Dissection of mitochondrial Ca2+ uptake and release fluxes in situ after depolarization-evoked [Ca2+](i) elevations in sympathetic neurons

We studied how mitochondrial Ca2+ transport influences [Ca2+](i) dynamics i n sympathetic neurons. Cells were treated with thapsigargin to inhibit Ca2 accumulation by SERCA pumps and depolarized to elevate [Ca2+](i); the reco very that followed repolarization was then examined. The total Ca2+ flux re sponsible for the [Ca2+](i) recovery was separated into mitochondrial and n onmitochondrial components based on sensitivity to the proton ionophore FCC P, a selective inhibitor of mitochondrial Ca2+ transport in these cells. Th e nonmitochondrial flux, representing net Ca2+ extrusion across the plasma membrane, has a simple dependence on [Ca2+](i), while the net mitochondrial flux (J(mito)) is bipbasic, indicative of Ca2+ accumulation during the ini tial phase of recovery when [Ca2+](i) is high, and net Ca2+ release during later phases of recovery. During each phase, mitochondrial Ca2+ transport h as distinct effects on recovery kinetics. J(mito) was separated into compon ents representing mitochondrial Ca2+ uptake and release based on sensitivit y to the specific mitochondrial Na+/Ca2+ exchange inhibitor; CGP 37157 (CGP ). The CGP-resistant (uptake) component of J(mito) increases steeply with [ Ca2+](i), as expected for transport by the mitochondrial uniporter. The CGP -sensitive (release) component is inhibited by lowering the intracellular N a+ concentration and depends on both intra- and extramitochondrial Ca2+ con centration, as expected for the Na+/Ca2+ exchanger. Above similar to 400 nM [Ca2+](i), net mitochondrial Ca2+ transport is dominated by uptake and is largely insensitive to CGP. When [Ca2+](i) is similar to 200-300 nM, the ne t 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 ma y provide a mechanism for quantitative control of ATP production after brie f or low frequency stimuli that raise [Ca2+](i) to levels below similar to 500 nM.