MiP2005: Session 4
Mitochondrial Physiology Network 10.9: 45 (2005) - download pdf
Reconstitution of kinase signaling in mitochondria - how mitoKATP opening inhibits permeability transition opening.
Keith D Garlid, ADT Costa
Dept. Biology, Portland State University, PO Box 751, Portland, Oregon 97207. - email@example.com
Pharmacological preconditioning can be triggered by an intracellular signaling pathway in which Gi-coupled surface receptors activate a cascade including PI3K, eNOS, guanylyl cyclase and protein kinase G (PKG) . Activated PKG opens the mitochondrial KATP channel (mitoKATP) and mitoKATP opening causes increased production of reactive oxygen species (ROS) [2,3], which then go on to activate other kinases. The steps between PKG and mitoKATP opening are unknown, as are the steps downstream of mitoKATP.
We found that exogenous PKG + cGMP induces mitoKATP opening in isolated heart mitochondria to the same extent as KATP channel openers such as diazoxide or cromakalim. This effect was blocked by mitoKATP blockers — 5-HD, glibenclamide, and TPP+, by the PKG-selective inhibitor KT5823, and by protein kinase C (PKC) inhibitors chelerythrine, Ro318220, and the highly selective PKC-peptide antagonist, V1-2. We also found that mitoKATP is opened by the PKC activators 12-phorbol 13-myristate acetate and H2O2. We conclude that PKG is the terminal cytosolic component of the signaling pathway and that it transmits the cardioprotective signal from cytosol to inner mitochondrial membrane by a pathway that includes PKC-.
KATP channel openers or activators of PKG or PKC inhibited MPT opening, and this effect was also mediated by a PKC-. Inhibition of MPT opening was prevented by MPG, indicating that the signal was transmitted by a mitoKATP-dependent increase in H2O2.
The effect of PKG + cGMP requires an intact outer membrane, whereas the effects of the two PKC-s do not. Indeed both effects of PKC- activation — mitoKATP opening and MPT inhibition — were observed in mitoplasts, implying that these PKCs are tightly bound to the inner membrane. This partial resolution of the mitochondrial portion of the cardioprotective signaling pathway should enable us to identify the kinase that phosphorylates mitoKATP.
1. Oldenburg O, Cohen MV, Downey JM (2003) Mitochondrial KATP channels in preconditioning. J. Mol. Cell. Cardiol. 35: 569-575.
2. Garlid KD (2000) Opening mitochondrial KATP in the heart - what happens, and what does not happen. Basic Res. Cardiol. 95: 275-279.
3. Andrukhiv A, Costa ADT, West IC, Garlid KD (2005) On the mechanism by which opening the mitochondrial ATP-dependent K+ channel (mitoKATP) increases mitochondrial production of reactive oxygen species. Am J Physiol Submitted and in review.