The role of the voltage-dependent anion channel (VDAC) in cell death was investigated using the expression of native and mutated murine VDAC1 in U-937 cells and VDAC inhibitors. Glutamate 72 in VDAC1, shown previously to bind dicyclohexylcarbodiimide (DCCD), which inhibits hexokinase isoform I (HK-I) binding to mitochondria, was mutated to glutamine. Binding of HK-I to mitochondria expressing E72Q-mVDAC1, as compared to native VDAC1, was decreased by B70% and rendered insensitive to DCCD. HK-I and ruthenium red (RuR) reduced the VDAC1 conductance but not that of E72Q-mVDAC1. Overexpression of native or E72Q-mVDAC1 in U-937 cells induced apoptotic cell death (80%). RuR or overexpression of HK-I prevented this apoptosis in cells expressing native but not E72Q-mVDAC1. Thus, a single amino-acid mutation in VDAC prevented HK-I-or RuR-mediated protection against apoptosis, suggesting the direct VDAC regulation of the mitochondria-mediated apoptotic pathway and that the protective effects of RuR and HK-I rely on their binding to VDAC.

In brain and tumor cells, the hexokinase isoforms HK-I and HK-II bind to the voltage-dependent anion channel (VDAC) in the outer mitochondrial membrane. We have previously shown that HK-I decreases murine VDAC1 (mVDAC1) channel conductance, inhibits cytochrome c release, and protects against apoptotic cell death. Now, we define mVDAC1 residues, found in two cytoplasmic domains, involved in the interaction with HK-I. Protection against cell death by HK-I, as induced by overexpression of native or mutated mVDAC1, served to identify the mVDAC1 amino acids required for interaction with HK-I. HK-I binding to mVDAC1 either in isolated mitochondria or reconstituted in a bilayer was inhibited upon mutation of specific VDAC1 residues. HK-I anti-apoptotic activity was also diminished upon mutation of these amino acids. HK-I-mediated inhibition of cytochrome c release induced by staurosporine was also diminished in cells expressing VDAC1 mutants. Our results thus offer new insights into the mechanism by which HK-I promotes tumor cell survival via inhibition of cytochrome c release through HK-I binding to VDAC1. These results, moreover, point to VDAC1 as a key player in mitochondrially mediated apoptosis and implicate an HK-I-VDAC1 interaction in the regulation of apoptosis. Finally, these findings suggest that interference with the binding of HK-I to mitochondria by VDAC1-derived peptides may offer a novel strategy by which to potentiate the efficacy of conventional chemotherapeutic agents.Accumulating evidence indicates that the mitochondrially bound isoforms of hexokinase, HK-I and HK-II, play pivotal roles in promoting cell growth and survival in rapidly growing, highly glycolytic tumors (1). As such, HK-I and HK-II were found to be overexpressed in many types of cancer, including colon, prostate, lymphoma, glioma, gastric adenomas, carcinomas, and breast cancers (2-5). The elevated levels of HK-I and HK-II allow tumor cells to evade apoptosis, thereby allowing proliferation to continue (6, 7). HK-I and HK-II dock onto the cytosolic surface of the outer mitochondrial membrane mainly through binding to the voltage-dependent anion channel (VDAC) 4 (8). It has been proposed that binding of HK to mitochondria allows a continuous ATP flux, providing energy for the phosphorylation of glucose, and thus an increased glycolytic rate (7). VDAC, also known as mitochondrial porin, functions as the major channel allowing passage of nucleotides, ions, Ca 2ϩ , and other metabolites between the intermembrane space and cytoplasm (9 -11).In vitro and in vivo studies have shown that HK-I and HK-II play a clear role in protecting against mitochondrially regulated apoptosis through direct interaction with mitochondria (3) and, more specifically, with VDAC (6). Several recent studies demonstrated that in tumor cells, HK-I (12-14) and HK-II (15, 16) not only augment cellular energy supply and levels of glucose 6-phosphate, an intermediate metabolic in many biosynthetic pathways, but also protect against cell death. The molecular mechanisms by...

Glucose entry into muscle cells is precisely regulated by insulin, through recruitment of GLUT4 (glucose transporter-4) to the membrane of muscle and fat cells. Work done over more than two decades has contributed to mapping the insulin signalling and GLUT4 vesicle trafficking events underpinning this response. In spite of this intensive scientific research, there are outstanding questions that continue to challenge us today. The present review summarizes the knowledge in the field, with emphasis on the latest breakthroughs in insulin signalling at the level of AS160 (Akt substrate of 160 kDa), TBC1D1 (tre-2/USP6, BUB2, cdc16 domain family member 1) and their target Rab proteins; in vesicle trafficking at the level of vesicle mobilization, tethering, docking and fusion with the membrane; and in the participation of the cytoskeleton to achieve optimal temporal and spatial location of insulin-derived signals and GLUT4 vesicles.

Mitochondria play a central role in energy metabolism, Ca2+ signalling, aging and cell death. To control cytosolic or mitochondrial Ca2+ concentration, mitochondria possess several Ca2+-transport systems across the inner membrane. However, the pathway for Ca2+ crossing the outer membrane has not been directly addressed. We report that purified voltage-dependent anion channel (VDAC) reconstituted into lipid bilayers or liposomes is highly permeable to Ca2+. VDAC contains Ca2+-binding sites that bind Ruthenium Red (RuR), La3+ and that RuR completely closed VDACs in single or multichannel experiments. Energized, freshly prepared mitochondria accumulate Ca2+ (500–700nmol/mg of protein), and subsequently released it. The release of Ca2+ is accompanied by cyclosporin A-inhibited swelling, suggesting activation of permeability transition pore (PTP). RuR and ruthenium amine binuclear complex, when added to mitochondria after Ca2+ accumulation has reached a maximal level and before PTP is activated, prevented the release of Ca2+ and the accompanied mitochondrial swelling. RuR also prevented PTP opening promoted by atractyloside, an adenine nucleotide translocase inhibitor. These results suggest that VDAC, located in the mitochondrial outer membrane, controls Ca2+ transport into and from the mitochondria, and that the inhibition of Ca2+ uptake by RuR and La3+ may result from their interaction with VDAC Ca2+-binding sites. Inhibition of PTP opening or assembly by RuR and ruthenium amine binuclear complex suggest the involvement of VDAC in PTP activity and/or regulation. The permeability of VDAC to Ca2+ and its binding of Ca2+, suggest that VDAC has a role in regulation of the mitochondrial Ca2+ homoeostasis.

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.