Analysis of Cardiac Mitochondrial Na+/Ca2+ Exchanger Kinetics with a Biophysical Model of Mitochondrial Ca2+ Handing Suggests a 3:1 Stoichiometry
Figure 1: Fitting of Ca2+ uniporter (CU) model (lines) to data (points) on CU fluxes. (A,B) The initial rates of Ca2+ uptake (points) were measured with varying external buffer [Ca2+] in pu-rified respiring mitochondria from rat liver (A) (Vinogradov & Scarpa, 1973) and rat heart (B) (Scarpa & Graziotti, 1973). Also shown are the model-simulated CU fluxes at 5 different levels of IMM ∆ψ (lines) in which the model is fitted to the data with IMM ∆ψ = 190 mV. The estimated values of the kinetic parameters were (A) KCa,CU = 48 μM and XCU = 1.35X10-2 nmol/mg/s for rat liver mitochondria, and (B) KCa,CU = 90 μM andXCU = 1.5×10-2 nmol/mg/s for rat heart mitochondria. (C) The fitting of the same kinetic model to another data set is shown where the initial rates of Ca2+uptake were measured in purified rat liver mitochondria with varying IMM ∆ψ for 3 different levels of extramitochondrial buffer [Ca2+] (Wingrove et al., 1984; Gunter & Pfeiffer, 1990; Gunter et al., 1994). To fit the model to this data set,XCU is adjusted to 2.025 nmol/mg/s while keeping KCa,CU fixed at 48 μM. (D) Model-simulated CU fluxes are shown for purified rat heart mitochondria with varying IMM ∆ψ for 4 different levels of extramitochon-drial buffer [Ca2+] with KCa,CU = 90 μM and XCU = 2.25 nmol/mg/s.
Figure 4: Characterization of the kinetics and stoichiometry of the mitochondrial Na+/Ca2+ exchanger (NCE). (A,B) Comparison of model predictions (lines) to measurements (points) of the time courses of Ca2+ decay in purified respiring mitochondria from rabbit heart with addition of varying levels of Na+ to the external buffer medium with the activity of CU blocked with ruthenium red (Cox & Matlib, 1993). The buffer medium contained 50 μM of EGTA to buffer the extruded Ca2+. The kinetic model of the NCE is based on a 2Na+/Ca2+ stoichiometry (electroneu-tral exchange) in plot-A and a 3Na+/Ca2+stoichiometry (electrogenic exchange) in plot-B. The solid lines are the model-fitted curves with a total of 1μM of Ca2+ in the buffer (negligible com-pared to the total EGTA of 50 μM) and the dashed lines are the model-simulated profiles with a total of 40 μM of Ca2+ in the buffer (comparable with the total EGTA of 50 μM). The model fit-ting provides estimates KNa,NCE = 3.45 mM, KCa,NCE = 2.1 μM, and XNCE = 1.76×10-3 nmol Ca2+/mg/s (3.52×10-3 nmol Na+/mg/s) for a 2Na+/Ca2+ exchange and KNa,NCE = 2.4 mM, KCa,NCE = 2.1 μM, and XNCE = 3.375×10-5 nmol Ca2+/mg/s (10.125×10-5 nmol Na+/mg/s) for a 3Na+/Ca2+exchange. (C,D) Comparison of the regions of model predictions (shaded area) to the measure-ments (points) of the initial rates of decrease of matrix free [Ca2+] (C), and matrix free [Ca2+] af-ter 3 minutes of addition of Na+ (D), both as functions of external buffer [Na+] with the total ex-ternal buffer [Ca2+] varying from 1 μM to 40 μM. The model predictions are shown for both the 2Na+/Ca2+ and 3Na+/Ca2+ stoichiometry models with model parameters as described above
Dash RK, Beard DA. Analysis of cardiac mitochondrial Na+ – Ca2+ exchanger kinetics with a biophysical model of mitochondrial Ca2+ handing suggests a 3 :1 stoichiometry. J Physiol 586.13: 3267–3285, 2008.