Mitochondrial Participation in the Intracellular Ca2+Network

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 Cacrequires 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 Camof 100-200 nM is similar to the resting Cacreported by the probes indo-1 and Calcium Green, or its dextran conjugate in the cytoplasm. Simultaneous monitoring of Camand Cacat high temporal resolution shows that, although Camincreases 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 Cacexcursions 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 Cacrecovery 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+.