Modeling the roles of Ca uniporter, Na/Ca exchanger and Na/H exchanger in regulating Ca, Na and pH flux in cardiac mitochondria using in vitro spectrofluorometry

Abstract

Ca mediates mitochondrial signaling pathways and modulates energy metabolism. Alteration of Ca homeostasis can lead to mitochondrial dysfunction. To gain a biophysical understanding of mitochondrial Ca regulation, we developed a thermodynamically-based model of mitochondrial energetics and cation balance by integrating kinetic models of Ca uniporter (CU), Na/Ca exchanger (NCE) and Na/H exchanger (NHE) across mitochondrial inner membrane to an existing model of oxidative phosphorylation (Beard, PLoS Comp Biol 1:, 2005). Kinetic flux expressions for CU, NCE and NHE were developed and parameterized using published data on fluxes measured in isolated cardiac mitochondria. We used the integrated model to understand the integrated roles of CU, NCE and NHE in regulating trans-membrane Ca, Na and H fluxes, and to determine the consequences of these fluxes on redox state and respiration. We measured Ca, Na and H in isolated cardiac mitochondria (by ion selective fluorescence spectrophotometry) and buffer (by ion selective electrodes) in response to changing buffer levels of these ions, ADP, and inhibitors of CU, NCE and NHE. Measurements of mitochondrial Ca dynamics match model predictions and support the hypothesis that NCE is electrogenic with stoichiometry of 3:1. This integrated model will help us to understand mechanisms by which Ca plays a role in mitochondrial signaling and energetics.