Sodium Effects on Calcium Dynamics in Mitochondrial ion Circuits

Abstract

Mitochondrial Ca2+ is modulated by the mitochondrial Ca2+ uniporter(mCU) and the mitochondrial Na+/Ca2+ exchanger(mNCE) to influence NADH production through Ca2+-sensitive dehydrogenases. Alterations in cytosolic Na+ will significantly impact the rate of mitochondrial Ca2+ accumulation by changing the driving force for Ca2+ efflux through mNCE when the amplitude or frequency of the cytosolic Ca2+ transient changes. In the present study, a protocol to selectively measure Ca2+ influx and efflux rates from guinea pig heart mitochondria was developed by utilizing an extra-mitochondrial Ca2+ indicator(CaGreen) and the inhibitors Ru360 and CGP-37157, while varying extramitochondrial free Ca2+ from 2 to 20μM, and extramitochondrial Na+ from 0 to 60mM. The extra-mitochondrial Ca2+, mitochondrial membrane potential, NADH, and mitochondrial volume were monitored simultaneously using a wavelength scanning fluorometer. Maximal mCU velocity with efflux blocked by either CGP or 0mM Na+ varied as a function of extramitochondrial Ca2+ (0.5 nmol/mg/sec with a pulse of 15uM free Ca2+) while Ca2+ efflux rates initiated by addition of Ru360 after Ca2+ uptake varied as a function of Na+, increasing from 0.02 to 0.07 nmol/mg/sec from 5 to 15 mM Na+ and then paradoxically decreasing at extramitochondrial Na+ of 30mM. The biphasic Na+ effect was hypothesized to be the result of high Na+ exceeds the capacity of the proton pumps to support the Na+ extrusion coupled to Na+/H+ exchange. To test this hypothesis, the measured calcium rates were incorporated into our isolated mitochondria computational model and a single calcium pulse was simulated in a closed compartment under varied sodium concentrations as in the cuvette conditions. The simulations showed that Na+/H+ exchange rate and proton pump rate decrease at high sodium concentrations, which verify the hypothesis. The study supports Na+ playing a regulatory role in mitochondria Ca2+ through coupled mitochondrial ion circuits.