Accumulating evidence implicates that the voltage-dependent anion channel (VDAC) functions in mitochondrion-mediated apoptosis and as a critical player in the release of apoptogenic proteins, such as cytochrome c, triggering caspase activation and apoptosis. The mechanisms regulating cytochrome c release and the molecular architecture of the cytochrome c-conducting channel remain unknown. Here the relationship between VDAC oligomerization and the induction of apoptosis was examined. We demonstrated that apoptosis induction by various stimuli was accompanied by highly increased VDAC oligomerization, as revealed by cross-linking and directly monitored in living cells using bioluminescence resonance energy transfer technology. VDAC oligomerization was induced in all cell types and with all apoptosis inducers used, including staurosporine, curcumin, As 2 O 3 , etoposide, cisplatin, selenite, tumor necrosis factor alpha (TNF-␣), H 2 O 2 , and UV irradiation, all acting through different mechanisms yet all involving mitochondria. Moreover, correlation between the levels of VDAC oligomerization and apoptosis was observed. Furthermore, the apoptosis inhibitor 4,4-diisothiocyanostilbene-2,2-disulfonic acid (DIDS) inhibited VDAC oligomerization. Finally, a caspase inhibitor had no effect on VDAC oligomerization and cytochrome c release. We propose that VDAC oligomerization is involved in mitochondrion-mediated apoptosis and may represent a general mechanism common to numerous apoptogens acting via different initiating cascades. Thus, targeting the oligomeric status of VDAC, and hence apoptosis, offers a therapeutic strategy for combating cancers and neurodegenerative diseases.It is well accepted that mitochondria serve as integrators and amplifiers of programmed cell death through the regulation of apoptosis, mediating the release of proapoptotic proteins and/or disrupting cellular energy metabolism (25). During the transduction of an apoptotic signal into the cell, an alteration in mitochondrial membrane permeability occurs, facilitating the release of apoptogenic proteins, such as cytochrome c (Cyto c), apoptosis-inducing factor (AIF), and Smac/DIABLO, from the intermembrane space into the cytosol (45). These proteins participate in complex processes resulting in the activation of proteases and nucleases, leading to protein and DNA degradation and, ultimately, cell death (25). However, it remains unclear how these apoptotic initiators cross the outer mitochondrial membrane (OMM) and are released into the cytosol. While some models predict that such release is facilitated by swelling of the mitochondrial matrix and subsequent rupture of the OMM, other models predict the formation of protein-conducting channels that are large enough to allow the passage of Cyto c and other proteins into the cytosol without compromising OMM integrity (15,44,47). The voltage-dependent anion channel (VDAC) offers such a route (45).Located in the OMM, the VDAC forms the main interface between the mitochondrial and cellular metabolisms by medi...

Mitochondria, central to basic life functions due to their generation of cellular energy, also serve as the venue for cellular decisions leading to apoptosis. A key protein in mitochondria-mediated apoptosis is the voltage-dependent anion channel (VDAC), which also mediates the exchange of metabolites and energy between the cytosol and the mitochondria. In this study, the functions played by the N-terminal region of VDAC1 and by VDAC1 oligomerization in the release of cytochrome c, Smac/Diablo and apoptosis-inducing factor (AIF) and subsequent apoptosis were addressed. We demonstrate that cells undergoing apoptosis induced by STS or cisplatin and expressing N-terminally truncated VDAC1 do not release cytochrome c, Smac/Diablo or AIF. Ruthenium red (RuR), AzRu, DIDS and hexokinase-I (HK-I), all known to interact with VDAC, inhibited the release of cytochrome c, Smac/Diablo and AIF, while RuR-mediated inhibition was not observed in cells expressing RuR-insensitive E72Q-VDAC1. These findings suggest that VDAC1 is involved in the release of not only cytochrome c but also of Smac/Diablo and AIF. We also demonstrate that apoptosis induction is associated with VDAC oligomerization, as revealed by chemical cross-linking and monitoring in living cells using Bioluminescence Resonance Energy Transfer. Apoptosis induction by STS, H2O2 or selenite augmented the formation of VDAC oligomers several fold. The results show VDAC1 to be a component of the apoptosis machinery and offer new insight into the functions of VDAC1 oligomerization in apoptosis and of the VDAC1 N-terminal domain in the release of apoptogenic proteins as well as into regulation of VDAC by anti-apoptotic proteins, such as HK and Bcl2.

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