Dissection of Mitochondrial Ca(2+) Uptake and Release Fluxes in Situ after Depolarization-Evoked [Ca(2+)](i) Elevations in Sympathetic Neurons

We studied how mitochondrial Ca(2+) transport influences [Ca(2+)](i) dynamics in sympathetic neurons. Cells were treated with thapsigargin to inhibit Ca(2+) accumulation by SERCA pumps and depolarized to elevate [Ca(2+)](i); the recovery that followed repolarization was then examined. The total Ca(2+) flux responsible for the [Ca(2+)](i) recovery was separated into mitochondrial and nonmitochondrial components based on sensitivity to the proton ionophore FCCP, a selective inhibitor of mitochondrial Ca(2+) transport in these cells. The nonmitochondrial flux, representing net Ca(2+) extrusion across the plasma membrane, has a simple dependence on [Ca(2+)](i), while the net mitochondrial flux (J(mito)) is biphasic, indicative of Ca(2+) accumulation during the initial phase of recovery when [Ca(2+)](i) is high, and net Ca(2+) release during later phases of recovery. During each phase, mitochondrial Ca(2+) transport has distinct effects on recovery kinetics. J(mito) was separated into components representing mitochondrial Ca(2+) uptake and release based on sensitivity to the specific mitochondrial Na(+)/Ca(2+) exchange inhibitor, CGP 37157 (CGP). The CGP-resistant (uptake) component of J(mito) increases steeply with [Ca(2+)](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 Ca(2+) concentration, as expected for the Na(+)/Ca(2+) exchanger. Above ∼400 nM [Ca(2+)](i), net mitochondrial Ca(2+) transport is dominated by uptake and is largely insensitive to CGP. When [Ca(2+)](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 Ca(2+) 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 [Ca(2+)](i) to levels below ∼500 nM.