Kinetic model for Ca2+-induced permeability transition in energized liver mitochondria discriminates between inhibitor mechanisms

Abstract

Cytotoxicity associated with pathophysiological Ca2+ overload (e.g. in stroke) appears mediated by an event termed the mitochondrial permeability transition (mPT). We built and solved a kinetic model of the mPT in populations of isolated rat liver mitochondria that quantitatively describes Ca2+-induced mPT as a two-step sequence of pre-swelling induction followed by Ca2+-driven, positive feedback, autocatalytic propagation. The model was formulated as two differential equations, each directly related to experimental parameters (Ca2+ flux/mitochondrial swelling). These parameters were simultaneously assessed using a spectroscopic approach to monitor multiple mitochondrial properties. The derived kinetic model correctly identifies a correlation between initial Ca2+ concentration and delay interval prior to mPT induction. Within the model's framework, Ru-360 (a ruthenium complex) and Mg2+ were shown to compete with the Ca2+-stimulated initiation phase of mPT induction, consistent with known inhibition at the phenomenological level of the Ca 2+ uniporter. The model further reveals that Mg2+, but not Ru-360, inhibits Ca2+-induced effects on a downstream stage of mPT induction at a site distinct from the uniporter. The analytical approach was then applied to promethazine, an FDA-approved drug previously shown to inhibit both mPT and ischemia-reperfusion injury. Kinetic analysis revealed that promethazine delayed mPT induction in a manner qualitatively distinct from that of lower concentrations of Mg2+. In summary, we have developed a kinetic model to aid in the quantitative characterization of mPT induction. This model is consistent with/informative about the biochemistry of several mPT inhibitors, and its success suggests that this kinetic approach can aid in the classification of agents or targets that modulate mPT induction. © 2008 by The American Society for Biochemistry and Molecular Biology, Inc.