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. -  garlid@pdx.edu

    Pharmacological preconditioning can be triggered by an intracellular signaling pathway in which G_i-coupled surface receptors activate a cascade including PI3K, eNOS,  guanylyl cyclase and protein kinase G (PKG) [1]. Activated PKG opens the mitochondrial K_ATP channel (mitoK_ATP) and mitoK_ATP opening causes increased production of reactive oxygen species (ROS) [2,3], which then go on to activate other kinases. The steps between PKG and mitoK_ATP opening are unknown, as are the steps downstream of mitoK_ATP. 

    We found that exogenous PKG + cGMP induces mitoK_ATP opening in isolated heart mitochondria to the same extent as K_ATP channel openers such as diazoxide or cromakalim.  This effect was blocked by mitoK_ATP 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, V_1-2.  We also found that mitoK_ATP 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-. 

    K_ATP 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 mitoK_ATP-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 — mitoK_ATP 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 mitoK_ATP.

1.  Oldenburg O, Cohen MV, Downey JM (2003) Mitochondrial K_ATP channels in preconditioning. J. Mol. Cell. Cardiol. 35: 569-575.

2.  Garlid KD (2000) Opening mitochondrial K_ATP 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.