Abstract 5420: Dephosphorylation of the Cardiac Mitochondrial Voltage-Dependent Anion Channel Prevents Inhibition by Hexokinase

Abstract

The mitochondrial permeability transition pore (mPTP) has been implicated as the end effector in ischemic and pharmacological preconditioning. Though the molecular composition of the mPTP is thought to consist of cyclophilin D located in the mitochondrial matrix, adenine nucleotide translocase on the inner mitochondrial membrane, and the voltage-dependent anion channel (VDAC) on the outer mitochondrial membrane, recent studies have raised the possibility that VDAC may be a regulatory, rather than a major, component of mPTP. Nevertheless, VDAC is likely to be a critical component of the preconditioning signaling pathway since it is the main conduit for metabolite diffusion across the mitochondrial outer membrane. Yet, the direct measurements of cardiac VDAC activity and modulation have been limited. In the present study, we purified VDAC from rat hearts using standard procedure and investigated its modulation by phosphatase and hexokinase. VDAC was incorporated into planar lipid bilayer for measurements of channel activities. The channel exhibited the reported voltage-dependent gating. Several conductance states were identified, with the most prevalent between 1.5 to 2 nS in 0.5 M NaCl. Koenig’s polyanion, a VDAC blocker, triggered channel flickering and decreased the mean current by 78±6%. In the presence of phosphatase (1 unit/ml), the mean conductance significantly increased from 1.81±0.03 to 3.68±0.61 nS (n=9; mean±SEM). However, the addition of a recombinant hexokinase (5 units/ml; GenWay Biotech) had no significant effect on the phosphatase-enhanced VDAC current (n=4). In contrast, recombinant hexokinase alone significantly decreased the mean conductance from 1.75±0.05 to 0.79±0.19 nS (n=4). The addition of phosphatase reversed the inhibitory effect of hexokinase and further enhanced VDAC activity, increasing the mean conductance to 2.69±0.19 nS (n=4). Our results suggest that the dephosphorylation of VDAC prevents the inhibitory effects of hexokinase. Furthermore, VDAC activity suppressed by hexokinase can be reversed by dephosphorylation of the channel. In conclusion, we have reported on a novel observation at the functional level that basal phosphorylation of the cardiac VDAC may be required for its modulation by hexokinase.