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 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 Ca+) 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 approximately 400 nM [Ca2+](i), net mitochondrial Ca2+ transport is dominated by uptake and is largely insensitive to CGP. When [Ca2+](i) is approximately 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 [Ca(2+)](i) to levels below approximately 500 nM.
Colegrove
S L
SL
Department of Neuroscience, Case Western Reserve University, Cleveland, Ohio 44106, USA.
Albrecht
M A
MA
Friel
D D
DD
D000818:Animals; D001692:Biological Transport / Q000187:drug effects / Q000502:physiology; D002118:Calcium / Q000493:pharmacokinetics*; D020013:Calcium Signaling / Q000187:drug effects / Q000502:physiology; D002259:Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone / Q000494:pharmacology; D002998:Clonazepam / Q000031:analogs & derivatives / Q000494:pharmacology; D008297:Male; D008928:Mitochondria / Q000737:chemistry* / Q000378:metabolism*; D009474:Neurons / Q000737:chemistry* / Q000378:metabolism*; D018408:Patch-Clamp Techniques; D011188:Potassium / Q000494:pharmacology; D011892:Rana catesbeiana; D012964:Sodium / Q000494:pharmacology; D013564:Sympathetic Nervous System / Q000166:cytology; D013841:Thiazepines / Q000494:pharmacology; D014475:Uncoupling Agents / Q000494:pharmacology
D013841:Thiazepines; D014475:Uncoupling Agents; D002259:Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; D002998:Clonazepam; C079032:CGP 37157; D012964:Sodium; D011188:Potassium; D002118:Calcium