Original Article

Dissection of Mitochondrial Ca²⁺ Uptake and Release Fluxes In Situ after Depolarization-evoked [Ca²⁺]_i Elevations in Sympathetic Neurons

Correspondence to: David D. Friel, Department of Neuroscience, Case Western Reserve University, 10900 Euclid Ave. Cleveland, OH 44106. Fax:216-368-4650 E-mail:ddf2{at}po.cwru.edu.

Released online: 28 February 2000

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 (J_mito) 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. J_mito 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 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 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.

Key Words: mitochondria, calcium, calcium signaling, neurons, CGP 37157

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