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+] (Ca-c) 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 Ca-c 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+] (Ca-c), The indicated resting Ca-m of 100-200 nM is similar to the resting Ca-c reported by the probes indo-1 and Calcium Green, or its dextran conjugate in the cytoplasm. Simultaneous monitoring of Ca-m and Ca-c at high temporal resolution shows that, although Ca-m increases less than Ca-c, 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 Ca-c excursions produced by Ca2+ entry or by mobilization of reticular stores. We also find that subsequent export of Ca2+ from mitochondria, seen as declining Ca-m prolongs complete Ca-c recovery and that suppressing export of Ca2+, by inhibition of the mitochondrial Na+/Ca2+ exchanger, reversibly hastens final recovery of Ca-c. 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+.