Nucleotide Interactions of the Human Voltage-dependent Anion Channel

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

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.     

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.     

VDAC3 gating is activated by suppression of disulfide-bond formation between the N-terminal region and the bottom of the pore

The voltage-dependent anion channels (VDACs), VDAC1, VDAC2, and VDAC3, are pore-forming proteins that control metabolite flux between mitochondria and cytoplasm. VDAC1 and VDAC2 have voltage-dependent gating activity, whereas VDAC3 is thought to have weak activity. The aim of this study was to analyze the channel properties of all three human VDAC isoforms and to clarify the channel function of VDAC3. Bacterially expressed recombinant human VDAC proteins were reconstituted into artificial planar lipid bilayers and their gating activities were evaluated. VDAC1 and VDAC2 had typical voltage-dependent gating activity, whereas the gating of VDAC3 was weak, as reported. However, gating of VDAC3 was evoked by dithiothreitol (DTT) and S-nitrosoglutathione (GSNO), which are thought to suppress disulfide-bond formation. Several cysteine mutants of VDAC3 also exhibited typical voltage-gating. Our results indicate that channel gating was induced by reduction of a disulfide-bond linking the N-terminal region to the bottom of the pore. Thus, channel gating of VDAC3 might be controlled by redox sensing under physiological conditions.     

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.     

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.     

Olesoxime,a cholesterol-like neuroprotectant restrains synaptic vesicle exocytosis in the mice motor nerve terminals: Possible role of VDACs

Olesoxime is a cholesterol-like neuroprotective compound that targets to mitochondrial voltage dependent anion channels (VDACs). VDACs were also found in the plasma membrane and highly expressed in the presynaptic compartment. Here, we studied the effects of olesoxime and VDAC inhibitors on neurotransmission in the mouse neuromuscular junction. Electrophysiological analysis revealed that olesoxime suppressed selectively evoked neurotransmitter release in response to a single stimulus and 20 Hz activity. Also olesoxime decreased the rate of FM1–43 dye loss (an indicator of synaptic vesicle exocytosis) at low frequency stimulation and 20 Hz. Furthermore, an increase in extracellular Cl⁻ enhanced the action of olesoxime on the exocytosis and olesoxime increased intracellular Cl⁻ levels. The effects of olesoxime on the evoked synaptic vesicle exocytosis and [Cl⁻]_i were blocked by membrane-permeable and impermeable VDAC inhibitors. Immunofluorescent labeling pointed on the presence of VDACs on the synaptic membranes. Rotenone-induced mitochondrial dysfunction perturbed the exocytotic release of FM1–43 and cell-permeable VDAC inhibitor (but not olesoxime or impermeable VDAC inhibitor) partially mitigated the rotenone-driven alterations in the FM1–43 unloading and mitochondrial superoxide production. Thus, olesoxime restrains neurotransmission by acting on plasmalemmal VDACs whose activation can limit synaptic vesicle exocytosis probably via increasing anion flux into the nerve terminals.     

Voltage-Dependent Anion Channel Proteins in Synaptosomes of the Torpedo Electric Organ: Immunolocalization, Purification, and Characterization

In this study, we purified and characterized the voltage-dependent anion channel (VDAC) from the Torpedo electric organ. Using immunogold labeling, VDAC was colocalized with the voltage-gated Ca²⁺ channel in the synaptic plasma membrane. By immunoblot analysis, five protein bands in synaptosomes isolated from the Torpedo electric organ cross reacted with two monoclonal anti-VDAC antibody. No more than about 7 to 10% mitochondrial contains could be detected in any synaptosomal membrane preparation tested. This was estimated by comparing the specific activity in mitochondria and synaptosomes of succinate–cytochrome-c oxidoreductase and antimycin-insensitive NADH–cytochrome-c oxidoreductase activities; mitochondrial inner and outer membrane marker enzymes, respectively. [¹⁴C]DCCD (dicyclohexylcarbodiimide), which specifically label mitochondrial VDAC, labeled four 30–35 kDa protein bands that were found to interact with the anti-VDAC antibody. The distribution of the Torpedo VDAC protein bands was different among membranes isolated from various tissues. VDAC was purified from synaptosomes and a separation between two of the proteins was obtained. The two purified proteins were characterized by their single channel activity and partial amino acid sequences. Upon reconstitution into a planar lipid bilayer, the purified VDACs showed voltage-dependent channel activity with properties similar to those of purified mitochondrial VDAC. Amino acid sequence of four peptides, derived from VDAC band II, exhibited high homology to sequences present in human VDAC1 (98%), VDAC2 (91.8%), and VDAC3 (90%), while another peptide, derived from VDAC band III, showed lower homology to either VDAC1 (88.4%) or VDAC2 (79%). Two more peptides show high homology to the sequence present in mouse brain VDAC3 (100 and 78%). In addition, we demonstrate the translocation of ATP into synaptosomes, which is inhibited by DCCD and by the anion transport inhibitor DIDS. The possible function of VDAC in the synaptic plasma membrane is discussed.     

Mitochondrial voltage-dependent anion channels (VDACs) as novel pharmacological targets for anti-cancer agents

Recently, it was demonstrated that some anti-cancer agents used mitochondrial voltage-dependent anion channels (VDAC1–3 isoforms) as their pharmacological target. VDACs are expressed more highly in cancer cells than normal cells; thus the VDAC-dependent cytotoxic agents can have cancer-selectivity. Furanonaphthoquinones (FNQs) induced caspase-dependent apoptosis via the production of NADH-dependent reactive oxygen species (ROS) by VDAC1. The ROS production and the anti-cancer activity of FNQs were increased by VDAC1 overexpression. Meanwhile, erastin induced RAS-RAF-MEK-dependent non-apoptotic cell death via VDAC2. On the other hand, VDACs were needed for transporting ATP to hexokinase (HK), which was highly expressed in cancer cells. We hypothesized that the high glycolysis might induce up-regulation of VDAC. In this review, we propose that VDACs are novel candidates for effective pharmacological targets of anti-cancer drugs.     

High-Level Expression, Refolding and Probing the Natural Fold of the Human Voltage-Dependent Anion Channel Isoforms I and II

The voltage-dependent anion channel (VDAC) is the major protein found in the outer membrane of mitochondria. The channel is responsible for the exchange of ATP/ADP and the translocation of ions and other small metabolites over the membrane. In order to obtain large amounts of pure and suitably folded human VDAC for functional and structural studies, the genes of the human isoforms I and II (HVDAC1 and HVDAC2) were cloned in Escherichia coli. High-level expression led to inclusion body formation. Both proteins could be refolded in vitro by adding denatured protein to a solution of zwitterionic or nonionic detergents. A highly efficient and fast protocol for refolding was developed that yielded more than 50 mg of pure human VDACs per liter of cell culture. The native and functional state of the refolded porins was probed by Fourier transform infrared spectroscopy to determine the secondary structure composition and by electrophysiological measurements, demonstrating the pore-forming activity of HVDAC1. Furthermore, binding of HVDAC1 to immobilized ATP was demonstrated. Limited proteolysis of HVDAC1 protein embedded in detergent micelles in combination with matrix-assisted laser desorption ionization mass spectrometric analysis was applied to identify micelle-exposed regions of the protein and to develop an improved topology model. Our analysis strongly suggests a 16-stranded, antiparallel beta-barrel with one large and seven short loops and turns. Initial crystallization trials of the protein yielded crystals diffracting to 8 Angstrom resolution.     

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.     

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.     

Correct folding of the beta-barrel of the human membrane protein VDAC requires a lipid bilayer

Spontaneous membrane insertion and folding of beta-barrel membrane proteins from an unfolded state into lipid bilayers has been shown previously only for few outer membrane proteins of Gram-negative bacteria. Here we investigated membrane insertion and folding of a human membrane protein, the isoform 1 of the voltage-dependent anion-selective channel (hVDAC1) of mitochondrial outer membranes. Two classes of transmembrane proteins with either alpha-helical or beta-barrel membrane domains are known from the solved high-resolution structures. VDAC forms a transmembrane beta-barrel with an additional N-terminal alpha-helix. We demonstrate that similar to bacterial OmpA, urea-unfolded hVDAC1 spontaneously inserts and folds into lipid bilayers upon denaturant dilution in the absence of folding assistants or energy sources like ATP. Recordings of the voltage-dependence of the single channel conductance confirmed folding of hVDAC1 to its active form. hVDAC1 developed first beta-sheet secondary structure in aqueous solution, while the alpha-helical structure was formed in the presence of lipid or detergent. In stark contrast to bacterial beta-barrel membrane proteins, hVDAC1 formed different structures in detergent micelles and phospholipid bilayers, with higher content of beta-sheet and lower content of alpha-helix when inserted and folded into lipid bilayers. Experiments with mixtures of lipid and detergent indicated that the content of beta-sheet secondary structure in hVDAC1 decreased at increased detergent content. Unlike bacterial beta-barrel membrane proteins, hVDAC1 was not stable even in mild detergents such as LDAO or dodecylmaltoside. Spontaneous folding of outer membrane proteins into lipid bilayers indicates that in cells, the main purpose of membrane-inserted or associated assembly factors may be to select and target beta-barrel membrane proteins towards the outer membrane instead of actively assembling them under consumption of energy as described for the translocons of cytoplasmic membranes.     

The major outer membrane protein of Legionella pneumophila Lpg1974 shows pore-forming characteristics similar to the human mitochondrial outer membrane pore,hVDAC1

Legionella pneumophila is an aerobic and nonspore-forming pathogenic Gram-negative bacterium of the genus Legionella. It is the causative agent of Legionnaires' disease, also known as Legionellosis. The hosts of this organism are diverse, ranging from simple water borne protozoans such as amoebae to more complex hosts such as macrophages in humans. Genome analyses have shown the presence of genes coding for eukaryotic like proteins in several Legionella species. The presence of these proteins may assist L. pneumophila in its adaptation to the eukaryotic host. We studied the characteristics of a protein (Lpg1974) of L. pneumophila that shows remarkable homologies in length of the primary sequence and for the identity/homology of many amino acids to the voltage dependent anion channel (human VDAC1, Porin 31HL) of human mitochondria. Two different forms of Lpg1974 were overexpressed in Escherichia coli and purified to homogeneity: the one containing a putative N-terminal signal sequence and one without it. Reconstituted protein containing the signal sequence formed ion-permeable pores in lipid bilayer membranes with a conductance of approximately 5.4 nS in 1 M KCl. When the predicted N-terminal signal peptide of Lpg1974 comprising an α-helical structure similar to that at the N-terminus of hVDAC1 was removed, the channels formed in reconstitution experiments had a conductance of 7.6 nS in 1 M KCl. Both Lpg1974 proteins formed pores that were voltage-dependent and anion-selective similar to the pores formed by hVDAC1. These results suggest that Lpg1974 of L. pneumophila is indeed a structural and functional homologue to hVDAC1.     

High Resolution Structure and Double Electron-Electron Resonance of the Zebrafish Voltage-dependent Anion Channel 2 Reveal an Oligomeric Population

In recent years, there has been a vast increase in structural and functional understanding of VDAC1, but VDAC2 and -3 have been understudied despite having many unique phenotypes. One reason for the paucity of structural and biochemical characterization of the VDAC2 and -3 isoforms stems from the inability of obtaining purified, functional protein. Here we demonstrate the expression, isolation, and basic characterization of zebrafish VDAC2 (zfVDAC2). Further, we resolved the structure of zfVDAC2 at 2.8 Å resolution, revealing a crystallographic dimer. The dimer orientation was confirmed in solution by double electron-electron resonance spectroscopy and by cross-linking experiments disclosing a dimer population of ∼20% in lauryldimethine amine oxide detergent micelles, whereas in lipidic bicelles a higher population of dimeric and higher order oligomers species were observed. The present study allows for a more accurate structural comparison between VDAC2 and its better-studied counterpart VDAC1.     

VDAC proteomics: Post-translation modifications

Voltage-dependent anion channels are abundant mitochondrial outer membrane proteins expressed in three isoforms, VDAC1-3, and are considered as "mitochondrial gatekeepers". Most tissues express all three isoforms. The functions of VDACs are several-fold, ranging from metabolite and energy exchange to apoptosis. Some of these functions depend on or are affected by interaction with other proteins in the cytosol and intermembrane space. Furthermore, the function of VDACs, as well as their interaction with other proteins, is affected by posttranslational modification, mainly phosphorylation. This review summarizes recent findings on posttranslational modification of VDACs and discusses the physiological outcome of these modifications. This article is part of a Special Issue entitled: VDAC structure, function, and regulation of mitochondrial metabolism.     

Each mammalian mitochondrial outer membrane porin protein is dispensable: effects on cellular respiration.

Voltage-dependent anion channels (VDACs, also known as mitochondrial porins) are small pore-forming proteins of the mitochondrial outer membrane found in all eukaryotes. Mammals harbor three distinct VDAC isoforms, with each protein sharing 65-70% sequence identity. Deletion of the yeast VDAC1 gene leads to conditional lethality that can be partially or completely complemented by the mammalian VDAC genes. In vitro, VDACs conduct a variety of small metabolites and in vivo they serve as a binding site for several cytosolic kinases involved in intermediary metabolism, yet the specific physiologic role of each isoform is unknown. Here we show that mouse embryonic stem cells lacking each isoform are viable but exhibit a 30% reduction in oxygen consumption. VDAC1 and VDAC2 deficient cells exhibit reduced cytochrome c oxidase activity, whereas VDAC3 deficient cells have normal activity. These results indicate that VDACs are not essential for cell viability and we speculate that reduced respiration in part reflects decreased outer membrane permeability for small metabolites necessary for oxidative phosphorylation.     

The BH4 Domain of Anti-apoptotic Bcl-XL,but Not That of the Related Bcl-2, Limits the Voltage-dependent Anion Channel 1 (VDAC1)-mediated Transfer of Pro-apoptotic Ca2+ Signals to Mitochondria

Excessive Ca²⁺ fluxes from the endoplasmic reticulum to the mitochondria result in apoptotic cell death. Bcl-2 and Bcl-XL proteins exert part of their anti-apoptotic function by directly targeting Ca²⁺-transport systems, like the endoplasmic reticulum-localized inositol 1,4,5-trisphosphate receptors (IP₃Rs) and the voltage-dependent anion channel 1 (VDAC1) at the outer mitochondrial membranes. We previously demonstrated that the Bcl-2 homology 4 (BH4) domain of Bcl-2 protects against Ca²⁺-dependent apoptosis by binding and inhibiting IP₃Rs, although the BH4 domain of Bcl-XL was protective independently of binding IP₃Rs. Here, we report that in contrast to the BH4 domain of Bcl-2, the BH4 domain of Bcl-XL binds and inhibits VDAC1. In intact cells, delivery of the BH4-Bcl-XL peptide via electroporation limits agonist-induced mitochondrial Ca²⁺ uptake and protects against staurosporine-induced apoptosis, in line with the results obtained with VDAC1^−/− cells. Moreover, the delivery of the N-terminal domain of VDAC1 as a synthetic peptide (VDAC1-NP) abolishes the ability of BH4-Bcl-XL to suppress mitochondrial Ca²⁺ uptake and to protect against apoptosis. Importantly, VDAC1-NP did not affect the ability of BH4-Bcl-2 to suppress agonist-induced Ca²⁺ release in the cytosol or to prevent apoptosis, as done instead by an IP₃R-derived peptide. In conclusion, our data indicate that the BH4 domain of Bcl-XL, but not that of Bcl-2, selectively targets VDAC1 and inhibits apoptosis by decreasing VDAC1-mediated Ca²⁺ uptake into the mitochondria.     

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.