| Mitochondria play a key role in cardiac ischemia and reperfusion (IR) injury. After prolonged ischemia, the onset of reperfusion results in mitochondrial Ca2+ overload and excessive reactive oxygen species generation. This triggers the opening of mitochondrial permeability transition pore (MPTP) opening, which leads to cell death through necrosis and apoptosis. Consequently, preservation of mitochondrial function is critical to the attenuation of cellular damage triggered during IR. This study focused on the modulation of the voltage-dependent anion channel (isoform 1; VDAC1) the most abundant protein on the outer membrane of the mitochondria (OMM), and the major conduit for the transport of metabolites between the OMM and the cytosol. Studies have suggested that VDAC1 undergoes post-translational modification (PTM) during IR injury, but the resultant functional impact on the channel, and subsequently on mitochondrial function has not been delineated. Our goal was to determine the impact of PTM on VDAC1 function, and its subsequent effects on mitochondrial respiration and cell death.;We identified a number of putative phosphorylation sites on VDAC1 by a proteomics approach. Of particular interest was a hexokinase (HK)-induced phosphorylation site, T70, since the binding of HK to VDAC1 is associated with cell survival. Phospho-mimetic mutants of VDAC1 were constructed, expressed, and purified for biophysical characterizations utilizing the planar lipid bilayers technique. The T70E mutant significantly decreased VDAC1 channel conductance and deprived VDAC1 of its unique voltage-dependent characteristics. In contrast, the S137E mutant displayed characteristics similar to the wild type. Hence the effect of VDAC1 phosphorylation on channel function was residue specific. Furthermore, the basally phosphorylated S137 is putatively located on the side facing the mitochondrial intermembrane space, while T70 is closer to the cytosol. Consequently, VDAC function appeared also to be dependent on the sidedness of phosphorylation.;The impact of VDAC1 phosphorylation on cellular function was then investigated. Plasmids containing GFP-tagged wild type VDAC1 (WT-VDAC1) or T70E mutant were transfected into HEK293 cells. Overexpression of GFPtagged VDAC1 was confirmed by western blotting and the co-localization of VDAC1 with mitochondria was confirmed by confocal microscopy. Based on a mitochondrial respiration assay, the T70E mutant transfected cells displayed a lower oxygen consumption rate in comparison to the WT-VDAC1. For further studies, we chose to utilize HL-1 cells, a cardiac muscle cell line, as it is the most cardiac relevant cell line currently available. HL-1 cells transfected with the T70E mutant showed a markedly lower basal cellular respiration rate when compared with HL-1 cells transfected with WT-VDAC1 or S137E mutant. These results are similar to the ones observed from HEK293 cells. To investigate cell viability after hypoxia-reoxygenation insult, LDH release assay was performed. The T70E mutant transfected HL-1 cells had significantly reduced LDH release compared to that of WT-VDAC1 and S137E. Moreover, the T70E mutant transfected HL-1 cells showed improved oxygen utilization at the onset of reoxygenation following hypoxia. Although decreased VDAC1 conductance may be protective, complete inhibition is likely not, as evidenced by the decreased cell viability we observed in VDAC1 knockout HL-1 cells. In conclusion, overexpression of the VDAC1 T70E mutant in a cardiac cell line is cytoprotective against hypoxic stress. Combining the results from the channel biophysical experiments, we showed that phosphorylation of a specific residue, T70, on VDAC1 that decreases channel conductance has cardioprotective properties.;We also examined the effects of other forms of PTM, specifically Snitrosylation (SNO) and nitration, on VDAC1 function. VDAC1 exhibited enhanced tyrosine nitration by excess peroxynitrite during IR injury and high concentration of peroxynitrite significantly increased VDAC1 channel conductance. Previous study has shown that, PAPA NONOate, an NO.. donor, had a biphasic effect on VDAC1 channel activity, decreasing channel conductance at low concentration while increasing it at high concentration, implying that SNO has a concentration-dependent effect on VDAC1 activity. VDAC1 can potentially be S-nitrosylated at two cysteine residues, C127 and C232. HL-1 cells transfected with cysteine-less C(127,232)A or C232A mutants, but not the C127A mutant, had significantly reduced LDH release after hypoxia/reoxygenation insult. These results suggested that C232 could be susceptible to modification by NO. donors and, subsequently, more likely to increase VDAC1 activity.;Overall, these findings suggest that various forms of PTM modify VDAC1 channel activity. Changes in VDAC1 channel activity impact cellular functions under physiological and pathophysiological conditions. During ischemia, phosphorylation triggered decrease in VDAC1 activity is likely to be protective, while activation of VDAC1 via tyrosine nitration is detrimental. However, Snitrosylating VDAC1 may potentially result in dual protective/detrimental effects.;Anion channels are also present on the inner membrane of mitochondria (IMM). We characterized a previously unreported novel anion channel when exploring channel activity on the IMM by patch clamping cardiac mitoplasts (mitochondria devoid of the OMM). The physiological role of this newly discovered anion channel is as yet unclear. However, our results suggest that the IMM exhibits a high activity of ionic fluxes. |
