The expression level of the voltage-dependent anion channel controls life and death of the cell

Abstract

Mitochondria not only generate cellular energy, but also act as the point for cellular decisions leading to apoptosis. The voltage-dependent anion channel (VDAC), as a major mitochondrial outer-membrane transporter, has an important role in energy production by controlling metabolite traffic and is also recognized as a key protein in mitochondria-mediated apoptosis. In this study, the role of VDAC1 in regulating cell survival and death was investigated by silencing endogenous human (h)VDAC1 expression by using a short hairpin RNA (shRNA)-expressing vector. The shRNA effectively down-regulated the expression in human T-REx-293 cells of hVDAC1 but not murine (m)VDAC1. Cells in which hVDAC1 expression was decreased by ≈90% proliferated extremely slowly. Normal growth was, however, restored upon expression of mVDAC1 in a tetracycline-regulated manner. Although low tetracycline concentrations promoted cell growth, high concentrations induced mVDAC1 overexpression, leading to cell death. Cells with low levels of VDAC1 showed 4-fold-lower ATP-synthesis capacity and contained low ATP and ADP levels, with a strong correlation between ATP levels and cell growth, suggesting limited metabolite exchange between mitochondria and cytosol. The possibility of suppressing endogenous hVDAC1 expression and introducing native and mutated mVDAC1 is used to further explore the involvement of VDAC1 in apoptosis. Cells suppressed for hVDAC1 but expressing either native mVDAC1 or an E72Q mutant underwent apoptosis induced by various stimuli that can be inhibited by ruthenium red in the native cells but not in the mutated cells, suggesting that VDAC1 regulates apoptosis independent of the apoptosis-inducing pathway.

VDAC1 selectively transfers apoptotic Ca2+ signals to mitochondria

“…Several observations support the notion that VDAC can finely tune cellular processes in an isoform-specific way: (i) selective genetic ablation of the three VDAC genes exhibits different phenotypes; 29 (ii) VDAC1 and VDAC2 exert diametrically opposite effects on apoptosis 15,16,18 and a compound acting through VDAC2, erastin, is effective in tumors harboring Ras mutations; 30 (iii) apoptotic challenges 31 and genomic programs, such as the PGC1-a pathway (De Stefani and Rizzuto, unpublished), differentially regulate the expression of VDAC isoforms; and (iv) the three isoforms are localized to different sub-domains of the OMM. 32 We thus investigated in greater detail the molecular mechanism underlying the different role of VDAC isoforms in apoptosis.…”

“…Indeed, VDACs have been shown to interact with cytoskeletal elements such as actin and tubulin, 4,5 metabolic enzymes, 6 Bcl2-family members including Bak, 7 Bad, 8 tBid 9 and Bcl-xL 10 or other channels such as ANT, 11 or the IP 3 R. 12,13 This scenario is further complicated by evidence showing that VDAC contribution to cell death can be isoform and stimulus dependent: VDAC1 acts predominantly as a pro-apoptotic protein [14][15][16][17] whereas VDAC2 protects from a number of apoptosis inducers, 18 but concurrently appears to be necessary for tBid-mediated cell-death. 9 However, these different effects are not supported by the apparent redundancy in the electrophysiological properties of the isoforms.…”

Antifungal drug itraconazole targets VDAC1 to modulate the AMPK/mTOR signaling axis in endothelial cells

“…Short hairpins (sh) targeting two nonoverlapping sequences within the coding region of human VDAC1 were designed based on previously published sequences (62,63). The shRNA was created using the following complementary sets of PAGE-purified oligonucleotides (Integrated DNA Technologies): VDAC1 sh1 forward (5′-TCACTAGGCACCGAGA-TTATTTCAAGAGAATAATCTCGGTGCCTAGTGTTTTTTC-3′), VDAC1 sh1 reverse (5′-TCGAGAAAAAACACTAGGCACCGAGATTATTCTCTTGAAATAATCTCGGTG-CCTAGTGA-3′); VDAC1 sh2 forward (5′-TGTGACGGGCAGTCTGGAATTTCAA-GAGAATTCCAGACTGCCCGTCACTTTTTTC-3′), VDAC1 sh2 reverse (5′-TCGA-GAAAAAAGTGACGGGCAGTCTGGAATTCTCTTGAAATTCCAGACTGCCCGTC-ACA-3′).…”

Proteomic characterization of a temperature-sensitive conditional lethal in Drosophila melanogaster

“…Ion channel kinetics in D. melanogaster have previously been shown to be sensitive to elevated temperatures (Peng et al, 2007), thereby distorting physiological and developmental processes essential for cellular functions. Porin is a pore-forming protein situated in the outer mitochondrial membrane having a central role in movement of metabolites across the outer membrane, and is known to affect energy metabolism and apoptosis (Komarov et al, 2004;Abu-Hamad et al, 2006;Lee et al, 2007). Previously, a recessive mutation in the porin gene has been shown to cause lethality or elevated early mortality and to be expressed in a sex-specific pattern (Oliva et al, 2002;Lee et al, 2007).…”