Genetic demonstration that the plasma membrane maxianion channel and voltage-dependent anion channels are unrelated proteins

The maxianion channel is widely expressed in many cell types, where it fulfills a general physiological function as an ATP-conductive gate for cell-to-cell purinergic signaling. Establishing the molecular identity of this channel is crucial to understanding the mechanisms of regulated ATP release. A mitochondrial porin (voltage-dependent anion channel (VDAC)) located in the plasma membrane has long been considered as the molecule underlying the maxianion channel activity, based upon similarities in the biophysical properties of these two channels and the purported presence of VDAC protein in the plasma membrane. We have deleted each of the three genes encoding the VDAC isoforms individually and collectively and demonstrate that maxianion channel (approximately 400 picosiemens) activity in VDAC-deficient mouse fibroblasts is unaltered. The channel activity is similar in VDAC1/VDAC3-double-deficient cells and in double-deficient cells with the VDAC2 protein depleted by RNA interference. VDAC deletion slightly down-regulated, but never abolished, the swelling-induced ATP release. The lack of correlation between VDAC protein expression and maxianion channel activity strongly argues against the long held hypothesis of plasmalemmal VDAC being the maxianion channel.     

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

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

VDAC is a conserved element of death pathways in plant and animal systems

Programmed cell death (PCD) is very much a part of plant life, although the underlying mechanisms are not so well understood as in animals. In animal cells, the voltage-dependent anion channel (VDAC), a major mitochondrial outer membrane transporter, plays an important role in apoptosis by participating in the release of intermembrane space proteins. To characterize plant PCD pathways by investigating the function of putative components in a mammalian apoptotic context, we have overexpressed a rice VDAC (osVDAC4) in the Jurkat T-cell line. Overexpression of osVDAC4 induces apoptosis, which can be blocked by Bcl-2 and the VDAC inhibitor DIDS. Modifying endogenous VDAC function by DIDS and hexokinase II (HxKII) in Jurkat cells inhibits mitochondria-mediated apoptotic pathways. Finally, we show that DIDS also abrogates heat-induced PCD in cucumber cotyledons. Our data suggest that VDAC is a conserved mitochondrial element of the death machinery in both plant and animal cells.     

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.     

Voltage-dependent anion channel (VDAC) participates in amyloid beta-induced toxicity and interacts with plasma membrane estrogen receptor α in septal and hippocampal neurons

Voltage-dependent anion channel (VDAC) is a porin known by its role in metabolite transport across mitochondria and participation in apoptotic processes. Although traditionally accepted to be located within mitochondrial outer membrane, some data has also reported its presence at the plasma membrane level where it seems to participate in regulation of normal redox homeostasis and apoptosis. Here, exposure of septal SN56 and hippocampal HT22 cells to specific anti-VDAC antibodies prior to amyloid beta (Aβ) peptide was observed to prevent neurotoxicity. In these cell lines, we identified a VDAC form associated with the plasma membrane that seems to be particularly abundant in caveolae. The two membrane-related isoforms of estrogen receptor α (mERα) (80 and 67 kDa), known in SN56 cells to participate in estrogen-induced neuroprotection against Aβ injury, were also observed to be present in caveolae. Interestingly, we demonstrated for the first time that both VDAC and mERα interact at the plasma membrane of these neurons as well as in microsomal fractions of the corresponding murine septal and hippocampal tissues. These proteins were also shown to associate with caveolin-1, thereby corroborating their presence in caveolar microdomains. Taken together, these results suggest that VDAC-mERα association at the plasma membrane level may participate in the modulation of Aβ-induced cell death.     

Voltage-dependent anion channel (VDAC) participates in amyloid beta-induced toxicity and interacts with plasma membrane estrogen receptor alpha in septal and hippocampal neurons

Voltage-dependent anion channel (VDAC) is a porin known by its role in metabolite transport across mitochondria and participation in apoptotic processes. Although traditionally accepted to be located within mitochondrial outer membrane, some data has also reported its presence at the plasma membrane level where it seems to participate in regulation of normal redox homeostasis and apoptosis. Here, exposure of septal SN56 and hippocampal HT22 cells to specific anti-VDAC antibodies prior to amyloid beta (Abeta) peptide was observed to prevent neurotoxicity. In these cell lines, we identified a VDAC form associated with the plasma membrane that seems to be particularly abundant in caveolae. The two membrane-related isoforms of estrogen receptor alpha (mERalpha) (80 and 67 kDa), known in SN56 cells to participate in estrogen-induced neuroprotection against Abeta injury, were also observed to be present in caveolae. Interestingly, we demonstrated for the first time that both VDAC and mERalpha interact at the plasma membrane of these neurons as well as in microsomal fractions of the corresponding murine septal and hippocampal tissues. These proteins were also shown to associate with caveolin-1, thereby corroborating their presence in caveolar microdomains. Taken together, these results suggest that VDAC-mERalpha association at the plasma membrane level may participate in the modulation of Abeta-induced cell death.     

Identification and modulation of a voltage-dependent anion channel in the plasma membrane of guard cells by high-affinity ligands.

Guard cell anion channels (GCAC1) catalyze the release of anions across the plasma membrane during regulated volume decrease and also seem to be involved in the targeting of the plant growth hormones auxins. We have analyzed the modulation and inhibition of these voltage-dependent anion channels by different anion channel blockers. Ethacrynic acid, a structural correlate of an auxin, caused a shift in activation potential and simultaneously a transient increase in the peak current amplitude, whereas other blockers shifted and blocked the voltage-dependent activity of the channel. Comparison of dose-response curves for shift and block imposed by the inhibitor, indicate two different sites within the channel which interact with the ligand. The capability to inhibit GCAC1 increases in a dose-dependent manner in the sequence: probenecid less than A-9-C less than ethacrynic acid less than niflumic acid less than IAA-94 less than NPPB. All inhibitors reversibly blocked the anion channel from the extracellular side. Channel block on the level of single anion channels is characterized by a reduction of long open transitions into flickering bursts, indicating an interaction with the open mouth of the channel. IAA-23, a structural analog of IAA-94, was used to enrich ligand-binding polypeptides from the plasma membrane of guard cells by IAA-23 affinity chromatography. From this protein fraction a 60 kDa polypeptide crossreacted specifically with polyclonal antibodies raised against anion channels isolated from kidney membranes. In contrast to guard cells, mesophyll plasma membranes were deficient in voltage-dependent anion channels and lacked crossreactivity with the antibody.     

Voltage-dependent anion channel as a resident protein of lipid rafts: post-transductional regulation by estrogens and involvement in neuronal preservation against Alzheimer's disease

The voltage-dependent anion channel, VDAC, is present at the neuronal membrane, where it appears to participate, among others, in the extrinsic apoptotic pathway and in the modulation of amyloid-beta induced injury, suggesting the involvement of this channel in Alzheimer's disease (AD) neurotoxicity. VDAC is also highly concentrated in neuronal lipid raft microdomains of different mouse and human cognitive areas, where it has been shown associated with estrogen receptor alpha (ERα), as a part of a `signalosome' that may activate some intracellular signal transduction. At the plasma membrane level, estrogens and antiestrogens (tamoxifen) have been demonstrated to exert rapid antagonist effects on the activation of VDAC, through their distinct effects on the channel post-transductional modulation. Therefore, part of the alternative mechanisms of estrogen related to neuroprotection against amyloid-beta may involve VDAC phosphorylation, in order to maintain the channel in an unactivated (closing) state. Interestingly, VDAC-ERα association has been shown to be disrupted in neuronal lipid rafts of AD brains, in correlation with the aberrant lipid composition observed in these microstructures, suggesting that disturbance of protein interactions may be related to variation in the physico-chemical properties of these microdomains.     

VDAC1 is a transplasma membrane NADH-ferricyanide reductase

Porin isoform 1 or VDAC (voltage-dependent anion-selective channel) 1 is the predominant protein in the outer mitochondrial membrane. We demonstrated previously that a plasma membrane NADH-ferricyanide reductase activity becomes up-regulated upon mitochondrial perturbation, and therefore suggested that it functions as a cellular redox sensor. VDAC1 is known to be expressed in the plasma membrane; however, its function there remained a mystery. Here we show that VDAC1, when expressed in the plasma membrane, functions as a NADH-ferricyanide reductase. VDAC1 preparations purified from both plasma membrane and mitochondria fractions exhibit NADH-ferricyanide reductase activity, which can be immunoprecipitated with poly- and monoclonal antibodies directed against VDAC(1). Transfecting cells with pl-VDAC1-GFP, which carries an N-terminal signal peptide, directs VDAC1 to the plasma membrane, as shown by confocal microscopy and FACS analysis, and significantly increases the plasma membrane NADH-ferricyanide reductase activity of the transfected cells. This novel enzymatic activity of the well known VDAC1 molecule may provide an explanation for its role in the plasma membrane. Our data suggest that a major function of VDAC1 in the plasma membrane is that of a NADH(-ferricyanide) reductase that may be involved in the maintenance of cellular redox homeostasis.     

The voltage-dependent anion channel is a receptor for plasminogen kringle 5 on human endothelial cells

Human plasminogen contains structural domains that are termed kringles. Proteolytic cleavage of plasminogen yields kringles 1-3 or 4 and kringle 5 (K5), which regulate endothelial cell proliferation. The receptor for kringles 1-3 or 4 has been identified as cell surface-associated ATP synthase; however, the receptor for K5 is not known. Sequence homology exists between the plasminogen activator streptokinase and the human voltage-dependent anion channel (VDAC); however, a functional relationship between these proteins has not been reported. A streptokinase binding site for K5 is located between residues Tyr252-Lys283, which is homologous to the primary sequence of VDAC residues Tyr224-Lys255. Antibodies against these sequences react with VDAC and detect this protein on the plasma membrane of human endothelial cells. K5 binds with high affinity (Kd of 28 nm) to endothelial cells, and binding is inhibited by these antibodies. Purified VDAC binds to K5 but only when reconstituted into liposomes. K5 also interferes with mechanisms controlling the regulation of intracellular Ca2+ via its interaction with VDAC. K5 binding to endothelial cells also induces a decrease in intracellular pH and hyperpolarization of the mitochondrial membrane. These studies suggest that VDAC is a receptor for K5.     

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

The voltage-dependent anion channel (VDAC), a major pore-forming protein in the outer membrane of mitochondria, is also found in the plasma membrane of a large number of cells where in addition to its role in regulating cellular ATP release and volume control it is important for maintaining redox homeostasis. Cell surface VDAC is a receptor for plasminogen kringle 5, which promotes partial closure of the channel. In this study, we demonstrate that VDAC binds tissue-type plasminogen activator (t-PA) on human neuroblastoma SK-N-SH cells. Binding of t-PA to VDAC induced a decrease in K_m and an increase in the V_max for activation of its substrate, plasminogen (Pg). This resulted in accelerated Pg activation when VDAC, t-PA, and Pg were bound together. VDAC is also a substrate for plasmin; hence, it mimics fibrin activity. Binding of t-PA to VDAC occurs between a t-PA fibronectin type I finger domain located between amino acids Ile⁵ and Asn³⁷ and a VDAC region including amino acids ²⁰GYGFG²⁴. These VDAC residues correspond to a GXXXG repeat motif commonly found in amyloid β peptides that is necessary for aggregation when these peptides form fibrillar deposits on the cell surface. Furthermore, we also show that Pg kringle 5 is a substrate for the NADH-dependent reductase activity of VDAC. This ternary complex is an efficient proteolytic complex that may facilitate removal of amyloid β peptide deposits from the normal brain and cell debris from injured brain tissue.     

Molecular and genetic characterization of the gene family encoding the voltage-dependent anion channel in Arabidopsis

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.     

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.     

VDAC activation by the 18 kDa translocator protein (TSPO), implications for apoptosis

The voltage dependent anion channel (VDAC), located in the outer mitochondrial membrane, functions as a major channel allowing passage of small molecules and ions between the mitochondrial inter-membrane space and cytoplasm. Together with the adenine nucleotide translocator (ANT), which is located in the inner mitochondrial membrane, the VDAC is considered to form the core of a mitochondrial multiprotein complex, named the mitochondrial permeability transition pore (MPTP). Both VDAC and ANT appear to take part in activation of the mitochondrial apoptosis pathway. Other proteins also appear to be associated with the MPTP, for example, the 18 kDa mitochondrial Translocator Protein (TSPO), Bcl-2, hexokinase, cyclophylin D, and others. Interactions between VDAC and TSPO are considered to play a role in apoptotic cell death. As a consequence, due to its apoptotic functions, the TSPO has become a target for drug development directed to find treatments for neurodegenerative diseases and cancer. In this context, TSPO appears to be involved in the generation of reactive oxygen species (ROS). This generation of ROS may provide a link between activation of TSPO and of VDAC, to induce activation of the mitochondrial apoptosis pathway. ROS are known to be able to release cytochrome c from cardiolipins located at the inner mitochondrial membrane. In addition, ROS appear to be able to activate VDAC and allow VDAC mediated release of cytochrome c into the cytosol. Release of cytochrome c from the mitochondria forms the initiating step for activation of the mitochondrial apoptosis pathway. These data provide an understanding regarding the mechanisms whereby VDAC and TSPO may serve as targets to modulate apoptotic rates. This has implications for drug design to treat diseases such as neurodegeneration and cancer.     

Mapping the ruthenium red-binding site of the voltage-dependent anion channel-1

We have previously shown that ruthenium red (RuR) binds to the voltage-dependent anion channel (VDAC) in the outer mitochondrial membrane, decreasing channel conductance and protecting against apoptotic cell death. In this report, we define the murine and yeast VDAC1 amino acid residues involved in the interaction with RuR. Binding of RuR to bilayer-reconstituted mVDAC1 and the resulting channel closure was inhibited upon mutation of specific VDAC1 residues. RuR protection against cell death, as induced by overexpression of native or mutated mVDAC1, was also diminished upon mutation of these amino acids. Moreover, RuR-mediated inhibition of cytochrome c release normally induced by staurosporine was not observed in cells expressing mutants VDAC1. We found that four glutamate residues, two each located in the first and third mVDAC1 cytosolic loops, are required for the interaction of VDAC1 with RuR and subsequent protection against cell death. Similar results were obtained with Q72E-yeast VDAC1, except that only three glutamate residues, located in two cytosolic loops were required. As a hexavalent reagent, RuR is expected to bind to more than one negatively charged group. Our results thus clearly indicate that RuR protects against cell death via a direct interaction with VDAC1 to inhibit cytochrome c release and subsequent cell death.     

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.     

Characterization and Functional Analysis of Voltage‐Dependent Anion Channel 1 (VDAC1) from Orange‐Spotted Grouper (Epinephelus coioides)

The voltage‐dependent anion channel (VDAC) is a highly conserved integral protein of mitochondria in different eukaryotic species. It forms a selective channel in the mitochondrial outer membrane that serves as the controlled pathway for small metabolites and ions. In this study, a VDAC gene, EcVDAC1, was isolated from orange‐spotted grouper (Epinephelus coioides). The EcVDAC1 exhibits ubiquitous expression in various tissues of orange‐spotted grouper and is upregulated in liver, gill, and spleen after stimulation with lipopolysaccharides (LPS). Subcellular localization analysis shows that the EcVDAC1 protein colocalized with the mitochondria. A caspase‐3 assay demonstrates that overexpression of the EcVDAC1 induced apoptotic cell death in fathead minnow cells. The data presented in this study provide new information regarding the relationship between LPS and the EcVDAC1 gene, suggesting that the fish VDAC1 gene may play an important role in antibacterial immune response.     

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.     

Prostacyclin receptor-mediated ATP release from erythrocytes requires the voltage-dependent anion channel

Erythrocytes have been implicated as controllers of vascular caliber by virtue of their ability to release the vasodilator ATP in response to local physiological and pharmacological stimuli. The regulated release of ATP from erythrocytes requires activation of a signaling pathway involving G proteins (G(i) or G(s)), adenylyl cyclase, protein kinase A, and the cystic fibrosis transmembrane conductance regulator as well as a final conduit through which this highly charged anion exits the cell. Although pannexin 1 has been shown to be the final conduit for ATP release from human erythrocytes in response to reduced oxygen tension, it does not participate in transport of ATP following stimulation of the prostacyclin (IP) receptor in these cells, which suggests that an additional protein must be involved. Using antibodies directed against voltage-dependent anion channel (VDAC)1, we confirm that this protein is present in human erythrocyte membranes. To address the role of VDAC in ATP release, two structurally dissimilar VDAC inhibitors, Bcl-x(L) BH4(4-23) and TRO19622, were used. In response to the IP receptor agonists, iloprost and UT-15C, ATP release was inhibited by both VDAC inhibitors although neither iloprost-induced cAMP accumulation nor total intracellular ATP concentration were altered. Together, these findings support the hypothesis that VDAC is the ATP conduit in the IP receptor-mediated signaling pathway in human erythrocytes. In addition, neither the pannexin inhibitor carbenoxolone nor Bcl-x(L) BH4(4-23) attenuated ATP release in response to incubation of erythrocytes with the β-adrenergic receptor agonist isoproterenol, suggesting the presence of yet another channel for ATP release from human erythrocytes.