Evidence for extra-mitochondrial localization of the VDAC/porin channel in eucaryotic cells

The expression of bacterial porin in outer membranes of gram-negative bacteria and of mitochondrial porin or voltage-dependent anion channel (VDAC) in outer mitochondrial membranes (OMM) of eucaryotic cells was demonstrated about 15 years ago. However, the expression of VDAC in the plasmalemma (PLM) of transformed human B lymphoblasts has recently been indicated by cytotoxicity and indirect immunofluorescence studies. New data suggest that the expression of VDAC may be even more widespread. Different cell types express porin channels in their PLM and in intracellular membranes other than OMM. The functional expression of these channels may differ in the various compartments since recent experiments have demonstrated that the voltage dependence and ion selectivity of mitochondrial VDAC may be altered by their interaction with modulators. The present paper proposes a unifying concept for the ion-selective channels of cell membranes, in particular, those whose regulation is affected in cystic fibrosis.     

Crystallization of the human, mitochondrial voltage-dependent anion-selective channel in the presence of phospholipids.

Overexpressed human voltage-dependent anion-selective channel VDAC or porin from mitochondrial outer membranes has been purified to homogeneity. Electron microscopic analysis of VDAC in detergent solution revealed a uniform particle population consisting of porin monomers. After dialysis of detergent-solubilized porin in the presence of dimyristoylphosphatidylcholine at lipid-to-protein ratios between 0.2 and 0.5 (percentage by weight), mostly multilamellar crystals were obtained. Crystals adsorbed to carbon films flattened during negative staining and air-drying and exhibited different structural features due to differences in the vertical stacking of several crystalline layers, each consisting of one membrane bilayer. Adsorbed, frozen-hydrated multilamellar membrane crystals revealed uniform diffraction patterns with sharp diffraction spots extending to 8.2 A. The surface structure of VDAC was reconstructed from freeze-dried and unidirectionally metal-shadowed crystals. Major protein protrusions were observed from two VDAC monomers present in the unit cell. Differences in the surface structural features indicate alternate orientations of VDAC molecules with respect to the lipid bilayer, allowing the simultaneous imaging of both the cytosolic and intramitochondrial surfaces. Each VDAC molecule consists of a pore lumen with a diameter of 17-20 A surrounded by a protein rim of nonuniform height, suggesting an asymmetrical distribution of protein mass around the diffusion channels.     

The Voltage-dependent Anion Channel in Endoplasmic/Sarcoplasmic Reticulum: Characterization,Modulation and Possible Function

In recent years, it has been recognized that there is a metabolic coupling between the cytosol, ER/SR and mitochondria. In this cross-talk, mitochondrial Ca²⁺ homeostasis and ATP production and supply play a major role. The primary transporter of adenine nucleotides, Ca²⁺and other metabolites into and out of mitochondria is the voltage-dependent anion channel (VDAC) located at the outer mitochondrial membrane, at a crucial position in the cell. VDAC has been established as a key player in mitochondrial metabolite and ion signaling and it has also been proposed that VDAC is present in extramitochondrial membranes. Thus, regulation of VDAC, as the main interface between mitochondrial and cellular metabolism, by other molecules is of utmost importance. This article reviews localization and function of VDAC, and focuses on VDAC as a skeletal muscle sarcoplasmic reticulum channel. The regulation of VDAC activity by associated proteins and by inhibitors is also presented. Several aspects of the physiological relevance of VDAC to Ca²⁺ homeostasis and mitochondria-mediated apoptosis will be discussed.     

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.     

The 'ins' and 'outs' of mitochondrial membrane channels.

The outer membrane of the mitochondrion contains thousands of copies of a pore-forming protein called VDAC or porin. Considerable progress has been made towards elucidating the molecular structure of this channel. Moreover, mounting evidence that the permeability of VDAC may be regulated is challenging the textbook notion of the outer membrane as a simple sieve. Numerous other channel activities have been detected by electrophysiol approaches in both the outer and inner mitochondrial membranes. The inner-membrane channels do not appear to be open under normal physiological conditions and so should not dissipate energy-transducing ion gradients. The biological functions of the different classes of mitochondrial channels are uncertain, but several possibilities (including protein translocation) are being explored.     

VDAC and peripheral channelling complexes in health and disease

VDAC changes its structure either voltage dependent in artificial membranes or physiologically by interaction with the c conformation of the adenine nucleotide translocator (ANT). This interaction creates contact sites and leads to a specific organisation of cytochrome c in the VDAC ANT complexes. The VDAC structure specific for contact sites thus generates a signal at the surface for several proteins in the cytosol to bind with high affinity such as hexokinase, glycerolkinase and Bax. If the VDAC binding site is not occupied by hexokinase, the VDAC ANT complex has two critical qualities: firstly, external Bax gets access to the cytochrome c and secondly the ANT stays in the c conformation that easily changes the structure to an unspecific uni-porter causing permeability transition. Activity of bound hexokinase protects against both, it hinders Bax binding and employs the ANT as specific anti-porter. The octamer of mitochondrial creatine kinase binds to VDAC from the inner surface of the outer membrane. This firstly hinders direct interaction between VDAC and ANT and secondly changes porin structure into low affinity for hexokinase and external Bax. Cytochrome c in the creatine kinase complex will be differently organised not accessible to Bax and the ANT is run as anti-porter by the active octamer. However, when free radicals cause dissociation of the octamer, VDAC interacts with the ANT with the same results as described above: Bax dependent cytochrome c release and risk of permeability transition pore opening.     

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.     

Transmembrane arrangement of mitochondrial porin or voltage-dependent anion channel (VDAC)

Porin or voltage-dependent anion-selective channel (VDAC) is the main protein responsible for the high permeability of the outer mitochondrial membrane. The mitochondrial porin is mainly composed of sided -strands, in analogy with bacterial porin, whose structure has been resolved at 1.8 Å resolution. In mitochondrial porins the N-terminal region forms an amphipathic -helix, a structure conserved in organisms very distant from the evolutionary point of view. This part of the protein is exposed to the water phase, as demonstrated by ELISA assays. Various extramembranous loops have been identified by specific proteolytic cleavages. These overall, combined results were used to draw a model of the transmembrane arrangement of mammalian porin. This model is compared to other mitochondrial and bacterial porin models.     

The VDAC channel as the mitochondria function regulator

Regulation of mitochondria physiology, indispensable for proper cell activity, requires an efficient exchange of molecules between mitochondria and cytoplasm at the level of the mitochondrial outer membrane. The common pathway for the metabolite exchange between mitochondria and cytoplasm is the VDAC channel (voltage dependent anion channel), known also as mitochondrial porin. The channel was identified for the first time in 1976 and since that time has been extensively studied. It has been recognized that the VDAC channel plays a crucial role in the regulation of metabolic and energetic functions of mitochondria. In this article we review the VDAC channel relevance to ATP rationing, Ca2+ homeostasis, protection against oxidative stress and apoptosis execution.     

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.     

Uncovering the role of VDAC in the regulation of cell life and death

Proper cell activity requires an efficient exchange of molecules between mitochondria and cytoplasm. Lying in the outer mitochondrial membrane, VDAC assumes a crucial position in the cell, forming the main interface between the mitochondrial and the cellular metabolisms. As such, it has been recognized that VDAC plays a crucial role in regulating the metabolic and energetic functions of mitochondria. Indeed, down-regulation of VDAC1 expression by shRNA leads to a decrease in energy production and cell growth. VDAC has also been recognized as a key protein in mitochondria-mediated apoptosis through its involvement in the release of apoptotic proteins located in the inter-membranal space and as the proposed target of pro- and anti-apoptotic members of the Bcl2-family and of hexokinase. Questions, however, remain as to if and how VDAC mediates the transfer of apoptotic proteins from the inter-membranal space to the cytosol. The diameter of the VDAC pore is only about 2.5–3 nm, insufficient for the passage of a folded protein like cytochrome c. New work, however, suggests that pore formation involves the assembly of homo-oligomers of VDAC or hetero-oligomers composed of VDAC and pro-apoptotic proteins, such as Bax. Thus, VDAC appears to represent a convergence point for a variety of cell survival and cell death signals. This review provides insight into the central role of VDAC in mammalian cell life and death, emphasizing VDAC function in the regulation of mitochondria-mediated apoptosis and, as such, its potential as a rational target for new therapeutics.     

Neisserial porin (PorB) causes rapid calcium influx in target cells and induces apoptosis by the activation of cysteine proteases.

The porin (PorB) of Neisseria gonorrhoeae is an intriguing bacterial factor owing to its ability to translocate from the outer bacterial membrane into host cell membranes where it modulates the infection process. Here we report on the induction of programmed cell death after prolonged infection of epithelial cells with pathogenic Neisseria species. The underlying mechanism we propose includes translocation of the porin, a transient increase in cytosolic Ca2+ and subsequent activation of the Ca2+ dependent protease calpain as well as proteases of the caspase family. Blocking the porin channel by ATP eliminates the Ca2+ signal and also abolishes its pro-apoptotic function. The neisserial porins share structural and functional homologies with the mitochondrial voltage-dependent anion channels (VDAC). The neisserial porin may be an analogue or precursor of the ancient permeability transition pore, the putative central regulator of apoptosis.     

Phylogenetic and coevolutionary analysis of the β-barrel protein family comprised of mitochondrial porin (VDAC) and Tom40

Beta-barrel proteins are the main transit points across the mitochondrial outer membrane. Mitochondrial porin, the voltage-dependent, anion-selective channel (VDAC), is responsible for the passage of small molecules between the mitochondrion and the cytosol. Through interactions with other mitochondrial and cellular proteins, it is involved in regulating organellar and cellular metabolism and likely contributes to mitochondrial structure. Tom40 is part of the translocase of the outer membrane, and acts as the channel for passage of preproteins during their import into the organelle. These proteins appear to share a common evolutionary origin and structure. In the current study, the evolutionary relationships between and within both proteins were investigated through phylogenetic analysis. The two groups have a common origin and have followed independent, complex evolutionary pathways, leading to the generation of paralogues in animals and plants. Structures of diverse representatives were modeled, revealing common themes rather than sites of high identity in both groups. Within each group, intramolecular coevolution was assessed, revealing a new set of sites potentially involved in structure-function relationships in these molecules. A weak link between Tom40 and proteins related to the mitochondrial distribution and morphology protein, Mdm10, was identified. This article is part of a Special Issue entitled: VDAC structure, function, and regulation of mitochondrial metabolism.     

The role of VDAC in cell death: Friend or foe?

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. This article is part of a Special Issue entitled: VDAC structure, function, and regulation of mitochondrial metabolism.     

Purification and characterization of the voltage-dependent anion channel from the outer mitochondrial membrane of yeast

Summary The outer mitochondrial membranes of all organisms so far examined contain a protein which forms voltage-dependent anion selective channels (VDAC) when incorporated into planar phospholipid membranes. Previous reports have suggested that the yeast (Saccharomyces cerevisiae) outer mitochondrial membrane component responsible for channel formation is a protein of 29,000 daltons which is also the major component of this membrane. In this report, we describe the purification of this 29,000-dalton protein to virtual homogeneity from yeast outer mitochondrial membranes. The purified protein readily incorporates into planar phospholipid membranes to produce ionic channels. Electrophysiological characterization of these channels has demonstrated they have a size, selectivity and voltage dependence similar to VDAC from other organisms. Biochemically, the purified protein has been characterized by determining its amino acid composition and isoelectric point (pI). In addition, we have shown that the purified protein, when reconstituted into liposomes, can bind hexokinase in a glucose-6-phosphate dependent manner, as has been shown for VDAC purified from other sources. Since physiological characterization suggests that the functional parameters of this protein have been conserved, antibodies specific to yeast VDAC have been used to assess antigenic conservation among mitochondrial proteins from a wide number of species. These experiments have shown that yeast VDAC antibodies will recognize single mitochondrial proteins fromDrosophila, Dictyostelium andNeurospora of the appropriate molecular weight to be VDAC from these organisms. No reaction was seen to any mitochondrial protein from rat liver, rainbow trout,Paramecium, or mung bean. In addition, yeast VDAC antibodies will recognize a 50-kDa mol wt protein present in tobacco chloroplasts. These results suggest that there is some antigenic as well as functional conservation among different VDACs.     

A 3D model of the voltage-dependent anion channel (VDAC)

Eukaryotic porins are a group of membrane proteins whose best known role is to form an aqueous pore channel in the mitochondrial outer membrane. As opposed to the bacterial porins (a large family of protein whose 3D structure has been determined by X-ray diffraction), the structure of eukaryotic porins (also termed VDACs, voltage-dependent anion-selective channels) is still a matter of debate. We analysed the secondary structure of VDAC from the yeast Saccharomyces cerevisiae, the fungus Neurospora crassa and the mouse with different types of neural network-based predictors. The predictors were able to discriminate membrane β-strands, globular -helices and membrane -helices and localised, in all three VDAC sequences, 16 β-strands along the chain. For all three sequences the N-terminal region showed a high propensity to form a globular -helix. The 16 β-strand VDAC motif was thus aligned to a bacterial porin-derived template containing a similar 16 β-strand motif. The alignment of the VDAC sequence with the bacterial porin sequence was used to compute a set of 3D coordinates, which constitutes the first 3D prediction of a eukaryotic porin. All the predicted structures assume a β-barrel structure composed of 16 β-strands with the N-terminus outside the membrane. Loops are shorter in this side of the membrane than in the other, where two long loops are protruding. The shape of the pore varies between almost circular for Neurospora and mouse and slightly oval for yeast. Average values between 3 and 2.5 nm at the C-carbon backbone are found for the diameter of the channels. In this model VDAC shows large portions of the structure exposed on both sides of the membrane. The architecture we determine allows speculation about the mechanism of possible interactions between VDAC and other proteins on both sides of the mitochondrial outer membrane. The computed 3D model is consistent with most of the experimental results so far reported.     

Does the voltage dependent anion channel modulate cardiac ischemia-reperfusion injury?

The voltage dependent anion channel (VDAC) provides exchange of metabolites, anions, and cations across the outer mitochondrial membrane. VDAC provides substrates and adenine nucleotides necessary for electron transport and therefore plays a key role in regulating mitochondrial bioenergetics. VDAC has also been suggested to regulate the response to cell death signaling. Emerging data show that VDAC is regulated by protein-protein interactions as well as by post-translational modifications. This review will focus on the regulation of VDAC and its potential role in regulating cell death in cardiac ischemia-reperfusion. This article is part of a Special Issue entitled: VDAC structure, function, and regulation of mitochondrial metabolism.     

The voltage-dependent anion channel as a biological transistor: theoretical considerations

The voltage-dependent anion channel (VDAC) is a porin of the mitochondrial outer membrane with a bell-shaped permeability-voltage characteristic. This porin restricts the flow of negatively charged metabolites at certain non-zero voltages, and thus might regulate their flux across the mitochondrial outer membrane. Here, we have developed a mathematical model illustrating the possibility of interaction between two steady-state fluxes of negatively charged metabolites circulating across the VDAC in a membrane. The fluxes interact by contributing to generation of the membrane electrical potential with subsequent closure of the VDAC. The model predicts that the VDAC might function as a single-molecule biological transistor and amplifier, because according to the obtained calculations a small change in the flux of one pair of different negatively charged metabolites causes a significant modulation of a more powerful flux of another pair of negatively charged metabolites circulating across the same membrane with the VDAC. Such transistor-like behavior of the VDAC in the mitochondrial outer membrane might be an important principle of the cell energy metabolism regulation under some physiological conditions.