Phosphorothioate oligonucleotides block the VDAC channel

Proapoptotic phosphorothioate oligonucleotides such as G3139 (an 18-mer) induce Bcl-2-independent apoptosis, perhaps partly via direct interaction with VDAC and reduction of metabolite flow across the mitochondrial outer membrane. Here, we analyzed the interactions at the molecular level. Ten micromolar G3139 induces rapid flickering of the VDAC conductance and, occasionally, a complete conductance drop. These phenomena occur only when VDAC is in the "open" conformation and therefore are consistent with pore blockage rather than VDAC closure. Blockage occurs preferentially from one side of the VDAC channel. It depends linearly on the [G3139] and is voltage-dependent with an effective valence of -3. The kinetics indicate at least a partial entry of G3139 into VDAC, forming an unstable bound state, which is responsible for the rapid flickering (approximately 0.1 ms). Subsequently, a long-lived blocked state is formed. An 8-mer phosphorothioate, polydeoxythymidine, induces partial blockage of VDAC and a change in selectivity from favoring anions to favoring cations. Thus, the oligonucleotide is close to the ion stream. The phosphodiester congener of G3139 is ineffective at the concentrations used, excluding a general polyanion effect. This shows the importance of sulfur atoms. The results are consistent with a binding-induced blockage rather than a permeation block.     

VDAC blockage by phosphorothioate oligonucleotides and its implication in apoptosis

Apoptosis is a crucial process that regulates the homeostasis of multicellular organisms. Impaired apoptosis contributes to cancer development, while enhanced apoptosis is detrimental in neurodegenerative diseases. The intrinsic apoptotic pathway is initiated by cytochrome c release from mitochondria. Research published in the recent decade has suggested that cytochrome c release can be influenced by the conducting states of VDAC, the channel in the mitochondrial outer membrane (MOM) responsible for metabolite flux. This review will describe the evidence that VDAC gating or blockage and subsequent changes in MOM permeability influence cytochrome c release and the onset of apoptosis. The blockage of VDAC by G3139, a proapoptotic phosphorothioate oligonucleotide, provides strong evidence for the role of VDAC in the initiation of apoptosis. The proapoptotic activity and VDAC blockage are linked in that both require the PS (phosphorothioate) modification, both are enhanced by an increase in oligonucleotide length, and both are insensitive to the nucleotide sequence. Thus, the mitochondrial outer membrane permeability regulated by VDAC gating may play an important role in mitochondrial function and in the control of apoptosis. This article is part of a Special Issue entitled: VDAC structure, function, and regulation of mitochondrial metabolism.     

Ethanol exposure decreases mitochondrial outer membrane permeability in cultured rat hepatocytes

Mitochondrial metabolism depends on movement of hydrophilic metabolites through the mitochondrial outer membrane via the voltage-dependent anion channel (VDAC). Here we assessed VDAC permeability of intracellular mitochondria in cultured hepatocytes after plasma membrane permeabilization with 8 μM digitonin. Blockade of VDAC with Koenig’s polyanion inhibited uncoupled and ADP-stimulated respiration of permeabilized hepatocytes by 33% and 41%, respectively. Tenfold greater digitonin (80 μM) relieved KPA-induced inhibition and also released cytochrome c, signifying mitochondrial outer membrane permeabilization. Acute ethanol exposure also decreased respiration and accessibility of mitochondrial adenylate kinase (AK) of permeabilized hepatocytes membranes by 40% and 32%, respectively. This inhibition was reversed by high digitonin. Outer membrane permeability was independently assessed by confocal microscopy from entrapment of 3 kDa tetramethylrhodamine-conjugated dextran (RhoDex) in mitochondria of mechanically permeabilized hepatocytes. Ethanol decreased RhoDex entrapment in mitochondria by 35% of that observed in control cells. Overall, these results demonstrate that acute ethanol exposure decreases mitochondrial outer membrane permeability most likely by inhibition of VDAC.     

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.     

The voltage-dependent anion channel: an essential player in apoptosis

The increase of outer mitochondrial membrane permeability is a central event in apoptotic cell death, since it releases several apoptogenic factors such as cytochrome c into the cytoplasm that activate the downstream destructive processes. The voltage-dependent anion channel (VDAC or mitochondrial porin) plays an essential role in the increase of mitochondrial membrane permeability, and it is regulated by the Bcl-2 family of proteins via direct interaction. Anti-apoptotic Bcl-2 family members close the VDAC, whereas some (but not all) pro-apoptotic members interact with the VDAC to generate a protein-conducting channel through which cytochrome c can pass. Although the VDAC is directly involved in the apoptotic increase of mitochondrial membrane permeability and is known to be a component of the permeability transition pore complex, its role in the regulation of outer membrane permeability can be separated from the occurrence of permeability transition events, such as mitochondrial swelling followed by rupture of the outer mitochondrial membrane. The VDAC not only interacts with Bcl-2 family members, but also with other proteins, and probably acts as a convergence point for a variety of life-or-death signals.     

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.     

Role of voltage-dependent anion channels of the mitochondrial outer membrane in regulation of cell metabolism

The role of voltage-dependent anion channels (VDAC/porins) of the mitochondrial outer membrane in the regulation of cell metabolism is assessed using an experimental model of ethanol toxicity in cultured hepatocytes. It is demonstrated that ethanol inhibits the phosphorylating and the uncoupled mitochondrial respiration, decreases the accessibility of mitochondrial adenylate kinase in the intermembrane space, and suppresses ureagenic respiration in the cells. Treatment with digitonin at high concentrations (>80 μM)—which creates pores in the mitochondrial outer membrane, allowing bypass of closed VDAC—restores all the processes suppressed with ethanol. It is concluded that the effect of ethanol in hepatocytes leads to global loss of mitochondrial function because of closure of VDAC, which limits the free diffusion of metabolites into the intermembrane space. Our studies also reveal the role of VDAC in the regulation of liver-specific intracellular processes such as ureagenesis. The data obtained can be used in development of pharmaceuticals that would prevent VDAC closure in mitochondria of ethanol-oxidizing liver, thus protecting liver tissue from the hepatotoxic action of alcohol.     

The role of the voltage-dependent anion channels in the outer membrane of mitochondria in the regulation of cellular metabolism

The role of the voltage-dependent anion channels (VDAC) harbored in the outer membrane of mitochondria in the regulation of cellular metabolism was investigated using an experimental model of ethanol toxicity in cultured hepatocytes. It was demonstrated that ethanol inhibits State 3 and uncoupled mitochondrial respirations, decreases the accessibility of mitochondrial adenylate kinase localized in the intermembrane space of mitochondria, and suppresses ureagenic respiration and synthesis of urea in cultured hepatocytes. Increasing the permeability of the outer mitochondrial membrane with closed VDAC with high concentrations of digitonin (> 80 microM), which creates pores in the membrane, allowing the alternative bypass of closed VDAC, and restores all reactions suppressed with ethanol. It is concluded that the effect of ethanol in hepatocytes leads to global loss of mitochondrial functions due to the closure of VDAC, which limits the free diffusion of metabolites into the intermembrane space of mitochondria. Our studies demonstrated that ethanol affects the main mitochondrial functions and revealed the role of VDAC channels in the outer mitochondrial membrane in the regulation of liver specific intracellular processes such as ureagenesis. The data obtained can be used for the development of pharmaceutical drugs that prevent the closure of VDAC in mitochondria of ethanol oxidizing liver, thus protecting liver tissue from the hepatotoxic action of alcohol.     

Is mPTP the gatekeeper for necrosis, apoptosis, or both?

Permeabilization of the mitochondrial membranes is a crucial step in apoptosis and necrosis. This phenomenon allows the release of mitochondrial death factors, which trigger or facilitate different signaling cascades ultimately causing the execution of the cell. The mitochondrial permeability transition pore (mPTP) has long been known as one of the main regulators of mitochondria during cell death. mPTP opening can lead to matrix swelling, subsequent rupture of the outer membrane, and a nonspecific release of intermembrane space proteins into the cytosol. While mPTP was purportedly associated with early apoptosis, recent observations suggest that mitochondrial permeabilization mediated by mPTP is generally more closely linked to events of late apoptosis and necrosis. Mechanisms of mitochondrial membrane permeabilization during cell death, involving three different mitochondrial channels, have been postulated. These include the mPTP in the inner membrane, and the mitochondrial apoptosis-induced channel (MAC) and voltage-dependent anion-selective channel (VDAC) in the outer membrane. New developments on mPTP structure and function, and the involvement of mPTP, MAC, and VDAC in permeabilization of mitochondrial membranes during cell death are explored. This article is part of a Special Issue entitled Mitochondria: the deadly organelle.     

Bcl-xL promotes the open configuration of the voltage-dependent anion channel and metabolite passage through the outer mitochondrial membrane

The diffusion of metabolites across the outer mitochondrial membrane is essential for coupled cellular respiration. The outer membrane of mitochondria isolated from growth factor-deprived cells is impaired in its ability to exchange metabolic anions. When added to mitochondria, recombinant Bcl-x(L) restores metabolite exchange across the outer membrane without inducing the loss of cytochrome c from the intermembrane space. Restoration of outer membrane permeability to anionic metabolites does not occur directly through Bcl-x(L) ion channels. Instead, recombinant Bcl-x(L) maintains the outer mitochondrial membrane channel, VDAC, in an open configuration. Consistent with these findings, when ADP-induced oxidative phosphorylation is limited by exogenous beta-NADH, recombinant Bcl-x(L) can sustain outer mitochondrial membrane permeability to ADP. beta-NADH limits respiration by promoting the closed configuration of VDAC. Together these results demonstrate that following an apoptotic signal, Bcl-x(L) can maintain metabolite exchange across the outer mitochondrial membrane by inhibiting VDAC closure.     

Closure of VDAC causes oxidative stress and accelerates the Ca-induced mitochondrial permeability transition in rat liver mitochondria

The electron transport chain of mitochondria is a major source of reactive oxygen species (ROS), which play a critical role in augmenting the Ca²⁺-induced mitochondrial permeability transition (MPT). Mitochondrial release of superoxide anions (O₂⁻) from the intermembrane space (IMS) to the cytosol is mediated by voltage dependent anion channels (VDAC) in the outer membrane. Here, we examined whether closure of VDAC increases intramitochondrial oxidative stress by blocking efflux of O₂⁻ from the IMS and sensitizing to the Ca²⁺-induced MPT. Treatment of isolated rat liver mitochondria with 5 μM G3139, an 18-mer phosphorothioate blocker of VDAC, accelerated onset of the MPT by 6.8 ± 1.4 min within a range of 100-250 μM Ca²⁺. G3139-mediated acceleration of the MPT was reversed by 20 μM butylated hydroxytoluene, a water soluble antioxidant. Pre-treatment of mitochondria with G3139 also increased accumulation of O₂⁻ in mitochondria, as monitored by dihydroethidium fluorescence, and permeabilization of the mitochondrial outer membrane with digitonin reversed the effect of G3139 on O₂⁻ accumulation. Mathematical modeling of generation and turnover of O₂⁻ within the IMS indicated that closure of VDAC produces a 1.55-fold increase in the steady-state level of mitochondrial O₂⁻. In conclusion, closure of VDAC appears to impede the efflux of superoxide anions from the IMS, resulting in an increased steady-state level of O₂⁻, which causes an internal oxidative stress and sensitizes mitochondria toward the Ca²⁺-induced MPT.     

Targeting of the pro-apoptotic VDAC-like porin (PorB) of Neisseria gonorrhoeae to mitochondria of infected cells

Infection of cell cultures with Neisseria gonorrhoeae results in apoptosis that is mediated by the PorB porin. During the infection process porin translocates from the outer bacterial membrane into host cell membranes where its channel activity is regulated by nucleotide binding and voltage-dependent gating, features that are shared by the mitochondrial voltage-dependent anion channel (VDAC). Here we show that porin is selectively and efficiently transported to mitochondria of infected cells. Prevention of porin translocation also blocked the induction of apoptosis. Mitochondria of cells treated with porin both in vitro and in vivo were depleted of cytochrome c and underwent permeability transition. Overexpression of Bcl-2 blocked porin-induced apoptosis. The release of cytochrome c occurred independently of active caspases but was completely prevented by Bcl-2. Our data suggest that the Neisseria porin can, like its eukaryotic homologue, function at the mitochondrial checkpoint to mediate apoptosis.     

Voltage-dependent anion-selective channel 1 (VDAC1)--a mitochondrial protein, rediscovered as a novel enzyme in the plasma membrane

The eukaryotic porin or voltage-dependent anion-selective channel (VDAC1) is a pore-forming protein discovered twenty five years ago in the mitochondrial outer membrane. Its gene in eukaryotes is known, but its tertiary structure has never been solved. Structure predictions highlight the presence of several amphipathic beta-strands possibly organised in a beta-barrel. VDAC1 has recently been described as being a NADH:ferricyanide reductase in the plasma membrane. There it affects the regulation of cell growth and death. Physiological cell death (apoptosis) has become a major research focus of biomedical research. Regulation of the enzyme will have impacts on cancer and autoimmune diseases (insufficient apoptosis) as well as neurodegenerative diseases (excessive apoptosis). VDAC1 in the plasma membrane establishes a novel level of apoptosis regulation putatively via its redox activity.     

Swapping of the N-terminus of VDAC1 with VDAC3 restores full activity of the channel and confers anti-aging features to the cell

Voltage-dependent anion-selective channels (VDACs) are pore-forming proteins allowing the permeability of the mitochondrial outer membrane. The VDAC3 isoform is the least abundant and least active in a complementation assay performed in a yeast strain devoid of porin-1. We swapped the VDAC3 N-terminal 20 amino acids with homologous sequences from the other isoforms. The substitution of the VDAC3 N-terminus with the VDAC1 N-terminus caused the chimaera to become more active than VDAC1. The VDAC2 N-terminus improved VDAC3 activity, though to a lesser extent. The VDAC3 carrying the VDAC1 N-terminus was able to complement the lack of the yeast porin in mitochondrial respiration and in modulation of reactive oxygen species (ROS). This chimaera increased life span, indicating a more efficient bioenergetic metabolism and/or a better protection from ROS.     

The role of the Bcl-2 family in the regulation of outer mitochondrial membrane permeability

Mitochondria are well known as sites of electron transport and generators of cellular ATP. Mitochondria also appear to be sites of cell survival regulation. In the process of programmed cell death, mediators of apoptosis can be released from mitochondria through disruptions in the outer mitochondrial membrane; these mediators then participate in the activation of caspases and of DNA degradation. Thus the regulation of outer mitochondrial membrane integrity is an important control point for apoptosis. The Bcl-2 family is made up of outer mitochondrial membrane proteins that can regulate cell survival, but the mechanisms by which Bcl-2 family proteins act remain controversial. Most metabolites are permeant to the outer membrane through the voltage dependent anion channel (VDAC), and Bcl-2 family proteins appear to be able to regulate VDAC function. In addition, many Bcl-2 family proteins can form channels in vitro, and some pro-apoptotic members may form multimeric channels large enough to release apoptosis promoting proteins from the intermembrane space. Alternatively, Bcl-2 family proteins have been hypothesized to coordinate the permeability of both the outer and inner mitochondrial membranes through the permeability transition (PT) pore. Increasing evidence suggests that alterations in cellular metabolism can lead to pro-apoptotic changes, including changes in intracellular pH, redox potential and ion transport. By regulating mitochondrial membrane physiology, Bcl-2 proteins also affect mitochondrial energy generation, and thus influence cellular bioenergetics. Cell Death and Differentiation (2000) 7, 1182 - 1191     

VDAC regulation by the Bcl-2 family of proteins

The Bcl-2 family of proteins consists of anti-apoptotic and pro-apoptotic members, which determine the life or death of cells by altering mitochondrial membrane permeability. Pro-apoptotic Bcl-2 family members increase mitochondrial membrane permeability, resulting in the release of mitochondrial apoptogenic factors such as cytochrome c that activates death proteases called caspases, whereas anti-apoptotic family members prevent this increase of mitochondrial membrane permeability. The release of cytochrome c is central to apoptotic signal transduction in mammals, and has been studied extensively, leading to the development of several models for cytochrome c release including rupture of the mitochondrial outer membrane and involvement of specific channels. This article describes the important role of a mitochondrial outer membrane channel, the voltage-dependent anion channel (VDAC), in apoptogenic cytochrome c release and its regulation by Bcl-2 family members, and also discusses the molecular architecture of the life - death switch in mammalian cells. Cell Death and Differentiation (2000) 7, 1174 - 1181     

The mitochondrial channel VDAC has a cation-selective open state

The mitochondrial channel VDAC is known to have two major classes of functional states, a large conductance "open" state that is anion selective, and lower conductance substates that are cation selective. The channel can reversibly switch between open and half-open states, with the latter predominant at increasing membrane voltages of either polarity. We report the presence of a new functional state of VDAC, a cation-selective state with conductance approximately equal to that of the canonical open state. This newly described state of VDAC can be reached from either the half-open cation-selective state or from the open anion-selective state. The latter transition implies that a mechanism exists for selectivity gating in VDAC that is separate from partial closure, which may be relevant to the physiological regulation of this channel and mitochondrial outer membrane permeability.     

VDAC closure increases calcium ion flux

VDAC is the major permeability pathway in the mitochondrial outer membrane and can control the flow of metabolites and ions. Therefore Ca²⁺ flux across the outer membrane occurs mainly through VDAC. Since both Ca²⁺ fluxes and VDAC are involved in apoptosis, we examined whether Ca²⁺ is required for channel formation by VDAC isolated from rat liver. The voltage gating of VDAC does not require Ca²⁺ and it functions normally with or without Ca²⁺. Additionally, VDAC generally shows a higher permeability to Ca²⁺ in the closed states (states with lower permeability to metabolites) than that in the open state. Thus VDAC closure, which induces apoptosis, also favors Ca²⁺ flux into mitochondria, which can also lead to permeability transition and cell death. These results are consistent with the view that VDAC closure is a pro-apoptotic signal.     

VDAC closure increases calcium ion flux

VDAC is the major permeability pathway in the mitochondrial outer membrane and can control the flow of metabolites and ions. Therefore Ca(2+) flux across the outer membrane occurs mainly through VDAC. Since both Ca(2+) fluxes and VDAC are involved in apoptosis, we examined whether Ca(2+) is required for channel formation by VDAC isolated from rat liver. The voltage gating of VDAC does not require Ca(2+) and it functions normally with or without Ca(2+). Additionally, VDAC generally shows a higher permeability to Ca(2+) in the closed states (states with lower permeability to metabolites) than that in the open state. Thus VDAC closure, which induces apoptosis, also favors Ca(2+) flux into mitochondria, which can also lead to permeability transition and cell death. These results are consistent with the view that VDAC closure is a pro-apoptotic signal.