Abstract 16370: Hexokinase-Induced Residue Specific Phosphorylation of Cardiac Mitochondrial Voltage-Dependent Anion Channel
Introduction: The signaling pathways that protect the heart during ischemia-reperfusion injury ultimately converge onto the mitochondria. Yet, the mitochondrial targets have not been clearly established. The voltage-dependent anion channel (VDAC1 isoform) serves as the gateway for apoptotic and anti-apoptotic signals. It is the receptor for hexokinase (HK), and the VDAC1-HK complex is known to significantly increase cell survival. In the present study, we have identified a novel HK-induced phosphorylated residue on VDAC1, and investigated its impact on channel activity.
Methods: Mitochondrial VDAC1 was purified from rat hearts. Identification of VDAC1 phosphorylated residues was conducted using a Thermofinnigan LTQ XL ion-trap mass spectrometer and analyzed by multistage-fragmentation analysis. VDAC1 mutants were constructed using site-directed mutagenesis. Channel activity was monitored by incorporating the VDAC1 into planar lipid bilayers. Recombinant HK (HK1+HK2) was obtained from GenWay Biotech (San Diego, CA).
Results: HK induced the phosphorylation of T70 in VDAC1. A phospho-mimetic VDAC1 mutant, T70E, revealed a significant decrease in the primary channel conductance from 1.75±0.02 nS (n=10) in the wild-type VDAC1 to 0.74±0.09 nS (n=3). This was accompanied by a loss of the characteristic VDAC1 voltage-dependence, whereby the mutant exhibited a linear current-voltage relationship between -80 to +80 mV. A basally phosphorylated residue, S137, was also identified. Compared to T70E, the phospho-mimetic S137E mutant exhibited a primary channel conductance (1.73±0.08 nS; n=3) similar to the wild-type, but with more frequent transitions to lower conductance states. The S137E mutant also maintained the characteristic wild-type voltage-dependence.
Conclusion: HK directly phosphorylated the cardiac VDAC1 at T70 and induced a significant decrease in channel conductance with the loss of the characteristic voltage-dependence. Thus, HK binding to VDAC1 regulates the flow of metabolites across the outer mitochondrial membrane. The differential modulation of cardiac VDAC1 by residue-specific phosphorylation could contribute to the maintenance of mitochondrial function during cellular stress as in ischemia-reperfusion.
- © 2011 by American Heart Association, Inc.