Mitochondrial Participation in the Intracellular Ca(2+) Network

Calcium can activate mitochondrial metabolism, and the possibility that mitochondrial Ca(2+) uptake and extrusion modulate free cytosolic [Ca(2+)] (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 Ca(2+) entry, and to monitor Ca(2+)-activated currents that report near-surface [Ca(2+)]. We show that rapid recovery from elevations of Ca(c) requires both the mitochondrial Ca(2+) uniporter and the mitochondrial energization that drives Ca(2+) 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 [Ca(2+)] (Ca(m)). 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 Ca(2+) is fast and has high capacity. We find that mitochondrial Ca(2+) uptake limits the rise and underlies the rapid decay of Ca(c) excursions produced by Ca(2+) entry or by mobilization of reticular stores. We also find that subsequent export of Ca(2+) from mitochondria, seen as declining Ca(m), prolongs complete Ca(c) recovery and that suppressing export of Ca(2+), by inhibition of the mitochondrial Na(+)/ Ca(2+) exchanger, reversibly hastens final recovery of Ca(c). We conclude that mitochondria are active participants in cellular Ca(2+) signaling, whose unique role is determined by their ability to rapidly accumulate and then release large quantities of Ca(2+).