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Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, California 94305-5426
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 mitochondria to modulate store-operated or "capacitative"
Ca2+ entry in Jurkat leukemic T cells and human T lymphocytes using fluorescence imaging techniques. Depletion of the ER Ca2+ store with thapsigargin (TG) activates 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 antimycin A1 + oligomycin, agents that are
known to inhibit mitochondrial Ca2+ import by dissipating the mitochondrial membrane potential. Conversely, intracellular Na+ depletion, which inhibits
Na+-dependent Ca2+ export from mitochondria, enhances store loading. In addition, we find that rhod-2
labels mitochondria in T cells, and it reports changes in
Ca2+ levels that are consistent with its localization in
the TG-insensitive store. Ca2+ uptake by the mitochondrial store is sensitive (threshold is <400 nM cytosolic
Ca2+), rapid (detectable within 8 s), and does not
readily saturate. The rate of mitochondrial Ca2+ uptake
is sensitive 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 these conditions, the rate of Ca2+ 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 Ca2+ that
enters T cells via store-operated Ca2+ channels, but
also play an active role in modulating the rate of capacitative Ca2+ entry.
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