Electrophysiological study of a novel large pore formed by Bax and the voltage-dependent anion channel that is permeable to cytochrome c

The Bcl-2 family of proteins, consisting of anti-apoptotic and pro-apoptotic members, regulates cell death by controlling mitochondrial membrane permeability that is crucial for apoptotic signal transduction. We have recently shown that some of these proteins, such as Bcl-x(L), Bax, and Bak, directly modulate the mitochondrial voltage-dependent anion channel (VDAC) and thus regulate apoptogenic cytochrome c release and potential loss. To elucidate the molecular mechanisms of VDAC regulation by Bcl-2 family proteins, an electrophysiological study was carried out. It was found that VDAC and pro-apoptotic Bax created a large pore, with conductance levels 4- and 10-fold greater than those of the VDAC and Bax channels, respectively. Although the VDAC and Bax channels both show ion selectivity and voltage-dependent modulation of their activity, the VDAC-Bax channel had neither of their properties. Anti-apoptotic Bcl-x(L) and its BH4 oligopeptide completely closed the VDAC, in contrast to the Bax. Cytochrome c passed through a single VDAC-Bax channel but not through the VDAC or Bax channel in a planar lipid bilayer. These data provide direct evidence that VDAC forms a novel large pore together with Bax.     

The endoplasmic reticulum gateway to apoptosis by Bcl-X(L) modulation of the InsP3R

Members of the Bcl-2 protein family modulate outer mitochondrial membrane permeability to control apoptosis. However, these proteins also localize to the endoplasmic reticulum (ER), the functional significance of which is controversial. Here we provide evidence that anti-apoptotic Bcl-2 proteins regulate the inositol 1,4,5-trisphosphate receptor (InsP(3)R) ER Ca(2+) release channel resulting in increased cellular apoptotic resistance and enhanced mitochondrial bioenergetics. Anti-apoptotic Bcl-X(L) interacts with the carboxyl terminus of the InsP(3)R and sensitizes single InsP(3)R channels in ER membranes to low [InsP(3)], enhancing Ca(2+) and InsP(3)-dependent regulation of channel activity in vitro and in vivo, reducing ER Ca(2+) content and stimulating mitochondrial energetics. The pro-apoptotic proteins Bax and tBid antagonize this effect by blocking the biochemical interaction of Bcl-X(L) with the InsP(3)R. These data support a novel model in which Bcl-X(L) is a direct effector of the InsP(3)R, increasing its sensitivity to InsP(3) and enabling ER Ca(2+) release to be more sensitively coupled to extracellular signals. As a consequence, cells are protected against apoptosis by a more sensitive and dynamic coupling of ER to mitochondria through Ca(2+)-dependent signal transduction that enhances cellular bioenergetics and preserves survival.     

PUMA Dissociates Bax and Bcl-X(L) to induce apoptosis in colon cancer cells

PUMA is a BH3-only Bcl-2 family protein that plays an essential role in DNA damage-induced apoptosis. PUMA interacts with anti-apoptotic Bcl-2 and Bcl-X(L) and is dependent on Bax to induce apoptosis. In this study, we investigated how the interactions of PUMA with the antiapoptotic proteins coordinate with Bax to initiate apoptosis in HCT116 colon cancer cells. We found that Bcl-X(L) was most effective among several antiapoptotic proteins in suppressing PUMA-induced apoptosis and PUMA-dependent apoptosis induced by the DNA-damaging agent adriamycin. Mutant Bcl-X(L) that cannot interact with Bax was unable to protect cells from PUMA-mediated apoptosis. Knockdown of Bcl-X(L) by RNA interference significantly enhanced PUMA-mediated apoptosis in HCT116 cells but not in PUMA-knockout cells. Furthermore, Bax was found to be dissociated preferentially from Bcl-X(L) in HCT116 cells but not in the PUMA-knockout cells, in response to PUMA induction and adriamycin treatment. PUMA inhibited the association of Bax and Bcl-X(L) in vitro by directly binding to Bcl-X(L) through its BH3 domain. Finally, we found that wild-type Bax, but not mutant Bax deficient in either multimerization or mitochondrial localization, was able to restore PUMA-induced apoptosis in the BAX-knockout cells. Together, these results indicate that PUMA initiates apoptosis in part by dissociating Bax and Bcl-X(L), thereby promoting Bax multimerization and mitochondrial translocation.     

Bcl-X(L) and calyculin A prevent translocation of Bax to mitochondria during apoptosis

During many forms of apoptosis, Bax, a pro-apoptotic protein of the Bcl-2 family, translocates from the cytosol to the mitochondria and induces cytochrome c release, followed by caspase activation and DNA degradation. Both Bcl-X(L) and the protein phosphatase inhibitor calyculin A have been shown to prevent apoptosis, and here we investigated their impact on Bax translocation. ML-1 cells incubated with either anisomycin or staurosporine exhibited Bax translocation, cytochrome c release, caspase 8 activation, and Bid cleavage; only the latter two events were caspase-dependent, confirming that they are consequences in this apoptotic pathway. Both Bcl-X(L) and calyculin A prevented Bax translocation and cytochrome c release. Bcl-X(L) is generally thought to heterodimerize with Bax to prevent cytochrome c release and yet they remain in different cellular compartments, suggesting that their heterodimerization at the mitochondria is not the primary mechanism of Bcl-X(L)-mediated protection. Using chemical cross-linking agents, Bax appeared to exist as a monomer in undamaged cells. Upon induction of apoptosis, Bax formed homo-oligomers in the mitochondrial fraction with no evidence for cross-linking to Bcl-2 or Bcl-X(L). Considering that both Bcl-X(L) and calyculin A inhibit Bax translocation, we propose that Bcl-X(L) may regulate Bax translocation through modulation of protein phosphatase or kinase signaling.     

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     

Differential regulation of Bax and Bak by anti-apoptotic Bcl-2 family proteins Bcl-B and Mcl-1

The pro-apoptotic members of the Bcl-2 family include initiator proteins that contain only BH3 domains and downstream effector multi-BH domain-containing proteins, including Bax and Bak. In this report, we compared the ability of the six human anti-apoptotic Bcl-2 family members to suppress apoptosis induced by overexpression of Bax or Bak, correlating findings with protein interactions measured by three different methods: co-immunoprecipitation, glutathione S-transferase pulldown, and fluorescence polarization assays employing synthetic BH3 peptides from Bax and Bak. Bcl-B and Mcl-1 showed strong preferences for binding to and suppression of Bax and Bak, respectively. In contrast, the other anti-apoptotic Bcl-2 family proteins (Bcl-2, Bcl-X(L), Bcl-W, and Bfl-1) suppressed apoptosis induced by overexpression of either Bax or Bak, and they displayed an ability to bind both Bax and Bak by at least one of the three protein interaction methods. Interestingly, however, full-length Bax and Bak proteins and synthetic Bax and Bak BH3 peptides exhibited discernible differences in their interactions with some anti-apoptotic members of the Bcl-2 family, cautioning against reliance on a single method for detecting protein interactions of functional significance. Altogether, the findings reveal striking distinctions in the behaviors of Bcl-B and Mcl-1 relative to the other anti-apoptotic Bcl-2 family members, where Bcl-B and Mcl-1 display reciprocal abilities to bind and neutralize Bax and Bak.     

Mitochondrially targeted Bcl-2 and Bcl-X(L) chimeras elicit different apoptotic responses

The Bcl-2 family of proteins interacts at the mitochondria to regulate apoptosis. However, the anti-apoptotic Bcl-2 and Bcl-X(L) are not completely localized to the mitochondria. In an attempt to generate Bcl-2 and Bcl-X(L) chimeras that are constitutively localized to the mitochondria, we substituted their C-terminal transmembrane tail or both the C-terminal transmembrane tail and the adjacent loop with the equivalent regions from Bak or Bax mutant (BaxS184V) as these regions determine the mitochondrial localization of Bak and Bax. The effects of these substitutions on subcellular localization and their activities were assessed following expression in HeLa and CHO K1 cells. The substitution of the C-terminal tail or the C-terminal tail and the adjacent loop of Bcl-2 with the equivalent regions from Bak or the Bax mutant resulted in its association with the mitochondria. This change in subcellular localization of Bcl-2 chimeras triggered cells to undergo apoptotic-like cell death. The localization of this Bcl-2 chimera to the mitochondria may be associated with the disruption of mitochondrial membrane potential. Unlike Bcl-2, the loop structure adjacent to the C-terminal tail in Bcl-X(L) is crucial for its localization. To localize the Bcl-X(L) chimeras to the mitochondria, the loop structure next to the C-terminal tail in Bcl-X(L) protein must remain intact and cannot be substituted by the loop from Bax or Bak. The chimeric Bcl-X(L) with both its C-terminal tail and the loop structure replaced by the equivalent regions of Bak or Bax mutant localized throughout the entire cytosol. The Bcl-X(L) chimeras that are targeted to the mitochondria and the wild type Bcl-X(L) provided same protection against cell death under several death inducing conditions.     

The BH3 alpha-helical mimic BH3-M6 disrupts Bcl-X(L), Bcl-2, and MCL-1 protein-protein interactions with Bax, Bak, Bad, or Bim and induces apoptosis in a Bax- and Bim-dependent manner

A critical hallmark of cancer cell survival is evasion of apoptosis. This is commonly due to overexpression of anti-apoptotic proteins such as Bcl-2, Bcl-X(L), and Mcl-1, which bind to the BH3 α-helical domain of pro-apoptotic proteins such as Bax, Bak, Bad, and Bim, and inhibit their function. We designed a BH3 α-helical mimetic BH3-M6 that binds to Bcl-X(L) and Mcl-1 and prevents their binding to fluorescently labeled Bak- or Bim-BH3 peptides in vitro. Using several approaches, we demonstrate that BH3-M6 is a pan-Bcl-2 antagonist that inhibits the binding of Bcl-X(L), Bcl-2, and Mcl-1 to multi-domain Bax or Bak, or BH3-only Bim or Bad in cell-free systems and in intact human cancer cells, freeing up pro-apoptotic proteins to induce apoptosis. BH3-M6 disruption of these protein-protein interactions is associated with cytochrome c release from mitochondria, caspase-3 activation and PARP cleavage. Using caspase inhibitors and Bax and Bak siRNAs, we demonstrate that BH3-M6-induced apoptosis is caspase- and Bax-, but not Bak-dependent. Furthermore, BH3-M6 disrupts Bcl-X(L)/Bim, Bcl-2/Bim, and Mcl-1/Bim protein-protein interactions and frees up Bim to induce apoptosis in human cancer cells that depend for tumor survival on the neutralization of Bim with Bcl-X(L), Bcl-2, or Mcl-1. Finally, BH3-M6 sensitizes cells to apoptosis induced by the proteasome inhibitor CEP-1612.     

Essential role of voltage-dependent anion channel in various forms of apoptosis in mammalian cells

Through direct interaction with the voltage-dependent anion channel (VDAC), proapoptotic members of the Bcl-2 family such as Bax and Bak induce apoptogenic cytochrome c release in isolated mitochondria, whereas BH3-only proteins such as Bid and Bik do not directly target the VDAC to induce cytochrome c release. To investigate the biological significance of the VDAC for apoptosis in mammalian cells, we produced two kinds of anti-VDAC antibodies that inhibited VDAC activity. In isolated mitochondria, these antibodies prevented Bax-induced cytochrome c release and loss of the mitochondrial membrane potential (Deltapsi), but not Bid-induced cytochrome c release. When microinjected into cells, these anti-VDAC antibodies, but not control antibodies, also prevented Bax-induced cytochrome c release and apoptosis, whereas the antibodies did not prevent Bid-induced apoptosis, indicating that the VDAC is essential for Bax-induced, but not Bid-induced, apoptogenic mitochondrial changes and apoptotic cell death. In addition, microinjection of these anti-VDAC antibodies significantly inhibited etoposide-, paclitaxel-, and staurosporine-induced apoptosis. Furthermore, we used these antibodies to show that Bax- and Bak-induced lysis of red blood cells was also mediated by the VDAC on plasma membrane. Taken together, our data provide evidence that the VDAC plays an essential role in apoptogenic cytochrome c release and apoptosis in mammalian cells.     

Mouse uterine epithelial apoptosis is associated with expression of mitochondrial voltage-dependent anion channels,release of cytochrome C from mitochondria,and the ratio of Bax to Bcl-2 or Bcl-X

The release of cytochrome c from mitochondria, which is regulated by Bcl-2 family members and is considered to take place through voltage-dependent anion channels (VDACs) on the outer membranes of mitochondria, results in activation of effector caspases, such as caspase-3, which induce apoptosis. We studied the involvement of the mitochondrial apoptosis pathway in uterine epithelial apoptosis. Estradiol-17beta pellets were implanted into ovariectomized mice and removed 4 days later (Day 0). The apoptotic index (percentage of apoptotic cells) of the luminal epithelium increased markedly, peaking on Day 2, whereas that of the glandular epithelium increased much less. Expression of VDAC1, 2, and 3 mRNAs increased in the luminal epithelium in correlation with the apoptotic index of the luminal epithelium. No increases in VDAC1, 2, and 3 mRNA levels were observed in the stroma or muscle, where no apoptosis occurs. VDAC1 protein levels in the uterus also correlated well with the apoptotic index of the luminal epithelium. In addition, the apoptotic index showed good correlation with the release of cytochrome c from mitochondria, activation of caspase-3, which was immunohistochemically detected only in the epithelium, and the mRNA and protein ratios of Bax:Bcl-2 and Bax:Bcl-X in the uterus. The present results suggest that the release of cytochrome c from mitochondria, which is regulated by Bcl-2 family members, plays a role in uterine epithelial apoptosis after estrogen deprivation. The increase in VDAC expression may facilitate the release of cytochrome c during apoptosis.     

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     

A novel inhibitory mechanism of mitochondrion-dependent apoptosis by a herpesviral protein

Upon viral infection, cells undergo apoptosis as a defense against viral replication. Viruses, in turn, have evolved elaborate mechanisms to subvert apoptotic processes. Here, we report that a novel viral mitochondrial anti-apoptotic protein (vMAP) of murine gamma-herpesvirus 68 (gammaHV-68) interacts with Bcl-2 and voltage-dependent anion channel 1 (VDAC1) in a genetically separable manner. The N-terminal region of vMAP interacted with Bcl-2, and this interaction markedly increased not only Bcl-2 recruitment to mitochondria but also its avidity for BH3-only pro-apoptotic proteins, thereby suppressing Bax mitochondrial translocation and activation. In addition, the central and C-terminal hydrophobic regions of vMAP interacted with VDAC1. Consequently, these interactions resulted in the effective inhibition of cytochrome c release, leading to the comprehensive inhibition of mitochondrion-mediated apoptosis. Finally, vMAP gene was required for efficient gammaHV-68 lytic replication in normal cells, but not in mitochondrial apoptosis-deficient cells. These results demonstrate that gammaHV-68 vMAP independently targets two important regulators of mitochondrial apoptosis-mediated intracellular innate immunity, allowing efficient viral lytic replication.     

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.     

BimS-induced apoptosis requires mitochondrial localization but not interaction with anti-apoptotic Bcl-2 proteins

Release of apoptogenic proteins such as cytochrome c from mitochondria is regulated by pro- and anti-apoptotic Bcl-2 family proteins, with pro-apoptotic BH3-only proteins activating Bax and Bak. Current models assume that apoptosis induction occurs via the binding and inactivation of anti-apoptotic Bcl-2 proteins by BH3-only proteins or by direct binding to Bax. Here, we analyze apoptosis induction by the BH3-only protein Bim(S). Regulated expression of Bim(S) in epithelial cells was followed by its rapid mitochondrial translocation and mitochondrial membrane insertion in the absence of detectable binding to anti-apoptotic Bcl-2 proteins. This caused mitochondrial recruitment and activation of Bax and apoptosis. Mutational analysis of Bim(S) showed that mitochondrial targeting, but not binding to Bcl-2 or Mcl-1, was required for apoptosis induction. In yeast, Bim(S) enhanced the killing activity of Bax in the absence of anti-apoptotic Bcl-2 proteins. Thus, cell death induction by a BH3-only protein can occur through a process that is independent of anti-apoptotic Bcl-2 proteins but requires mitochondrial targeting.     

RNAi suppression of Bax and Bak enhances viability in fed-batch cultures of CHO cells

Bcl-2 family proteins play a crucial role in the regulation of the mitochondrial pathway that leads to apoptosis. Members of the Bcl-2 family can be divided into the anti-apoptotic proteins such as Bcl-2 and Bcl-X(L), and the pro-apoptotic proteins such as Bax and Bak and the BH3-only proteins. In this study, siRNA constructs to silence the Bax and Bak genes in Chinese hamster ovary (CHO) cells were generated. Stable CHO cell lines in which the expression of Bax and Bak were significantly knocked down were screened by Western blot analysis and confirmed by RT-PCR. CHO cells with both Bax and Bak knocked down showed a clear resistance against cytotoxic lectins and UV irradiation-induced apoptosis. Compared to original CHO-K1 cells, these cells also survived longer when cultured under extreme conditions such as complete nutrient depletion or in high-osmolality medium. CHO cells with both Bax and Bak genes knocked down displayed an extended lifespan as well as higher viable cell densities in fed-batch cultures, both in adherent form on microcarrier beads and in suspension. The IFN-gamma productivity by a rCHO IFN-gamma cell line in which both Bak and Bax were knocked down increased by 35% compared to the control cells. These results indicate that the genetic inactivation of Bax and Bak in recombinant CHO cells can be an effective strategy in delaying the onset of apoptosis in batch and fed-batch cultures.     

Cleavage of Bax enhances its cell death function

Members of the Bcl-2 family of proteins are key regulators of apoptosis. Some of these proteins undergo posttranslational modification, such as phosphorylation or proteolysis, that serves to alter their function. Caspases are known to cleave Bid, a proapoptotic family member, as well as Bcl-2 and Bcl-X(L), two prosurvival family members, which activate their cytotoxic activity resulting in the release of cytochrome c from mitochondria. Previously we showed that Bax was cleaved by calpain rather than by caspases from full-length 21 kDa to generate a cleavage fragment of 18 kDa. Since cleavage of Bid serves to activate its cytotoxic activity, we wanted to determine if the p18 form of Bax exhibited increased cytotoxicity compared to p21 Bax. Using a transient transfection system in human embryonic kidney 293T cells we show that the p18 form of Bax displays a more potent ability to induce cell death. The pancaspase inhibitor Z-VAD-fmk completely blocked apoptosis induced by p21 Bax but only partially inhibited apoptosis induced by p18 Bax. Cyclosporin A, an inhibitor of the mitochondrial permeability transition (PT) pore, had no effect on Bax-mediated apoptosis of 293T cells suggesting that apoptosis was independent of the PT. Thus cleavage of p21 Bax during apoptosis to the p18 form may serve to increase the intrinsic cytotoxic properties of this proapoptotic molecule and enhance its cell death function at the mitochondria.     

A Bax/Bak-independent mechanism of cytochrome c release

Bax and Bak are multidomain pro-apoptotic members of the Bcl-2 family of proteins that regulate mitochondria-mediated apoptosis by direct modulation of mitochondrial membrane permeability. Since double-knock-out mouse embryonic fibroblasts with deficiency of Bax and Bak are resistant to multiple apoptotic stimuli, Bax and Bak are considered to be an essential gateway for various apoptotic signals. Here we showed that the combination of calcium ionophore A23187 and arachidonic acid induced cytochrome c release and caspase-dependent death of double-knock-out mouse embryonic fibroblasts, indicating that other mechanisms of cytochrome c release exist. Furthermore, A23187/arachidonic acid (ArA)-induced caspase-dependent death was significantly suppressed by the treatment of several serine protease inhibitors including 4-(2-aminoethyl)benzenesulfonylfluoride and l-1-chloro-3-(4-tosylamido)-4-phenyl-2-butanone but not the overexpression of anti-apoptotic Bcl-2 family of proteins or the inhibition of mitochondrial membrane permeability transition. These results indicate that there are at least two mechanisms of cytochrome c release leading to caspase activation, a Bax/Bak-dependent mechanism and a Bax/Bak-independent, but serine protease(s)-dependent, mechanism.     

Selenium as a modulator of membrane stability parameters and surface changes during the initiation phase of 1,2-dimethylhydrazine induced colorectal carcinogenesis

Epithelial ovarian cancer (EOC) represents the most challenging of gynecological malignancies. Defective apoptosis is a major causative factor in the development and progression of cancer. The two important pathways of apoptosis are extrinsic death receptor pathway (Fas family) and intrinsic mitochondrial pathway (Bcl-2 family). In this study, differential protein expression of the major Fas family members (Fas, FasL, and FAP-1) and Bcl-2 family members (Bax, Bcl-2, and Bcl-X(L)) in benign versus malignant surface epithelial ovarian tumors was evaluated at the protein level by immunohistochemistry. The expression of these molecules was compared in 30 benign versus 35 malignant surface epithelial ovarian tumors. The findings of the present study showed that there was no significant difference in the expression of the Fas family members in benign and malignant ovarian tumors. However, benign tumors showed higher levels of anti-apoptotic Bcl-2 protein levels (p < 0.009), whereas malignant tumors showed higher levels of pro-apoptotic Bax (p < 0.001). In general, there was no significant difference in Bcl-X(L) protein levels. The observations made in the present study suggest that alterations in expression of the Fas family and the Bcl-2 family members occur and play a key role in the deregulated growth of epithelial ovarian cancer.     

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.