VDAC1 serves as a mitochondrial binding site for hexokinase in oxidative muscles

Voltage-dependent anion channels (VDACs), also known as mitochondrial porins, are the main pathway for metabolites across the mitochondrial outer membrane and may serve as binding sites for kinases, including hexokinase. We determined that mitochondria-bound hexokinase activity is significantly reduced in oxidative muscles (heart and soleus) in vdac1(-/-) mice. The activity data were supported by western blot analysis using HK2 specific antibody. To gain more insight into the physiologic mean of the results with the activity data, VDAC deficient mice were subjected to glucose tolerance testing and exercise-induced stress, each of which involves tissue glucose uptake via different mechanisms. vdac1(-/-) mice exhibit impaired glucose tolerance whereas vdac3(-/-) mice have normal glucose tolerance and exercise capacity. Mice lacking both VDAC1 and VDAC3 (vdac1(-/-)/vdac3(-/-)) have reduced exercise capacity together with impaired glucose tolerance. Therefore, we demonstrated a link between VDAC1 mediated mitochondria-bound hexokinase activity and the capacity for glucose clearance.     

Immotile sperm and infertility in mice lacking mitochondrial voltage-dependent anion channel type 3

Voltage-dependent anion channels (VDACs), also known as mitochondrial porins, are small channel proteins involved in the translocation of metabolites across the mitochondrial outer membrane. A single channel-forming protein is found in yeast, whereas higher eukaryotes express multiple VDACs, with humans and mice each harboring three distinct channels (VDAC1-3) encoded by separate genes. To begin to assess the functions of each of the three isoforms, the VDAC3 gene was inactivated by targeted disruption in embryonic stem cells. Here we show that mice lacking VDAC3 are healthy, but males are infertile. Although there are normal sperm numbers, the sperm exhibit markedly reduced motility. Structural defects were found in two-thirds of epididymal axonemes, with the most common abnormality being loss of a single microtubule doublet at a conserved position within the axoneme. In testicular sperm, the defect was only rarely observed, suggesting that instability of a normally formed axoneme occurs with sperm maturation. In contrast, tracheal epithelial cilia showed no structural abnormalities. In addition, skeletal muscle mitochondria were abnormally shaped, and activities of the respiratory chain complexes were reduced. These results demonstrate that axonemal defects may be caused by associated nonaxonemal components such as mitochondrial channels and illustrate that normal mitochondrial function is required for stability of the axoneme.     

Mitochondrial VDAC2 and cell homeostasis: highlighting hidden structural features and unique functionalities

Voltage‐dependent anion channels (VDACs) are the gateway to mitochondrial processes, interlinking the cytosolic and mitochondrial compartments. The mitochondrion acts as a storehouse for cytochrome c, the effector of apoptosis, and hence VDACs become intricately involved in the apoptotic pathway. Isoform 1 of VDAC is abundant in the outer mitochondrial membrane of many cell types, while isoform 2 is the preferred channel in specialized cells including brain and some cancer cells. The primary role of VDACs is metabolite flux. The pro‐ and anti‐apoptotic role of VDAC1 and VDAC2, respectively, are secondary, and are influenced by external factors and interacting proteins. Herein, we focus on the less‐studied VDAC2, and shed light on its unique functions and features. VDAC2, along with sharing many of its functions with VDAC1, such as metabolite and Ca²⁺ transport, also has many delineating functions. VDAC2 is closely engaged in the gametogenesis and steroidogenesis pathways and in protection from oxidative stress as well as in neurodegenerative diseases like Alzheimer's and epilepsy. A closer examination of the functional pathways of VDACs indicates that the unique functions of VDAC2 are a result of the different interactome of this isoform. We couple functional differences to the structural and biophysical evidence obtained for the VDACs, and present a testament of why the two VDAC isoforms with >90% sequence similarity, are functionally diverse. Based on these differences, we suggest that the VDAC isoforms now be considered as paralogs. An in‐depth understanding of VDAC2 will help us to design better biomolecule targets for cancer and neurodegenerative diseases.     

Molecular and cell biology of a family of voltage-dependent anion channel porins in Lotus japonicus

Voltage-dependent anion channels (VDACs) are generally considered as the main pathway for metabolite transport across the mitochondrial outer membrane. Recent proteomic studies on isolated symbiosome membranes from legume nodules indicated that VDACs might also be involved in transport of nutrients between plants and rhizobia. In an attempt to substantiate this, we carried out a detailed molecular and cellular characterization of VDACs in Lotus japonicus and soybean (Glycine max). Database searches revealed at least five genes encoding putative VDACs in each of the legumes L. japonicus, Medicago truncatula, and soybean. We obtained and sequenced cDNA clones from L. japonicus encoding five full-length VDAC proteins (LjVDAC1.1-1.3, LjVDAC2.1, and LjVDAC3.1). Complementation of a yeast (Saccharomyces cerevisiae) mutant impaired in VDAC1, a porin of the mitochondrial outer membrane, showed that LjVDAC1.1, LjVDAC1.2, LjVDAC2.1, and LjVDAC3.1, but not LjVDAC1.3, are functional and targeted to the mitochondrial outer membrane in yeast. Studies of the expression pattern of the five L. japonicus VDAC genes revealed largely constitutive expression of each throughout the plant, including nodules. Antibodies to LjVDAC1.1 of L. japonicus and the related POM36 protein of potato (Solanum tuberosum) recognized several proteins between 30 and 36 kD on western blots, including LjVDAC1.1, LjVDAC1.2, LjVDAC1.3, and LjVDAC2.1. Immunolocalization of VDACs in L. japonicus and soybean root nodules demonstrated their presence on not only mitochondria but also on numerous, small vesicles at the cell periphery. No evidence was found for the presence of VDACs on the symbiosome membrane. Nonetheless, the data indicate that VDACs may play more diverse roles in plants than suspected previously.     

VDAC1 serves as a mitochondrial binding site for hexokinase in oxidative muscles

Voltage-dependent anion channels (VDACs), also known as mitochondrial porins, are the main pathway for metabolites across the mitochondrial outer membrane and may serve as binding sites for kinases, including hexokinase. We determined that mitochondria-bound hexokinase activity is significantly reduced in oxidative muscles (heart and soleus) in vdac1^−/− mice. The activity data were supported by western blot analysis using HK2 specific antibody. To gain more insight into the physiologic mean of the results with the activity data, VDAC deficient mice were subjected to glucose tolerance testing and exercise-induced stress, each of which involves tissue glucose uptake via different mechanisms. vdac1^−/− mice exhibit impaired glucose tolerance whereas vdac3^−/− mice have normal glucose tolerance and exercise capacity. Mice lacking both VDAC1 and VDAC3 (vdac1^−/−/vdac3^−/−) have reduced exercise capacity together with impaired glucose tolerance. Therefore, we demonstrated a link between VDAC1 mediated mitochondria-bound hexokinase activity and the capacity for glucose clearance.     

Functional model of metabolite gating by human voltage-dependent anion channel 2

Voltage-dependent anion channels (VDACs) are critical regulators of outer mitochondrial membrane permeability in eukaryotic cells. VDACs have also been postulated to regulate cell death mechanisms. Erastin, a small molecule quinazolinone that is selectively lethal to tumor cells expressing mutant RAS, has previously been reported as a ligand for hVDAC2. While significant efforts have been made to elucidate the structure and function of hVDAC1, structural and functional characterization of hVDAC2 remains lacking. Here, we present an in vitro system that provides a platform for both functional and structural investigation of hVDAC2 and its small molecule modulator, erastin. Using this system, we found that erastin increases permeability of VDAC2 liposomes to NADH in a manner that requires the amino-terminal region of VDAC2. Furthermore, we confirmed that this VDAC2-lipsome sample is folded using solid-state NMR.     

VDAC as a voltage-dependent mitochondrial gatekeeper under physiological conditions

Mitochondria, composed of two membranes, play a key role in energy production in eukaryotic cells. The main function of the inner membrane is oxidative phosphorylation, while the mitochondrial outer membrane (MOM) seems to control the energy flux and exchange of various charged metabolites between mitochondria and the cytosol. Metabolites cross MOM via the various isoforms of voltage-dependent anion channel (VDAC). In turn, VDACs interact with some enzymes, other proteins and molecules, including drugs. This work aimed to analyze various literature experimental data related to targeting mitochondrial VDACs and VDAC-kinase complexes on the basis of the hypothesis of generation of the outer membrane potential (OMP) and OMP-dependent reprogramming of cell energy metabolism. Our previous model of the VDAC-hexokinase-linked generation of OMP was further complemented in this study with an additional regulation of the MOM permeability by the OMP-dependent docking of cytosolic proteins like tubulin to VDACs. Computational analysis of the model suggests that OMP changes might be involved in the mechanisms of apoptosis promotion through the so-called transient hyperpolarization of mitochondria. The high concordance of the performed computational estimations with many published experimental data allows concluding that OMP generation under physiological conditions is highly probable and VDAC might function as an OMP-dependent gatekeeper of mitochondria, controlling cell life and death. The proposed model of OMP generation allows understanding in more detail the mechanisms of cancer death resistance and anticancer action of various drugs and treatments influencing VDAC voltage-gating properties, VDAC content, mitochondrial hexokinase activity and VDAC-kinase interactions in MOM.     

Genetic strategies for dissecting mammalian and Drosophila voltage-dependent anion channel functions

Voltage-dependent anion channels (VDACs), also known as mitochondrial porins, are a family of small pore-forming proteins of the mitochondrial outer membrane that are found in all eukaryotes. VDACs are thought to play important roles in the regulated flux of metabolites between the cytosolic and mitochondrial compartments, in overall energy metabolism via interactions with cytosolic kinases, and a debated role in programmed cell death (apoptosis). The mammalian genome contains three VDAC loci termed Vdac1, Vdac2, and Vdac3, raising the question as to what function each isoform may be performing. Based upon expression studies of the mouse VDACs in yeast, biophysical differences can be identified but the physiologic significance of these differences remains unclear. Creation of “knockout” cell lines and mice that lack one or more VDAC isoforms has led to the characterization of distinct phenotypes that provide a different set of insights into function which must be interpreted in the context of complex physiologic systems. Functions in male reproduction, the central nervous system and glucose homeostasis have been identified and require a deeper and more mechanistic examination. Annotation of the genome sequence of Drosophila melanogaster has recently revealed three additional genes (CG17137, CG17139, CG17140) with homology to porin, the previously described gene that encodes the VDAC of D. melanogaster. Molecular analysis of these novel VDACs has revealed a complex pattern of gene organization and expression. Sequence comparisons with other insect VDAC homologs suggest that this gene family evolved through a mechanism of duplication and divergence from an ancestral VDAC gene during the radiation of the genus Drosophila. Striking similarities to mouse VDAC mutants can be found that emphasize the conservation of function over a long evolutionary time frame.     

VDAC1, having a shorter N-terminus than VDAC2 but showing the same migration in an SDS-polyacrylamide gel, is the predominant form expressed in mitochondria of various tissues

The voltage-dependent anion channel (VDAC) is a pore-forming protein expressed in the outer membrane of eukaryotic mitochondria. Three isoforms of it, i.e., VDAC1, VDAC2, and VDAC3, are known to be expressed in mammals; however, the question as to which is the main isoform in mitochondria is still unanswered. To address this question, we first prepared standard VDACs by using a bacterial expression system and raised various antibodies against them by using synthetic peptides as immunogens. Of the three bacterially expressed VDAC isoforms, VDAC3 showed faster migration in SDS-polyacrylamide gels than VDAC1 and VDAC2, although VDAC2 is longer than VDAC1 and VDAC3, due to a 12-amino acid extension of its N-terminal region. Even with careful structural characterization of the expressed VDACs by LC-MS/MS analysis, serious structural modifications of VDACs causing changes in their migration in SDS-polyacrylamide gels were not detected. Next, immunoreactivities of the raised antibodies toward these bacterially expressed VDAC isoforms were evaluated. Trials to prepare specific antibodies against the three individual VDAC isoforms were not successful except in the case of VDAC1. However, using a synthetic peptide corresponding to the highly conserved region among the three VDACs, we were successful in preparing an antibody showing essentially equal immunoreactivities toward all three VDACs. When mitochondrial outer membrane proteins of various rat tissues were subjected to 2-dimensional electrophoresis followed by immunoblotting with this antibody, six immunoreactive protein spots were detected. These spots were characterized by LC-MS/MS analysis, and the signal intensities among the spots were compared. As a result, the signal intensity of the spot representing VDAC1 was the highest, and thus, VDAC1 was concluded to be the most abundantly expressed of the three VDAC isoforms in mammalian mitochondria.     

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.     

Voltage-dependant anion channels: Novel insights into isoform function through genetic models

Voltage-dependant Anion Channels, also known as mitochondrial porins, are pore-forming proteins located in the mitochondrial outer membrane (MOM) that, in addition to forming complexes with other proteins that localize to the MOM, also function as the main conduit for transporting metabolites between the cytoplasm and mitochondria. VDACs are encoded by a multi-member gene family, and the number of isoforms and specific functions of VDACs varies between species. Translating the well-described in vitro characteristics of the VDAC isoforms into in vivo functions has been a challenge, with the generation of animal models of VDAC deficiency providing much of the available information about isoform-specific roles in biology. Here, we review the approaches used to create these insect and mammalian animal models, and the conclusions reached by studying the consequences of loss of function mutations on the genetic, physiologic, and biochemical properties of the resulting models. This article is part of a Special Issue entitled: VDAC structure, function, and regulation of mitochondrial metabolism.     

Voltage-dependent anion channels: their roles in plant defense and cell death

The voltage-dependent anion channels (VDACs), mitochondrial outer membrane components, are present in organisms from fungi to animals and plants. They are thought to function in the regulation of metabolite transport between mitochondria and the cytoplasm. Sufficient knowledge on plant VDACs has been accumulated, so that we can here summarize the current information. Then, the involvement of mitochondria in plant defense and cell death is overviewed. While, in mammals, it is suggested that VDAC, also known as a component of the permeability transition pore (PTP) complex formed in the junction site of mitochondrial outer and inner membrane, is a key player in mitochondria-mediated cell death, little is known about the role of plant VDACs in this process. We have shown that plant VDACs are involved in mitochondria-mediated cell death and in defense against a non-host pathogen. In light of the current findings, we discuss the role of the PTP complex and VDAC as its component in plant pathogen defense and cell death.     

Post-translational modifications of VDAC1 and VDAC2 cysteines from rat liver mitochondria

VDACs three isoforms (VDAC1, VDAC2, VDAC3) are integral proteins of the outer mitochondrial membrane whose primary function is to permit the communication and exchange of molecules related to the mitochondrial functions. We have recently reported about the peculiar over-oxidation of VDAC3 cysteines. In this work we have extended our analysis, performed by tryptic and chymotryptic proteolysis and UHPLC/High Resolution ESI-MS/MS, to the other two isoforms VDAC1 and VDAC2 from rat liver mitochondria, and we have been able to find also in these proteins over-oxidation of cysteines. Further PTM of cysteines as succination has been found, while the presence of selenocysteine was not detected. Unfortunately, a short sequence stretch containing one genetically encoded cysteine was not covered both in VDAC2 and in VDAC3, raising the suspect that more, unknown modifications of these proteins exist. Interestingly, cysteine over-oxidation appears to be an exclusive feature of VDACs, since it is not present in other transmembrane mitochondrial proteins eluted by hydroxyapatite. The assignment of a functional role to these modifications of VDACs will be a further step towards the full understanding of the roles of these proteins in the cell.     

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.     

VDAC isoforms in mammals

VDACs (Voltage Dependent Anion selective Channels) are a family of pore-forming proteins discovered in the mitochondrial outer membrane. In the animal kingdom, mammals show a conserved genetic organization of the VDAC genes, corresponding to a group of three active genes. Three VDAC protein isoforms thus exist. From a historically point of view most of the data collected about this protein refer to the VDAC1 isoform, the first to be identified and also the most abundant in the organisms. In this work we compare the information available about the three VDAC isoforms, with a special emphasis upon the human proteins, here considered prototypical of the group, and we try to shed some light on specific functional roles of this apparently redundant group of proteins. A new hypothesis about the VDAC(s) involvement in ROS control is proposed. This article is part of a Special Issue entitled: VDAC structure, function, and regulation of mitochondrial metabolism.     

VDAC isoforms in mammals

VDACs (Voltage Dependent Anion selective Channels) are a family of pore-forming proteins discovered in the mitochondrial outer membrane. In the animal kingdom, mammals show a conserved genetic organization of the VDAC genes, corresponding to a group of three active genes. Three VDAC protein isoforms thus exist. From a historically point of view most of the data collected about this protein refer to the VDAC1 isoform, the first to be identified and also the most abundant in the organisms. In this work we compare the information available about the three VDAC isoforms, with a special emphasis upon the human proteins, here considered prototypical of the group, and we try to shed some light on specific functional roles of this apparently redundant group of proteins. A new hypothesis about the VDAC(s) involvement in ROS control is proposed. This article is part of a Special Issue entitled: VDAC structure, function, and regulation of mitochondrial metabolism.     

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.     

Isoforms of voltage-dependent anion channel of the outer mitochondrial membrane and experimental models to study their physiological role

The review outlines our current understanding of the role of porins, the proteins forming voltage-dependent anion channels (VDAC), in regulation of permeability of the outer mitochondrial membrane. Recent data on the porin structure, amino acid sequence, and isoforms are discussed. The existence of three different VDAC isoforms in mammalian cells suggests that each isoform may play a specific physiological role that remains unknown so far. Different model systems and methods used for studies of functional differences between VDAC isoforms are overviewed. Particular attention is paid to studies of mammalian VDAC isoforms by means of expression of the corresponding genes in yeast and human cells as well as creation of stem cell clones and animals with genetically deficient isoforms of VDAC. It is concluded that permeability of the outer membrane plays a crucial role in the mechanisms of metabolic regulation and that porins are vitally important in the physiology of mammals. The data on the functional role of the VDAC isoforms can be useful for under-standing the mechanisms of such pathologies as cancer, diabetes, and neuromuscular diseases.     

Mouse VDAC Isoforms Expressed in Yeast: Channel Properties and Their Roles in Mitochondrial Outer Membrane Permeability

The channel-forming protein called VDAC forms the major pathway in the mitochondrial outer membrane and controls metabolite flux across that membrane. The different VDAC isoforms of a species may play different roles in the regulation of mitochondrial functions. The mouse has three VDAC isoforms (VDAC1, VDAC2 and VDAC3). These proteins and different versions of VDAC3 were expressed in yeast cells (S. cerevisiae) missing the major yeast VDAC gene and studied using different approaches. When reconstituted into liposomes, each isoform induced a permeability in the liposomes with a similar molecular weight cutoff (between 3,400 and 6,800 daltons based on permeability to polyethylene glycol). In contrast, electrophysiological studies on purified proteins showed very different channel properties. VDAC1 is the prototypic version whose properties are highly conserved among other species. VDAC2 also has normal gating activity but may exist in 2 forms, one with a lower conductance and selectivity. VDAC3 can also form channels in planar phospholipid membranes. It does not insert readily into membranes and generally does not gate well even at high membrane potentials (up to 80 mV). Isolated mitochondria exhibit large differences in their outer membrane permeability to NADH depending on which of the mouse VDAC proteins was expressed. These differences in permeability could not simply be attributed to different amounts of each protein present in the isolated mitochondria. The roles of these different VDAC proteins are discussed. Received: 19 June 1998/Revised: 1 April 1999