Comparative kinetic analysis reveals that inducer-specific ion release precedes the mitochondrial permeability transition

Abstract

Relationships among the multiple events that precede the mitochondrial membrane permeability transition (MPT) are not yet clearly understood. A combination of newly developed instrumental and computational approaches to this problem is described. The instrumental innovation is a high-resolution digital apparatus for the simultaneous, real-time measurement of four mitochondrial parameters as indicators of the respiration rate, membrane potential, calcium ion transport, and mitochondrial swelling. A computational approach is introduced that tracks the fraction of mitochondria that has undergone pore opening. This approach allows multiple comparisons on a single time scale. The validity of the computational approach for studying complex mitochondrial phenomena was evaluated with mitochondria undergoing an MPT induced by Ca(2+), phenylarsine oxide or alamethicin. Selective ion leaks were observed that precede the permeability transition and that are inducer specific. These results illustrate the occurrence of inducer-specific sequential changes associated with the induction of the permeability transition. Analysis of the temporal relationship among the multiple mitochondrial parameters of isolated mitochondria should provide insights into the mechanisms underlying these responses.

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

“…its rate is slower than the Ca 2ϩ absorption step (k 2 Ͻ k 1 ). Our proposed model offers several improvements as compared with the earlier models by Massari and others (13,14,19). First, the rate of Ca 2ϩ consumption by mitochondria (the k 1 step) is assumed to be a second order reaction; therefore, the rate changes in proportion with [Ca 2ϩ ] out .…”

“…One major advantage of the current system as compared with the previously used potentiometric method is that this system enables measurement of parameters of interest at up to 62.5 Hz (versus 0.5 Hz in the previous method (14)). This ability facilitates the measurement of parameters with relatively fast kinetics, such as net Ca 2ϩ uptake by populations of isolated mitochondria.…”

“…Our recent work has demonstrated the possibility of simultaneously measuring Ca 2ϩ fluxes and changes in the ⌬ m of isolated mitochondria using an ion-selective electrode system. Coupling these electrodes with a Clark-type O 2 -electrode and a light-emitting diode/photodiode pair enabled the set-up to simultaneously measure four essential mitochondrial parameters (14). Electrode-based measurement systems, however, are limited by sensor response times as well as chemical properties of the measurement chamber.…”

“…This feedback-like Ca 2ϩ reuptake cycle makes it impossible to find an analytical solution for the Ca 2ϩ -induced mPT kinetic model. In practice, previous workers have attempted to avoid this complication, using approaches such as focusing on de-energized mitochondria that do not take up Ca 2ϩ (19) or adding high concentrations of Ca 2ϩ , such that the process of reuptake of Ca 2ϩ is negligible relative to the initial uptake (13,14). We extended the modeling efforts of these groups to address these conditions by applying a numerical approach, which enables use of these systems under more (patho)physiological conditions.…”

Mechanisms of non-apoptotic programmed cell death in diabetes and heart failure

“…It is ironic that John Foxton Ross Kerr, the father of modern apoptosis, originally used the term "programmed cell necrosis" to describe his concept review review MPTP dissipates the normal mitochondrial pH gradient and inner membrane electrochemical potential (Δψ m ) that are required for ATP production through oxidative phosphorylation. 20 MPTP opening therefore reverses ATP synthesis, and the mitochondrion becomes a net ATP consumer. The loss of cellular ATP subsequent to MPTP opening ignites a cascade of metabolic dysfunction that can ultimately lead to cell necrosis.…”