MITOCHONDRIAL REGULATION OF STORE-OPERATED CALCIUM SIGNALING IN T-LYMPHOCYTES

Mitochondria act as potent buffers of intracellular Ca2+ in many cells , but a more active role in modulating the generation of Ca2+ signals is not well established. We have investigated the ability of mitochond ria to modulate store-operated or ''capacitative'' Ca2+ entry in Jurka t leukemic T cells and human T lymphocytes using fluorescence imaging techniques. Depletion of the ER Ca2+ store with thapsigargin (TG) acti vates Ca2+ release-activated Ca2+ (CRAC) channels in T cells, and the ensuing influx of Ca2+ 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 antimy cin Al + oligomycin, agents that are known to inhibit mitochondrial Ca 2+ import by dissipating the mitochondrial membrane potential. Convers ely, intracellular Nat depletion, which inhibits Nat-dependent Ca2+ ex port from mitochondria, enhances store loading. In addition, we find t hat rhod-2 labels mitochondria in T cells, and it reports changes in C a2+ levels that are consistent with its localization in the TG-insensi tive store. Ca2+ uptake by the mitochondrial store is sensitive (thres hold is <400 nM cytosolic Ca2+), rapid (detectable within 8 s), and do es not readily saturate. The rate of mitochondrial Ca2+ uptake is sens itive to extracellular [Ca2+], indicating that mitochondria sense Ca2 gradients near CRAC channels. Remarkably, mitochondrial uncouplers or Na+ depletion prevent the ability of T cells to maintain a high rate of capacitative Ca2+ entry over prolonged periods of >10 min. Under th ese conditions, the rate of Ca2+ influx in single cells undergoes abru pt transitions from a high influx to a low influx state. These results demonstrate that mitochondria not only buffer the Ca2+ that enters T cells via store-operated Ca2+ channels, but also play an active role i n modulating the rate of capacitative Ca2+ entry.