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

Abstract

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.