Dissection of Mitochondrial Ca2+ Uptake and Release Fluxes in Situ after Depolarization-Evoked [Ca2+]i Elevations in Sympathetic Neurons

Abstract

We studied how mitochondrial Ca2+ transport influences [Ca2+]i dynamics in sympathetic neurons. Cells were treated with thapsigargin to inhibit Ca2+ accumulation by SERCA pumps and depolarized to elevate [Ca2+]i; the recovery that followed repolarization was then examined. The total Ca2+ flux responsible for the [Ca2+]i recovery was separated into mitochondrial and nonmitochondrial components based on sensitivity to the proton ionophore FCCP, a selective inhibitor of mitochondrial Ca2+ transport in these cells. The nonmitochondrial flux, representing net Ca2+ extrusion across the plasma membrane, has a simple dependence on [Ca2+]i, while the net mitochondrial flux (Jmito) is biphasic, indicative of Ca2+ accumulation during the initial phase of recovery when [Ca2+]i is high, and net Ca2+ release during later phases of recovery. During each phase, mitochondrial Ca2+ transport has distinct effects on recovery kinetics. Jmito was separated into components representing mitochondrial Ca2+ uptake and release based on sensitivity to the specific mitochondrial Na+/Ca2+ exchange inhibitor, CGP 37157 (CGP). The CGP-resistant (uptake) component of Jmito increases steeply with [Ca2+]i, as expected for transport by the mitochondrial uniporter. The CGP-sensitive (release) component is inhibited by lowering the intracellular Na+ concentration and depends on both intra- and extramitochondrial Ca2+ concentration, as expected for the Na+/Ca2+ exchanger. Above ∼400 nM [Ca2+]i, net mitochondrial Ca2+ transport is dominated by uptake and is largely insensitive to CGP. When [Ca2+]i is ∼200–300 nM, the net mitochondrial flux is small but represents the sum of much larger uptake and release fluxes that largely cancel. Thus, mitochondrial Ca2+ transport occurs in situ at much lower concentrations than previously thought, and may provide a mechanism for quantitative control of ATP production after brief or low frequency stimuli that raise [Ca2+]i to levels below ∼500 nM.