Abstract 3024: Binding of Hexokinase to Mitochondria is Involved in the Regulation of Mitochondrial Permeability Transition Pore by Glycogen Synthase Kinase-3β
Background: Although the mitochondrial permeability transition pore (mPTP) is one of the down stream targets of glycogen synthase kinase-3β (GSK3β), the detailed mechanisms of the effect of GSK3β on mPTP remain elusive. We hypothesized that the opening of mPTP by activated GSK3β requires a persistent activation of hexokinase II (HKII) on mitochondrial membrane.
Method: (1) In permeabilized rat myocytes, we assessed the opening of mPTP by measuring calcein leakage from mitochondria, and mitochondrial membrane potential (ΔΨm) with a voltage sensitive dye, TMRE, using a laser scanning confocal microscopy (LSCM). (2) For the immunogold labeling of mitochondria-bound HKII with electron microscopy (EM) and western blotting, the isolated mitochondria were prepared by the centrifugal method.
GSK3β (10 nM) decreased calcein intensity (79.2±1.3 % of the baseline, p<0.01), indicating the opening of the mPTP, and this was prevented by cyclosporine A (CsA, an inhibitor of mPTP: 100 nM, 90.4±1.8 %, p<0.01). Application of SB216763 (an inhibitor of GSK3β; 3 μM) inhibited the GSK3β-induced calcein leakage (92.1±1.2 %, p<0.01).
PKA catalytic subunit (PKAcat, 10 U/ml) inhibited the GSK3β-induced calcein leakage (94.5±1.5 %, p<0.01).
GSK3β depolarized ΔΨm (78.3±2.4 %, p<0.01), and this effect was abolished by CsA, SB216763, and PKAcat.
Reduction of HKII activity (by glucose deprivation or a negative feed back with glucose-6-phosphate) inhibited GSK3β-induced mPTP opening (91.5±1.5 % and 88.7±1.4 %, respectively, p<0.01).
Enhancement of HKII binding to mitochondria by dextran (1%) ameliorated GSK3β-induced mPTP opening and ΔΨm depolarization (92.8±1.6 % and 87.0±4.2%, respectively, p<0.01).
DCCD (1 μM), which detached HKII from the mitochondria, enhanced GSK3β-induced mPTP opening and ΔΨm depolarization (59.3±1.5 % and 59.5±1.7%, respectively, p<0.01).
Both immunogold labeling of HKII in EM and the western blot analysis showed that GSK3β significantly reduced HKII-mitochondrial binding (5.6±0.8 golds/mitochondria, and 83.2±3.8 arbitrary unit, respectively, p<0.01).
Conclusion: Opening of the mPTP and depolarization of ΔΨm by GSK3β depend on the activity and the binding of HKII to mitochondria.