Mitochondrial Regulation of Store-operated Calcium Signaling in T Lymphocytes

Mitochondria act as potent buffers of intracellular Ca(2+) in many cells, but a more active role in modulating the generation of Ca(2+) signals is not well established. We have investigated the ability of mitochondria to modulate store-operated or “capacitative” Ca(2+) entry in Jurkat leukemic T cells and human T lymphocytes using fluorescence imaging techniques. Depletion of the ER Ca(2+) store with thapsigargin (TG) activates Ca(2+) release-activated Ca(2+) (CRAC) channels in T cells, and the ensuing influx of Ca(2+) loads a TG- insensitive intracellular store that by several criteria appears to be mitochondria. Loading of this store is prevented by carbonyl cyanide m-chlorophenylhydrazone or by antimycin A1 + oligomycin, agents that are known to inhibit mitochondrial Ca(2+) import by dissipating the mitochondrial membrane potential. Conversely, intracellular Na(+) depletion, which inhibits Na(+)-dependent Ca(2+) export from mitochondria, enhances store loading. In addition, we find that rhod-2 labels mitochondria in T cells, and it reports changes in Ca(2+) levels that are consistent with its localization in the TG-insensitive store. Ca(2+) uptake by the mitochondrial store is sensitive (threshold is <400 nM cytosolic Ca(2+)), rapid (detectable within 8 s), and does not readily saturate. The rate of mitochondrial Ca(2+) uptake is sensitive to extracellular [Ca(2+)], indicating that mitochondria sense Ca(2+) gradients near CRAC channels. Remarkably, mitochondrial uncouplers or Na(+) depletion prevent the ability of T cells to maintain a high rate of capacitative Ca(2+) entry over prolonged periods of >10 min. Under these conditions, the rate of Ca(2+) influx in single cells undergoes abrupt transitions from a high influx to a low influx state. These results demonstrate that mitochondria not only buffer the Ca(2+) that enters T cells via store-operated Ca(2+) channels, but also play an active role in modulating the rate of capacitative Ca(2+) entry.