Calcium can activate mitochondrial metabolism, and the possibility that mitochondrial Ca2+ uptake and extrusion modulate free cytosolic [Ca2+] (Cac) now has renewed interest. We use whole-cell and perforated patch clamp methods together with rapid local perfusion to introduce probes and inhibitors to rat chromaffin cells, to evoke Ca2+ entry, and to monitor Ca2+-activated currents that report near-surface [Ca2+]. We show that rapid recovery from elevations of Cac requires both the mitochondrial Ca2+ uniporter and the mitochondrial energization that drives Ca2+ uptake through it. Applying imaging and single-cell photometric methods, we find that the probe rhod-2 selectively localizes to mitochondria and uses its responses to quantify mitochondrial free [Ca2+] (Cam). The indicated resting Cam of 100–200 nM is similar to the resting Cac reported by the probes indo-1 and Calcium Green, or its dextran conjugate in the cytoplasm. Simultaneous monitoring of Cam and Cac at high temporal resolution shows that, although Cam increases less than Cac, mitochondrial sequestration of Ca2+ is fast and has high capacity. We find that mitochondrial Ca2+ uptake limits the rise and underlies the rapid decay of Cac excursions produced by Ca2+ entry or by mobilization of reticular stores. We also find that subsequent export of Ca2+ from mitochondria, seen as declining Cam, prolongs complete Cac recovery and that suppressing export of Ca2+, by inhibition of the mitochondrial Na+/ Ca2+ exchanger, reversibly hastens final recovery of Cac. We conclude that mitochondria are active participants in cellular Ca2+ signaling, whose unique role is determined by their ability to rapidly accumulate and then release large quantities of Ca2+.

Cytosolic Ca2+ (Ca2+c) clearance from adrenal chromaffin cells was studied by whole-cell patch clamp and indo-1 Ca2+ photometry after influx of Ca2+ through voltage-dependent Ca2+ channels. We isolated the rates of Ca2+c clearance by several mechanisms using combinations of the following agents (with their expected targets): Li+ or TEA substituted for Na+ (Na(+)-Ca2+ exchange), 1 mM La3+ applied after the depolarization (Na(+)-Ca2+ exchange and plasma membrane Ca(2+)-ATPase), 1 microM thapsigargin (pumping into reticular stores), and 2 microM carbonyl cyanide m-chlorophenylhydrazone (uptake into mitochondria). Remarkably, whenever [Ca2+]c rose above approximately 500 nM, Ca2+c clearance by mitochondria exceeded clearance by either Na(+)-Ca2+ exchange or the Ca2+ pumps of the plasma and reticular membranes. As [Ca2+]c fell again, Ca2+ reemerged from mitochondria, prolonging the final return to basal levels.

1. Intracellular Ca2+ clearance mechanisms were studied in rat adrenal chromaffin cells, by measuring slow tail currents through small-conductance Ca2+-activated K+ channels and using indo-1 photometry following depolarization-induced Ca2+ loading.