Key regions of VDAC1 functioning in apoptosis induction and regulation by hexokinase

The voltage-dependent anion channel (VDAC), located in the mitochondrial outer membrane, functions as gatekeeper for the entry and exit of mitochondrial metabolites, and thus controls cross-talk between mitochondria and the cytosol. VDAC also serves as a site for the docking of cytosolic proteins, such as hexokinase, and is recognized as a key protein in mitochondria-mediated apoptosis. The role of VDAC in apoptosis has emerged from various studies showing its involvement in cytochrome c release and apoptotic cell death as well as its interaction with proteins regulating apoptosis, including the mitochondria-bound isoforms of hexokinase (HK-I, HK-II). Recently, the functional HK-VDAC association has shifted from being considered in a predominantly metabolic light to the recognition of its major impact on the regulation of apoptotic responsiveness of the cell. Here, we demonstrate that the HK-VDAC1 interaction can be disrupted by mutating VDAC1 and by VDAC1-based peptides, consequently leading to diminished HK anti-apoptotic activity, suggesting that disruption of HK binding to VDAC1 can decrease tumor cell survival. Indeed, understanding structure-function relationships of VDAC is critical for deciphering how this channel can perform such a variety of differing functions, all important for cell life and death. By expressing VDAC1 mutants and VDAC1-based peptides, we have identified VDAC1 amino acid residues and domains important for interaction with HK and protection against apoptosis. These include negatively- and positively-charged residues, some of which are located within β-strands of the protein. The N-terminal region of VDAC1 binds HK-I and prevents HK-mediated protection against apoptosis induced by STS, while expression of a VDAC N-terminal peptide detaches HK-I-GFP from mitochondria. These findings indicate that the interaction of HK with VDAC1 involves charged residues in several β-strands and in the N-terminal domain. Displacing HK, serving as the ‘guardian of the mitochondrion’, from its binding site on VDAC1 may thus be exploited as an approach to cancer therapy.     

Hexokinase-I protection against apoptotic cell death is mediated via interaction with the voltage-dependent anion channel-1: mapping the site of binding

In brain and tumor cells, the hexokinase isoforms HK-I and HK-II bind to the voltage-dependent anion channel (VDAC) in the outer mitochondrial membrane. We have previously shown that HK-I decreases murine VDAC1 (mVDAC1) channel conductance, inhibits cytochrome c release, and protects against apoptotic cell death. Now, we define mVDAC1 residues, found in two cytoplasmic domains, involved in the interaction with HK-I. Protection against cell death by HK-I, as induced by overexpression of native or mutated mVDAC1, served to identify the mVDAC1 amino acids required for interaction with HK-I. HK-I binding to mVDAC1 either in isolated mitochondria or reconstituted in a bilayer was inhibited upon mutation of specific VDAC1 residues. HK-I anti-apoptotic activity was also diminished upon mutation of these amino acids. HK-I-mediated inhibition of cytochrome c release induced by staurosporine was also diminished in cells expressing VDAC1 mutants. Our results thus offer new insights into the mechanism by which HK-I promotes tumor cell survival via inhibition of cytochrome c release through HK-I binding to VDAC1. These results, moreover, point to VDAC1 as a key player in mitochondrially mediated apoptosis and implicate an HK-I-VDAC1 interaction in the regulation of apoptosis. Finally, these findings suggest that interference with the binding of HK-I to mitochondria by VDAC1-derived peptides may offer a novel strategy by which to potentiate the efficacy of conventional chemotherapeutic agents.     

The voltage-dependent anion channel-1 modulates apoptotic cell death

The role of the voltage-dependent anion channel (VDAC) in cell death was investigated using the expression of native and mutated murine VDAC1 in U-937 cells and VDAC inhibitors. Glutamate 72 in VDAC1, shown previously to bind dicyclohexylcarbodiimide (DCCD), which inhibits hexokinase isoform I (HK-I) binding to mitochondria, was mutated to glutamine. Binding of HK-I to mitochondria expressing E72Q-mVDAC1, as compared to native VDAC1, was decreased by approximately 70% and rendered insensitive to DCCD. HK-I and ruthenium red (RuR) reduced the VDAC1 conductance but not that of E72Q-mVDAC1. Overexpression of native or E72Q-mVDAC1 in U-937 cells induced apoptotic cell death (80%). RuR or overexpression of HK-I prevented this apoptosis in cells expressing native but not E72Q-mVDAC1. Thus, a single amino-acid mutation in VDAC prevented HK-I- or RuR-mediated protection against apoptosis, suggesting the direct VDAC regulation of the mitochondria-mediated apoptotic pathway and that the protective effects of RuR and HK-I rely on their binding to VDAC.     

Voltage-dependent Anion Channel 1-based Peptides Interact with Bcl-2 to Prevent Antiapoptotic Activity

The antiapoptotic proteins of the Bcl-2 family are expressed at high levels in many types of cancer. However, the mechanism by which Bcl-2 family proteins regulate apoptosis is not fully understood. Here, we demonstrate the interaction of Bcl-2 with the outer mitochondrial membrane protein, voltage-dependent anion channel 1 (VDAC1). A direct interaction of Bcl-2 with bilayer-reconstituted purified VDAC was demonstrated, with Bcl-2 decreasing channel conductance. Expression of Bcl-2-GFP prevented apoptosis in cells expressing native but not certain VDAC1 mutants. VDAC1 sequences and amino acid residues important for interaction with Bcl-2 were defined through site-directed mutagenesis. Synthetic peptides corresponding to the VDAC1 N-terminal region and selected sequences bound specifically, in a concentration- and time-dependent manner, to immobilized Bcl-2, as revealed by the real-time surface plasmon resonance. Moreover, expression of the VDAC1-based peptides in cells over-expressing Bcl-2 prevented Bcl-2-mediated protection against staurosporine-induced apoptotic cell death. Similarly, a cell-permeable VDAC1-based synthetic peptide was also found to prevent Bcl-2-GFP-mediated protection against apoptosis. These results point to Bcl-2 as promoting tumor cell survival through binding to VDAC1, thereby inhibiting cytochrome c release and apoptotic cell death. Moreover, these findings suggest that interfering with the binding of Bcl-2 to mitochondria by VDAC1-based peptides may serve to potentiate the efficacy of conventional chemotherapeutic agents.     

Mediation of the antiapoptotic activity of Bcl-xL protein upon interaction with VDAC1 protein

The mitochondrial protein, the voltage-dependent anion channel (VDAC), is implicated in the control of apoptosis, including via its interaction with the pro- and antiapoptotic proteins. We previously demonstrated the direct interaction of Bcl2 with VDAC, leading to reduced channel conductance. VDAC1-based peptides interacted with Bcl2 to prevent its antiapoptotic activity. Here, using a variety of approaches, we show the interaction of the antiapoptotic protein, Bcl-xL, with VDAC1 and reveal that this interaction mediates Bcl-xL protection against apoptosis. C-terminally truncated Bcl-xL(Δ21) interacts with purified VDAC1, as revealed by microscale thermophoresis and as reflected in the reduced channel conductivity of bilayer-reconstituted VDAC1. Overexpression of Bcl-xL prevented staurosporine-induced apoptosis in cells expressing native VDAC1 but not certain VDAC1 mutants. Having identified mutations in VDAC1 that interfere with the Bcl-xL interaction, certain peptides representing VDAC1 sequences, including the N-terminal domain, were designed and generated as recombinant and synthetic peptides. The VDAC1 N-terminal region and two internal sequences were found to bind specifically, and in a concentration- and time-dependent manner, to immobilized Bcl-xL(Δ21), as revealed by surface plasmon resonance. Moreover, expression of the recombinant peptides in cells overexpressing Bcl-xL prevented protection offered by the protein against staurosporine-induced apoptosis. These results point to Bcl-xL acting as antiapoptotic protein, promoting tumor cell survival via binding to VDAC1. These findings suggest that interfering with Bcl-xL binding to the mitochondria by VDAC1-based peptides may serve to induce apoptosis in cancer cells and to potentiate the efficacy of conventional chemotherapeutic agents.     

Inhibition of Bak Activation by VDAC2 Is Dependent on the Bak Transmembrane Anchor

Bax and Bak are pro-apoptotic factors that are required for cell death by the mitochondrial or intrinsic pathway. Bax is found in an inactive state in the cytosol and upon activation is targeted to the mitochondrial outer membrane where it releases cytochrome c and other factors that cause caspase activation. Although Bak functions in the same way as Bax, it is constitutively localized to the mitochondrial outer membrane. In the membrane, Bak activation is inhibited by the voltage-dependent anion channel isoform 2 (VDAC2) by an unknown mechanism. Using blue native gel electrophoresis, we show that in healthy cells endogenous inactive Bak exists in a 400-kDa complex that is dependent on the presence of VDAC2. Activation of Bak is concomitant with its release from the 400-kDa complex and the formation of lower molecular weight species. Furthermore, substitution of the Bak transmembrane anchor with that of the mitochondrial outer membrane tail-anchored protein hFis1 prevents association of Bak with the VDAC2 complex and increases the sensitivity of cells to an apoptotic stimulus. Our results suggest that VDAC2 interacts with the hydrophobic tail of Bak to sequester it in an inactive state in the mitochondrial outer membrane, thereby raising the stimulation threshold necessary for permeabilization of the mitochondrial outer membrane and cell death.     

Alterations in the mitochondrial proteome of neuroblastoma cells in response to complex 1 inhibition

Increasing evidence points to mitochondrial dysfunction in Parkinson's disease (PD) associated with complex I dysfunction, but the exact pathways which lead to cell death have not been resolved. 2D-gel electrophoresis profiles of isolated mitochondria from neuroblastoma cells treated with subcytotoxic concentrations of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), a well-characterized complex I inhibitor, were assessed to identify associated targets. Up to 27 differentially expressed proteins were observed, of which 16 were identified using peptide mass fingerprinting. Changes in protein levels were validated by immunoprobing 1D blots, confirming increases in heat shock cognate 71 kDa (Hsc70), 60 kDa heat shock protein (Hsp60), fumarase, glutamate oxaloacetate transaminase 2, ATP synthase subunit d, and voltage-dependent anion-channel 1 (VDAC1). Immunoprobing of 2D blots revealed isoform changes in Hsc70, Hsp60, and VDAC1. Subcytotoxic concentrations of MPTP modulated a host of mitochondrial proteins including chaperones, metabolic enzymes, oxidative phosphorylation-related proteins, an inner mitochondrial protein (mitofilin), and an outer mitochondrial membrane protein (VDAC1). Early changes in chaperones suggest a regulated link between complex 1 inhibition and protein folding. VDAC1, a multifunctional protein, may have a key role in signaling between mitochondria and the rest of the cell prior to cell death. Our work provides new important information of relevance to PD.     

VDAC, a multi-functional mitochondrial protein as a pharmacological target

Regulation of mitochondrial physiology requires an efficient exchange of molecules between mitochondria and the cytoplasm via the outer mitochondrial membrane (OMM). The voltage-dependent anion channel (VDAC) lies in the OMM and forms a common pathway for the exchange of metabolites between the mitochondria and the cytosol, thus playing a crucial role in the regulation of metabolic and energetic functions of mitochondria. VDAC is also recognized to function in mitochondria-mediated apoptosis and in apoptosis regulation via interaction with anti-apoptotic proteins, namely members of Bcl-2 family, and the pro-survival protein, hexokinase, overexpressed in many cancer types. Thus, VDAC appears to be a convergence point for a variety of cell survival and cell death signals, mediated by its association with various ligands and proteins. In this article, we review mammalian VDAC, specifically focusing on VDAC1, addressing its functions in cell life and the regulation of apoptosis and its involvement in several diseases. Additionally, we provide insight into the potential of VDAC1 as a rational target for novel therapeutics.     

The voltage dependent anion channel affects mitochondrial cholesterol distribution and function

We have observed abnormally high membrane cholesterol levels and a subsequent deficiency of oxidative energy production in mitochondria from cultured Morris hepatoma cells (MH7777). Using cholesterol affinity chromatography and MALDI-TOF Mass Spectrometry, we have identified the voltage dependent anion channel (VDAC) as a necessary component of a protein complex involved in mitochondrial membrane cholesterol distribution. VDAC is known to associate strongly with hexokinase, particularly in glycolytic cancers. By constructing an E72Q mutant form of VDAC that inhibits its binding of hexokinase, we report an increase in oxidative phosphorylation activity of MH7777 cells, as well as reduced membrane cholesterol ratios to levels near that of normal liver mitochondria. This paper demonstrates that the ability of VDAC to influence mitochondrial membrane cholesterol distribution may have implications on mitochondrial characteristics such as oxidative phosphorylation and induction of apoptosis, as well as the propensity of cancer cells to exhibit a glycolytic phenotype.     

Hexokinase-binding properties of the mitochondrial VDAC protein: Inhibition by DCCD and location of putative DCCD-binding sites

The outer mitochondrial membrane receptor for hexokinase binding has been identified as the VDAC protein, also known as mitochondrial porin. The ability of the receptor to bind hexokinase is inhibited by pretreatment with dicyclohexylcarbodiimide (DCCD). At low concentrations, DCCD inhibits hexokinase binding by covalently labeling the VDAC protein, with no apparent effect on VDAC channel-forming activity. The stoichiometry of [¹⁴C]-DCCD labeling is consistent with one to two high-affinity DCCD-binding sites per VDAC monomer. A comparison between the sequence of yeast VDAC and a conserved sequence found at DCCD-binding sites of several membrane proteins showed two sites where the yeast VDAC amino acid sequence appears to be very similar to the conserved DCCD-binding sequence. Both of these sites are located near the C-terminal end of yeast VDAC (residues 257–265 and 275–283). These results are consistent with a model in which the C-terminal end of VDAC is involved in binding to the N-terminal end of hexokinase.     

Mitochondrial membrane cholesterol,the voltage dependent anion channel (VDAC), and the Warburg effect

Normal cells of aerobic organisms synthesize the energy they require in the form of ATP via the process of oxidative phosphorylation. This complex system resides in the mitochondria of cells and utilizes oxygen to produce a majority of cellular ATP. However, in most tumors, especially those with elevated cholesterogenesis, there is an increased reliance on glycolysis for energy, even in conditions where oxygen is available. This aerobic glycolysis (the Warburg effect) has far reaching ramifications on the tumor itself and the cells that surround it. In this brief review, we will discuss how abnormally high membrane cholesterol levels can result in a subsequent deficiency of oxidative energy production in mitochondria from cultured Morris hepatoma cells (MH-7777). We have identified the voltage dependent anion channel (VDAC) as a necessary component of a protein complex involved in mitochondrial membrane cholesterol distribution and transport. Work in our laboratory demonstrates that the ability of VDAC to influence mitochondrial membrane cholesterol distribution may have implications on mitochondrial characteristics such as oxidative phosphorylation and induction of apoptosis, as well as the propensity of cancer cells to exhibit a glycolytic phenotype.     

Strategies for maximizing ATP supply in the microsporidian Encephalitozoon cuniculi: direct binding of mitochondria to the parasitophorous vacuole and clustering of the mitochondrial porin VDAC

Microsporidia are obligate intracellular parasites with extremely reduced genomes and a dependence on host‐derived ATP. The microsporidium Encephalitozoon cuniculi proliferates within a membranous vacuole and we investigated how the ATP supply is optimized at the vacuole–host interface. Using spatial EM quantification (stereology), we found a single layer of mitochondria coating substantial proportions of the parasitophorous vacuole. Mitochondrial binding occurred preferentially over the vegetative ‘meront’ stages of the parasite, which bulged into the cytoplasm, thereby increasing the membrane surface available for mitochondrial interaction. In a broken cell system mitochondrial binding was maintained and was typified by electron dense structures (< 10 nm long) bridging between outer mitochondrial and vacuole membranes. In broken cells mitochondrial binding was sensitive to a range of protease treatments. The function of directly bound mitochondria, as measured by the membrane potential sensitive dye JC‐1, was indistinguishable from other mitochondria in the cell although there was a generalized depression of the membrane potential in infected cells. Finally, quantitative immuno‐EM revealed that the ATP‐delivering mitochondrial porin, VDAC, was concentrated atthe mitochondria‐vacuole interaction site. Thus E. cuniculi appears to maximize ATP supply by direct binding of mitochondria to the parasitophorous vacuole bringing this organelle within 0.020 microns of the growing vegetative form of the parasite. ATP‐delivery is further enhanced by clustering of ATP transporting porins in those regions of the outer mitochondrial membrane lying closest to the parasite.     

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.     

Direct modulation of the outer mitochondrial membrane channel,voltage-dependent anion channel 1 (VDAC1) by cannabidiol: a novel mechanism for cannabinoid-induced cell death

Cannabidiol (CBD) is a non-psychoactive plant cannabinoid that inhibits cell proliferation and induces cell death of cancer cells and activated immune cells. It is not an agonist of the classical CB1/CB2 cannabinoid receptors and the mechanism by which it functions is unknown. Here, we studied the effects of CBD on various mitochondrial functions in BV-2 microglial cells. Our findings indicate that CBD treatment leads to a biphasic increase in intracellular calcium levels and to changes in mitochondrial function and morphology leading to cell death. Density gradient fractionation analysis by mass spectrometry and western blotting showed colocalization of CBD with protein markers of mitochondria. Single-channel recordings of the outer-mitochondrial membrane protein, the voltage-dependent anion channel 1 (VDAC1) functioning in cell energy, metabolic homeostasis and apoptosis revealed that CBD markedly decreases channel conductance. Finally, using microscale thermophoresis, we showed a direct interaction between purified fluorescently labeled VDAC1 and CBD. Thus, VDAC1 seems to serve as a novel mitochondrial target for CBD. The inhibition of VDAC1 by CBD may be responsible for the immunosuppressive and anticancer effects of CBD.     

Regulation of mitochondrial function by voltage dependent anion channels in ethanol metabolism and the Warburg effect

Voltage dependent anion channels (VDAC) are highly conserved proteins that are responsible for permeability of the mitochondrial outer membrane 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 metabolism in apoptotic cells and also in cells that are not dying. During hepatic ethanol oxidation to acetaldehyde, VDAC closure suppresses exchange of mitochondrial metabolites, resulting in inhibition of ureagenesis. In vivo, VDAC closure after ethanol occurs coordinately with mitochondrial uncoupling. Since acetaldehyde passes through membranes independently of channels and transporters, VDAC closure and uncoupling together foster selective and more rapid oxidative metabolism of toxic acetaldehyde to nontoxic acetate by mitochondrial aldehyde dehydrogenase. In single reconstituted VDAC, tubulin decreases VDAC conductance, and in HepG2 hepatoma cells, free tubulin negatively modulates mitochondrial membrane potential, an effect enhanced by protein kinase A. Tubulin-dependent closure of VDAC in cancer cells contributes to suppression of mitochondrial metabolism and may underlie the Warburg phenomenon of aerobic glycolysis. This article is part of a Special Issue entitled: VDAC structure, function, and regulation of mitochondrial metabolism.     

An N-terminal nucleotide-binding site in VDAC1: involvement in regulating mitochondrial function

In a previous study, we presented evidence for the existence of a nucleotide-binding site (NBS) in the N-terminal region of the voltage-dependent anion channel (VDAC1). In this study, further localization and possible roles of the proposed VDAC1-NBS were investigated using site-directed mutagenesis. The predicated NBS of murine VDAC1 (mVDAC1) was mutated by replacing two glycine residues with alanines or a conserved lysine residue with a serine. Expression of the G21A,G23A- and K20S-mVDAC1s in human T-REx-293 cells in which endogenous VDAC1 expression had been silenced affected cell growth and cytosolic ATP levels. While G21A,G23A-mVDAC1-expressing cells displayed growth rates similar to native-mVDAC1-expressing cells, the K20S-mVDAC1-expressing cells displayed significantly retarded growth and increased resistance to cell death. Cells expressing either mVDAC1 mutant also displayed significantly reduced cellular ATP levels. When K20S-mutant mVDAC1 was expressed in porin1-less yeast, the transformed cells grew slower on non-fermentable carbon sources, while isolated mitochondria expressing either mVDAC1 mutant showed significant reduction in ATP synthesis. Purified K20S-mVDAC1 displayed a significant decrease in [alpha-(32)P]BzATP-binding and altered channel properties, that is, reduced ion selectivity, while the G21A,G23A-mutant protein displayed only a mild reduction in channel selectivity. These results suggest that mutations in the proposed VDAC1-NBS, particularly the K20S, altered channel activity, thereby interfering with VDAC function as the major pathway for the transport of metabolites and adenine nucleotides across the outer mitochondrial membrane. Finally, involvement of the VDAC1-NBS in the control of mitochondrial ATP synthesis, cell growth and viability is discussed.     

VDAC1-based peptides: novel pro-apoptotic agents and potential therapeutics for B-cell chronic lymphocytic leukemia

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.     

NMR structural investigation of the mitochondrial outer membrane protein VDAC and its interaction with antiapoptotic Bcl-xL

Bcl-2 family proteins are essential regulators of cell death and exert their primary pro- or antiapoptotic roles at the mitochondrial outer membrane. Previously, pro- and antiapoptotic 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 antiapoptotic 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 one or two VDAC molecules forming heterodimers and heterotrimers. Our characterization of the VDAC/Bcl-xL complex offers initial structural insight into the role of antiapoptotic Bcl-xL in regulating apoptotic events in the mitochondrial outer membrane.     

Nucleotide Interactions of the Human Voltage-dependent Anion Channel

The voltage-dependent anion channel (VDAC) mediates and gates the flux of metabolites and ions across the outer mitochondrial membrane and is a key player in cellular metabolism and apoptosis. Here we characterized the binding of nucleotides to human VDAC1 (hVDAC1) on a single-residue level using NMR spectroscopy and site-directed mutagenesis. We find that hVDAC1 possesses one major binding region for ATP, UTP, and GTP that partially overlaps with a previously determined NADH binding site. This nucleotide binding region is formed by the N-terminal α-helix, the linker connecting the helix to the first β-strand and adjacent barrel residues. hVDAC1 preferentially binds the charged forms of ATP, providing support for a mechanism of metabolite transport in which direct binding to the charged form exerts selectivity while at the same time permeation of the Mg²⁺-complexed ATP form is possible.     

Bcl-2 family of proteins: life-or-death switch in mitochondria

An increase in the permeability of outer mitochondrial membrane is central to apoptotic cell death, and results in the release of several apoptogenic factors such as cytochrome c into the cytoplasm to activate downstream destructive programs. The voltage-dependent anion channel (VDAC or mitochondrial porin) plays an essential role in disrupting the mitochondrial membrane barrier and is regulated directly by members of the Bcl-2 family proteins. Anti-apoptotic Bcl-2 family members interact with and close the VDAC, whereas some, but not all, proapoptotic members interact with VDAC to open protein-conducting pore through which apoptogenic factors pass. Although the VDAC is involved directly in breaking the mitochondrial membrane barrier and is a known component of the permeability transition pore complex, VDAC-dependent increase in outer membrane permeability can be independent of the permeability transition event such as mitochondrial swelling followed by rupture of the outer mitochondrial membrane. VDAC interacts not only with Bcl-2 family members but also with proteins such as gelsolin, an actin regulatory protein, and appears to be a convergence point for a variety of cell survival and cell death signals.