. Second, ATP release was compared in cells isolated from VDAC-1 knockout [VDAC-1 (−/−)] and wild-type (WT) mice. Fibroblasts from VDAC-1 (−/−) mice released less ATP than WT mice in response to a medium change. Well-differentiated cultures from nasal and tracheal epithelia of VDAC-1 (−/−) mice exhibited less ATP release in response to luminal hypotonic challenge than WT mice. Confocal microscopy studies revealed that cell volume acutely increased in airway epithelia from both VDAC-1 (−/−) and WT mice after luminal hypotonic challenge, but VDAC-1 (−/−) cells exhibited a slower regulatory volume decrease (RVD) than WT cells. Addition of ATP or apyrase to the luminal surface of VDAC-1 (−/−) or WT cultures with hypotonic challenge produced similar initial cell height responses and RVD kinetics in both cell types, suggesting that involvement of VDAC-1 in RVD is through ATP release. Taken together, these studies suggest that VDAC-1, directly or indirectly, contributes to ATP release from murine cells. However, the observation that VDAC-1 knockout cells released a significant amount of ATP suggests that other molecules also play a role in this function.
doi:10.1085/jgp.200409154
PMCID: PMC2234005  PMID: 15477379
voltage-dependent anion channel; ATP release; osmotic cell swelling; regulatory volume decrease; airway epithelia
14.  RAS–RAF–MEK-dependent oxidative cell death involving voltage-dependent anion channels 
Nature  2007;447(7146):864-868.
Therapeutics that discriminate between the genetic makeup of normal cells and tumour cells are valuable for treating and understanding cancer. Small molecules with oncogene-selective lethality may reveal novel functions of oncoproteins and enable the creation of more selective drugs1. Here we describe the mechanism of action of the selective anti-tumour agent erastin, involving the RAS–RAF–MEK signalling pathway functioning in cell proliferation, differentiation and survival. Erastin exhibits greater lethality in human tumour cells harbouring mutations in the oncogenes HRAS, KRAS or BRAF. Using affinity purification and mass spectrometry, we discovered that erastin acts through mitochondrial voltage-dependent anion channels (VDACs)—a novel target for anti-cancer drugs. We show that erastin treatment of cells harbouring oncogenic RAS causes the appearance of oxidative species and subsequent death through an oxidative, non-apoptotic mechanism. RNA-interference-mediated knockdown of VDAC2 or VDAC3 caused resistance to erastin, implicating these two VDAC isoforms in the mechanism of action of erastin. Moreover, using purified mitochondria expressing a single VDAC isoform, we found that erastin alters the permeability of the outer mitochondrial membrane. Finally, using a radiolabelled analogue and a filter-binding assay, we show that erastin binds directly to VDAC2. These results demonstrate that ligands to VDAC proteins can induce non-apoptotic cell death selectively in some tumour cells harbouring activating mutations in the RAS–RAF–MEK pathway.
doi:10.1038/nature05859
PMCID: PMC3047570  PMID: 17568748
15.  VDAC Inhibition by Tubulin and its Physiological Implications 
Biochimica et Biophysica Acta  2011;1818(6):1526-1535.
Regulation of mitochondrial outer membrane (MOM) permeability has dual importance: in normal metabolite and energy exchange between mitochondria and cytoplasm, and thus in control of respiration, and in apoptosis by release of apoptogenic factors into the cytosol. However, the mechanism of this regulation involving the voltage-dependent anion channel (VDAC), the major channel of MOM, remains controversial. For example, one of the long-standing puzzles was that in permeabilized cells, adenine nucleotide translocase is less accessible to cytosolic ADP than in isolated mitochondria. Still another puzzle was that, according to channel-reconstitution experiments, voltage regulation of VDAC is limited to potentials exceeding 30 mV, which are believed to be much too high for MOM. We have solved these puzzles and uncovered multiple new functional links by identifying a missing player in the regulation of VDAC and, hence, MOM permeability – the cytoskeletal protein tubulin. We have shown that, depending on VDAC phosphorylation state and applied voltage, nanomolar to micromolar concentrations of dimeric tubulin induce functionally important reversible blockage of VDAC reconstituted into planar phospholipid membranes. The voltage sensitivity of the blockage equilibrium is truly remarkable. It is described by an effective “gating charge” of more than ten elementary charges, thus making the blockage reaction as responsive to the applied voltage as the most voltage-sensitive channels of electrophysiology are. Analysis of the tubulin-blocked state demonstrated that although this state is still able to conduct small ions, it is impermeable to ATP and other multi-charged anions because of the reduced aperture and inversed selectivity.
The findings, obtained in a channel reconstitution assay, were supported by experiments with isolated mitochondria and human hepatoma cells. Taken together, these results suggest a previously unknown mechanism of regulation of mitochondrial energetics, governed by VDAC interaction with tubulin at the mitochondria-cytosol interface. Immediate physiological implications include new insights into serine/threonine kinase signaling pathways, Ca2+ homeostasis, and cytoskeleton/microtubule activity in health and disease, especially in the case of the highly dynamic microtubule network which is characteristic of cancerogenesis and cell proliferation. In the present review, we speculate how these findings may help to identify new mechanisms of mitochondria-associated action of chemotherapeutic microtubule-targeting drugs, and also to understand why and how cancer cells preferentially use inefficient glycolysis rather than oxidative phosphorylation (Warburg effect).
doi:10.1016/j.bbamem.2011.11.004
PMCID: PMC3302949  PMID: 22100746
Voltage-dependent anion channel; mitochondria; microtubules; phosphorylation; signaling networks; selectivity
16.  The Role of VDAC in Cell Death: Friend or Foe? 
Biochimica et Biophysica Acta  2011;1818(6):1444-1450.
As the voltage-dependent anion channel (VDAC) forms the interface between mitochondria and the cytosol, its importance in metabolism is well understood. However, research on VDAC’s role in cell death is a rapidly growing field, unfortunately with much confusing and contradictory results. The fact that VDAC plays a role in outer mitochondrial membrane permeabilization is undeniable, however, the mechanisms behind this remain very poorly understood. In this review, we will summarize the studies that show evidence of VDAC playing a role in cell death. To begin, we will discuss the evidence for and against VDAC’s involvement in mitochondrial permeability transition (MPT) and attempt to clarify that VDAC is not an essential component of the MPT pore (MPTP). Next, we will evaluate the remaining literature on VDAC in cell death which can be divided into three models: proapoptotic agents escaping through VDAC, VDAC homo- or hetero-oligomerization, or VDAC closure resulting in outer mitochondrial membrane permeabilization through an unknown pathway. We will then discuss the growing list of modulators of VDAC activity that have been associated with induction/protection against cell death.
doi:10.1016/j.bbamem.2011.10.025
PMCID: PMC3288473  PMID: 22062421
VDAC; mitochondria; permeability transition; apoptosis; necrosis
17.  The electrostatics of VDAC: implications for selectivity and gating 
Journal of molecular biology  2009;396(3):580-592.
The voltage-dependent anion channel (VDAC) is the major pathway mediating the transfer of metabolites and ions across the mitochondrial outer membrane. Two hallmarks of the channel in the open state are high metabolite flux and anion selectivity, while the partially closed state blocks metabolites and is cation selective. Here we report the results from electrostatics calculations carried out on the recently determined high-resolution structure of murine VDAC1 (mVDAC1). Poisson-Boltzmann (PB) calculations show that the ion transfer free energy through the channel is favorable for anions, suggesting that mVDAC1 represents the open state. This claim is buttressed by Poisson-Nernst-Planck (PNP) calculations that predict a high single-channel conductance indicative of the open state and an anion selectivity of 1.75 – nearly a 2-fold selectivity for anions over cations. These calculations were repeated on mutant channels and gave selectivity changes in accord with experimental observations. We were then able to engineer an in silico mutant channel with three point mutations that converted mVDAC1 into a channel with a preference for cations. Finally, we investigated two proposals for how the channel gates between the open and closed state. Both models involve the movement of the N-terminal helix, but neither motion produced the observed voltage sensitivity, nor did either model result in a cation selective channel, which is observed experimentally. Thus, we were able to rule out certain models for channel gating, but the true motion has yet to be determined.
doi:10.1016/j.jmb.2009.12.006
PMCID: PMC3736979  PMID: 20005234
VDAC; ion channel; continuum electrostatics; gating charge; PNP
18.  The role of solution NMR in the structure determinations of VDAC-1 and other membrane proteins 
The voltage dependent anion channel (VDAC) is an essential protein in the eukaryotic outer mitochondrial membrane, providing the pore for substrate diffusion. Three high-resolution structures of the isoform 1 of VDAC in detergent micelles and bicelles have recently been published, using solution NMR and X-ray crystallography. They resolve longstanding discussions about the membrane topology of VDAC and provide the first eukaryotic β-barrel membrane protein structure. The structure contains a surprising feature that had not been observed in an integral membrane protein before: A parallel β-strand pairing and thus an odd number of strands. The studies also give a structural and functional basis for the voltage gating mechanism of VDAC and its modulation by NADH, however they do not fully explain these functions yet. With the de novo structure of VDAC-1, as well as those of half a dozen other proteins, the number of integral membrane protein structures solved by solution NMR has doubled in the past two years. Numerous further structural and functional studies on many different membrane proteins show that solution NMR has become an important tool for membrane protein molecular biology.
doi:10.1016/j.sbi.2009.07.013
PMCID: PMC2739811  PMID: 19665886
19.  The Use of Anti-VDAC2 Antibody for the Combined Assessment of Human Sperm Acrosome Integrity and Ionophore A23187-Induced Acrosome Reaction 
PLoS ONE  2011;6(2):e16985.
Voltage-dependent anion channel (VDAC) is mainly located in the mitochondrial outer membrane and participates in many biological processes. In mammals, three VDAC subtypes (VDAC1, 2 and 3) have been identified. Although VDAC has been extensively studied in various tissues and cells, there is little knowledge about the distribution and function of VDAC in male mammalian reproductive system. Several studies have demonstrated that VDAC exists in mammalian spermatozoa and is implicated in spermatogenesis, sperm maturation, motility and fertilization. However, there is no knowledge about the respective localization and function of three VDAC subtypes in human spermatozoa. In this study, we focused on the presence of VDAC2 in human spermatozoa and its possible role in the acrosomal integrity and acrosome reaction using specific anti-VDAC2 monoclonal antibody for the first time. The results exhibited that native VDAC2 existed in the membrane components of human spermatozoa. The co-incubation of spermatozoa with anti-VDAC2 antibody did not affect the acrosomal integrity and acrosome reaction, but inhibited ionophore A23187-induced intracellular Ca2+ increase. Our study suggested that VDAC2 was located in the acrosomal membrane or plasma membrane of human spermatozoa, and played putative roles in sperm functions through mediating Ca2+ transmembrane transport.
doi:10.1371/journal.pone.0016985
PMCID: PMC3036732  PMID: 21347391
20.  VDAC3 has differing mitochondrial functions in two types of striated muscles 
Biochimica et biophysica acta  2010;1807(1):150-156.
Voltage-dependent anion channel (VDAC) is an abundant mitochondrial outer membrane protein. In mammals, three VDAC isoforms have been characterized. We have previously reported alterations in the function of mitochondria when assessed in situ in different muscle types in VDAC1 deficient mice (Anflous, K., Armstrong, D., Craigen, W.J., 2001, J. Biol. Chem. 276, 1954-1960). In the present report we extend the study to VDAC3 deficient muscles and measure the respiratory enzyme activity in both VDAC1 and VDAC3 deficient muscles. While in the heart the absence of VDAC3 causes a decrease in the apparent affinity of in situ mitochondria for ADP, in the gastrocnemius, a mixed glycolytic/oxidative muscle, the affinity of in situ mitochondria for ADP remains unchanged. The absence of VDAC1 causes multiple defects in respiratory complex activities in both types of muscle. However, in VDAC3 deficient mice the defect is restricted to the heart and only to complex IV. These functional alterations correlate with structural aberrations of mitochondria. These results demonstrate that, unlike VDAC1, there is muscle-type specificity for VDAC3 function and therefore in vivo these two isoforms may fulfill different physiologic functions.
doi:10.1016/j.bbabio.2010.09.007
PMCID: PMC2998388  PMID: 20875390
mitochondrial outer membrane; VDAC; ADP; mitochondrial inner membrane
21.  Structural and functional characterization of the integral membrane protein VDAC-1 in lipid bilayer nanodiscs 
Journal of the American Chemical Society  2009;131(49):17777-17779.
Biophysical studies of membrane proteins are often impeded by the requirement for a membrane mimicking environment. Detergent micelles are the most common choice, but the denaturing properties make them unsatisfactory for studies of many membrane proteins and their interactions. In the present work, we explore phospholipid bilayer nanodiscs as membrane mimics and employ electron microscopy and solution NMR spectroscopy to characterize the structure and function of the human voltage dependent anion channel (VDAC-1) as an example of a polytopic integral membrane protein. Electron microscopy reveals the formation of VDAC-1 multimers, an observation that is consistent with results obtained in native mitochondrial outer membranes. High-resolution NMR spectroscopy demonstrates a well folded VDAC-1 protein and native NADH binding functionality. The observed chemical shift changes upon addition of the native ligand NADH to nanodisc-embedded VDAC-1 resemble those of micelle-embedded VDAC-1, indicating a similar structure and function in the two membrane-mimicking environments. Overall, the ability to study integral membrane proteins at atomic resolution with solution NMR in phospholipid bilayers, rather than in detergent micelles, offers exciting novel possibilities to approach the biophysical properties of membrane proteins under non-denaturing conditions, which makes this technology particular suitable for protein–protein interactions and other functional studies.
doi:10.1021/ja907918r
PMCID: PMC2793270  PMID: 19916553
22.  VDAC1-based peptides: novel pro-apoptotic agents and potential therapeutics for B-cell chronic lymphocytic leukemia 
Cell Death & Disease  2013;4(9):e809-.
The voltage-dependent anion channel 1 (VDAC1), localized in the outer mitochondrial membrane, mediates metabolic cross-talk between the mitochondrion and the cytoplasm and thus serves a fundamental role in cell energy metabolism. VDAC1 also plays a key role in mitochondria-mediated apoptosis, interacting with anti-apoptotic proteins. Resistance of cancer cells to apoptosis involves quenching the mitochondrial apoptotic pathway by over-expression of anti-apoptotic/pro-survival hexokinase (HK) and Bcl-2 family proteins, proteins that mediate their anti-apoptotic activities via interaction with VDAC1. Using specifically designed VDAC1-based cell-penetrating peptides, we targeted these anti-apoptotic proteins to prevent their pro-survival/anti-apoptotic activities. Anti-apoptotic proteins are expressed at high levels in B-cell chronic lymphocytic leukemia (CLL), an incurable disease requiring innovative new approaches to improve therapeutic outcome. CLL is characterized by a clonal accumulation of mature neoplastic B cells that are resistant to apoptosis. Specifically, we demonstrate that the VDAC1-based peptides (Antp-LP4 and N-Terminal-Antp) selectively kill peripheral blood mononuclear cells (PBMCs) obtained from CLL patients, yet spare those obtained from healthy donors. The cell death induction competence of the peptides was well correlated with the amount of double positive CD19/CD5 cancerous CLL PBMCs, further illustrating their selectivity toward cancer cells. Moreover, these VDAC1-based peptides induced apoptosis by activating the mitochondria-mediated pathway, reflected in membrane blebbing, condensation of nuclei, DNA fragmentation, release of mitochondrial cytochrome c, loss of mitochondrial membrane potential, decreased cellular ATP levels and detachment of HK, all leading to apoptotic cell death. Thus, the mode of action of the peptides involves decreasing energy production and inducing apoptosis. Over 27 versions of cell-penetrating VDAC1-based peptides were designed and screened to identify the most stable, short and apoptosis-inducing peptides toward CLL-derived lymphocytes. In this manner, three optimized peptides suitable for in vivo studies were identified. This study thus reveals the potential of VDAC1-based peptides as an innovative and effective anti-CLL therapy.
doi:10.1038/cddis.2013.316
PMCID: PMC3789174  PMID: 24052077
apoptosis; CLL; metabolism; mitochondria; peptides; VDAC1
23.  Molecular and genetic characterization of the gene family encoding the voltage-dependent anion channel in Arabidopsis 
Journal of Experimental Botany  2011;62(14):4773-4785.
The voltage-dependent anion channel (VDAC), a major outer mitochondrial membrane protein, is thought to play an important role in energy production and apoptotic cell death in mammalian systems. However, the function of VDACs in plants is largely unknown. In order to determine the individual function of plant VDACs, molecular and genetic analysis was performed on four VDAC genes, VDAC1–VDAC4, found in Arabidopsis thaliana. VDAC1 and VDAC3 possess the eukaryotic mitochondrial porin signature (MPS) in their C-termini, while VDAC2 and VDAC4 do not. Localization analysis of VDAC–green fluorescent protein (GFP) fusions and their chimeric or mutated derivatives revealed that the MPS sequence is important for mitochondrial localization. Through the functional analysis of vdac knockout mutants due to T-DNA insertion, VDAC2 and VDAC4 which are expressed in the whole plant body are important for various physiological functions such as leaf development, the steady state of the mitochondrial membrane potential, and pollen development. Moreover, it was demonstrated that VDAC1 is not only necessary for normal growth but also important for disease resistance through regulation of hydrogen peroxide generation.
doi:10.1093/jxb/err113
PMCID: PMC3192994  PMID: 21705391
Arabidopsis thaliana; defence response; mitochondrial porin signature; mitochondrial membrane potential; pollen germination; voltage-dependent anion channel
24.  Solution structure of the integral human membrane protein VDAC-1 in detergent micelles** 
Science (New York, N.Y.)  2008;321(5893):1206-1210.
The voltage-dependent anion channel (VDAC) mediates trafficking of small molecules and ions across the eukaryotic outer mitochondrial membrane. VDAC also interacts with anti-apoptotic proteins from the Bcl-2 family and this interaction inhibits release of apoptogenic proteins from the mitochondrion. We present the NMR solution structure of recombinant human VDAC-1 reconstituted in detergent micelles. It forms a 19-stranded β-barrel with the first and last strand parallel. The hydrophobic outside perimeter of the barrel is covered by detergent molecules in a belt-like fashion. In the presence of cholesterol recombinant VDAC-1 can form voltage-gated channels in phospholipid bilayers similar to the native protein. NMR measurements revealed the binding sites of VDAC-1 for the Bcl-2 protein Bcl-xL, for β-NADH and for cholesterol. Bcl-xL interacts with the VDAC barrel laterally at strands 17 and 18.
doi:10.1126/science.1161302
PMCID: PMC2579273  PMID: 18755977
25.  Mcl-1 promotes lung cancer cell migration by directly interacting with VDAC to increase mitochondrial Ca2+ uptake and reactive oxygen species generation 
Cell Death & Disease  2014;5(10):e1482-.
Mcl-1 is an antiapoptotic member of the Bcl-2 family frequently upregulated in non-small cell lung carcinoma (NSCLC). We now report the physiological significance of an interaction between Mcl-1 and the mitochondrial outer membrane-localized voltage-dependent anion channel (VDAC) in NSCLC cell lines. Mcl-1 bound with high affinity to VDAC1 and 3 isoforms but only very weakly to VDAC2 and binding was disrupted by peptides based on the VDAC1 sequence. In A549 cells, reducing Mcl-1 expression levels or application of VDAC-based peptides limited Ca2+ uptake into the mitochondrial matrix, the consequence of which was to inhibit reactive oxygen species (ROS) generation. In A549, H1299 and H460 cells, both Mcl-1 knockdown and VDAC-based peptides attenuated cell migration without affecting cell proliferation. Migration was rescued in Mcl-1 knockdown cells by experimentally restoring ROS levels, consistent with a model in which ROS production drives increased migration. These data suggest that an interaction between Mcl-1 and VDAC promotes lung cancer cell migration by a mechanism that involves Ca2+-dependent ROS production.
doi:10.1038/cddis.2014.419
PMCID: PMC4237246  PMID: 25341036

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