Kinetic Model for Ca2+-induced Permeability Transition in Energized Liver Mitochondria Discriminates between Inhibitor Mechanisms

Sergei V Baranov,Irina G Stavrovskaya,Abraham M Brown,Alexei M Tyryshkin,Bruce S Kristal

doi:10.1074/jbc.m703484200

Abstract

Cytotoxicity associated with pathophysiological Ca(2+) 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 Ca(2+)-induced mPT as a two-step sequence of pre-swelling induction followed by Ca(2+)-driven, positive feedback, autocatalytic propagation. The model was formulated as two differential equations, each directly related to experimental parameters (Ca(2+) 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 Ca(2+) concentration and delay interval prior to mPT induction. Within the model's framework, Ru-360 (a ruthenium complex) and Mg(2+) were shown to compete with the Ca(2+)-stimulated initiation phase of mPT induction, consistent with known inhibition at the phenomenological level of the Ca(2+) uniporter. The model further reveals that Mg(2+), but not Ru-360, inhibits Ca(2+)-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 Mg(2+). 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.

Highlights

Cellular Ca2ϩ concentration and a mitochondria-associated process
From a kinetic point of view, at least three conceptually distinct phases must be resolved during the mitochondrial permeability transition (mPT) induction: (i) the initiation phase, which includes the initial intake of excess Ca2ϩ via the Ca2ϩ uniporter; (ii) the “lag” phase, during which Ca2ϩ cycling induces or accompanies a series of reactions in mitochondria; and (iii) the propagation or termination phase
In the analysis that follows, only the kinetic data for Ca2ϩ concentration and mitochondria swelling were used to formulate the kinetic model of the mPT process

Summary

Introduction

Cellular Ca2ϩ concentration and a mitochondria-associated process. These events have been hypothesized to be linked biochemically, because mitochondria are a major site for Ca2ϩ sequestration, normally absorbing excess cytosolic Ca2ϩ through the Ca2ϩ uniporter (which is driven by the membrane potential ⌬␸m) and releasing it via the Naϩ/Ca2ϩ and Ca2ϩ/Hϩ exchangers, resulting in a slow, continuous cycling of Ca2ϩ across the inner mitochondrial membrane. From a kinetic point of view, at least three conceptually distinct phases must be resolved during the mPT induction: (i) the initiation phase, which includes the initial intake of excess Ca2ϩ via the Ca2ϩ uniporter; (ii) the “lag” phase, during which Ca2ϩ cycling induces or accompanies a series of reactions in mitochondria (e.g. the pore constituents are recruited and possibly assembled); and (iii) the propagation or termination phase This final phase is associated with a decrease in membrane potential observable at the population level, release of mitochondrially sequestered Ca2ϩ (and a resultant increase in the steady-state Ca2ϩ concentration in the medium, which fuels subsequent Ca2ϩ uptake by the remaining intact mitochondria, creating a positive feedback loop for damage propagation), final pore assembly, and pore opening. The first order kinetic models developed by Massari [13] and used by Krasnikov et al [14] are only applicable at saturating concentrations of Ca2ϩ and fail to account for the lag phase often observed at lower, more (patho)physiologically relevant Ca2ϩ concentrations

Methods

Results

Conclusion