Mouse VDAC Isoforms Expressed in Yeast: Channel Properties and Their Roles in Mitochondrial Outer Membrane Permeability

Abstract

The channel-forming protein called VDAC forms the major pathway in the mitochondrial outer membrane and controls metabolite flux across that membrane. The different VDAC isoforms of a species may play different roles in the regulation of mitochondrial functions. The mouse has three VDAC isoforms (VDAC1, VDAC2 and VDAC3). These proteins and different versions of VDAC3 were expressed in yeast cells (S. cerevisiae) missing the major yeast VDAC gene and studied using different approaches. When reconstituted into liposomes, each isoform induced a permeability in the liposomes with a similar molecular weight cutoff (between 3,400 and 6,800 daltons based on permeability to polyethylene glycol). In contrast, electrophysiological studies on purified proteins showed very different channel properties. VDAC1 is the prototypic version whose properties are highly conserved among other species. VDAC2 also has normal gating activity but may exist in 2 forms, one with a lower conductance and selectivity. VDAC3 can also form channels in planar phospholipid membranes. It does not insert readily into membranes and generally does not gate well even at high membrane potentials (up to 80 mV). Isolated mitochondria exhibit large differences in their outer membrane permeability to NADH depending on which of the mouse VDAC proteins was expressed. These differences in permeability could not simply be attributed to different amounts of each protein present in the isolated mitochondria. The roles of these different VDAC proteins are discussed.

RAS–RAF–MEK-dependent oxidative cell death involving voltage-dependent anion channels

“…We found little NADH oxidation in mitochondria expressing VDAC3, suggesting minimal intrinsic membrane permeability ( Supplementary Fig. 17); this is consistent with previous reports that VDAC3 does not gate well in vitro 20 . Finally, we found that an inactive analogue of erastin (erastin A8, Fig.…”

“…20 ). As a control for mitochondrial intactness, parallel assays were run using hypotonically shocked mitochondria.…”

“…To test the hypothesis that erastin alters mitochondrial outer membrane permeability, we monitored the function of VDACs using purified mitochondria from yeast engineered to express a single mouse VDAC isoform in place of yeast VDAC 20 . A previous report demonstrated that the rate of NADH uptake through the outer membrane of such mitochondria is dependent on the specific VDAC expressed in yeast.…”

VDAC1 selectively transfers apoptotic Ca2+ signals to mitochondria

“…9 However, these different effects are not supported by the apparent redundancy in the electrophysiological properties of the isoforms. 19,20 Mitochondrial [Ca 2 þ ] is commonly regarded as an important determinant in cell sensitivity to apoptotic stimuli. 21 Indeed, mitochondrial Ca 2 þ accumulation acts as a 'priming signal' sensitizing the organelle and promoting the release of caspase cofactors, both in isolated mitochondria as well as in intact cells.…”

Modulation of Human Mitochondrial Voltage-dependent Anion Channel 2 (hVDAC-2) Structural Stability by Cysteine-assisted Barrel-lipid Interactions

“…Channels formed by mouse VDAC-2 are noisier than VDAC-1 (4,23), suggesting alternate functions for the less abundant hVDAC-2 and hVDAC-3 isoforms other than the "ion conductance" usually associated with these proteins. The abundance of cysteine residues in hVDAC-2 is particularly intriguing (supplemental Fig.…”