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Mitochondrial regulation of store-operated calcium signaling in T lymphocytes.

Hoth M, Fanger CM, Lewis RS - J. Cell Biol. (1997)

Bottom Line: Ca2+ uptake by the mitochondrial store is sensitive (threshold is <400 nM cytosolic Ca2+), rapid (detectable within 8 s), and does not readily saturate.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cellular Physiology, Stanford University School of Medicine, California 94305-5426, USA. mhoth@leland.stanford.edu

ABSTRACT
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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A hypothesis for  mitochondrial modulation of  capacitative Ca2+ entry. (A)  When Ca2+ uptake by mitochondria is blocked, Ca2+ accumulates near CRAC channels and inhibits them (bold  dashed line). A steady-state  [Ca2+]i near the channels is  reached as a result of this negative feedback. (B) Sequestration of Ca2+ by mitochondria  in the absence of export reduces the local [Ca2+]i, thereby  partially relieving inactivation of ICRAC (thin dashed line). At steady state, ICRAC is larger than in A, but much of the Ca2+ from this increased influx is sequestered and hence is not available to enhance global [Ca2+]i. (C) In the presence of normal mitochondrial uptake  and release of Ca2+, mitochondria redistribute the Ca2+ derived from enhanced ICRAC to distant sites where it can elevate global [Ca2+]i.
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Figure 10: A hypothesis for mitochondrial modulation of capacitative Ca2+ entry. (A) When Ca2+ uptake by mitochondria is blocked, Ca2+ accumulates near CRAC channels and inhibits them (bold dashed line). A steady-state [Ca2+]i near the channels is reached as a result of this negative feedback. (B) Sequestration of Ca2+ by mitochondria in the absence of export reduces the local [Ca2+]i, thereby partially relieving inactivation of ICRAC (thin dashed line). At steady state, ICRAC is larger than in A, but much of the Ca2+ from this increased influx is sequestered and hence is not available to enhance global [Ca2+]i. (C) In the presence of normal mitochondrial uptake and release of Ca2+, mitochondria redistribute the Ca2+ derived from enhanced ICRAC to distant sites where it can elevate global [Ca2+]i.

Mentions: Inhibition of either mitochondrial Ca2+ uptake (by CCCP or antimycin A1) or export (by Na+ depletion) attenuates the high Ca2+ plateau, suggesting that both uptake and export of Ca2+ are necessary to maintain a high rate of capacitative Ca2+ entry. A model based on Ca2+-dependent inhibition of CRAC channels (Hoth and Penner, 1993; Zweifach and Lewis, 1995a,b) may provide a way to reconcile these results. As shown in Fig. 10 A, in the absence of functional mitochondria (e.g., with CCCP or antimycin + oligomycin present), Ca2+ entering the cell through CRAC channels accumulates near the plasma membrane and inhibits Ca2+ entry, leading eventually to a steadystate level of ICRAC and [Ca2+]i. Intracellular Ca2+ inhibits ICRAC in several ways. Fast inactivation is thought to involve binding of Ca2+ to sites located within several nanometers of the intracellular mouth of the channels (Zweifach and Lewis, 1995a), while slow inactivation occurs via store refilling as well as by a store-independent process that persists in the presence of TG (Zweifach and Lewis, 1995b). Slow inactivation can be prevented by intracellular EGTA, implying that the Ca2+ binding sites underlying this process are located further than ∼100 nm from the channels (Zweifach and Lewis, 1995a,b). Considering the evidence that mitochondria can sense [Ca2+]i gradients near CRAC channels (Fig. 3), it seems reasonable that they may reduce [Ca2+]i at locations that influence slow inactivation. Effects on fast inactivation are less likely given the extreme constraints that the inactivation site would place on mitochondrial location and uptake rates. Thus, when mitochondria are allowed to take up but not export Ca2+ (e.g., in Na+-depleted cells; Fig. 10 B), they may act as a sink to reduce the local [Ca2+]i. The reduction of local [Ca2+]i would lessen CRAC channel inactivation until a new steady state is reached, in which ICRAC is somewhat greater than it was before and the local [Ca2+]i is somewhat less than it was before. Thus, the paradoxical result would be that, without export, mitochondrial uptake would enhance ICRAC but not necessarily global [Ca2+]i. Only if Ca2+ export from mitochondria is also permitted (e.g., in untreated cells; Fig. 10 C) can the increased Ca2+ influx contribute effectively to the global [Ca2+]i. Thus, mitochondria may serve two functions; as Ca2+ buffers, they remove Ca2+ from sites where it inhibits CRAC channels, and through Ca2+ export, they redistribute it to distant sites where it does not inhibit ICRAC. To explain why transitions are uncommon in normal cells, we would speculate that the redistribution of Ca2+ by mitochondria helps to prevent transitions. As discussed above, further study will be required to test this hypothesis.


Mitochondrial regulation of store-operated calcium signaling in T lymphocytes.

Hoth M, Fanger CM, Lewis RS - J. Cell Biol. (1997)

A hypothesis for  mitochondrial modulation of  capacitative Ca2+ entry. (A)  When Ca2+ uptake by mitochondria is blocked, Ca2+ accumulates near CRAC channels and inhibits them (bold  dashed line). A steady-state  [Ca2+]i near the channels is  reached as a result of this negative feedback. (B) Sequestration of Ca2+ by mitochondria  in the absence of export reduces the local [Ca2+]i, thereby  partially relieving inactivation of ICRAC (thin dashed line). At steady state, ICRAC is larger than in A, but much of the Ca2+ from this increased influx is sequestered and hence is not available to enhance global [Ca2+]i. (C) In the presence of normal mitochondrial uptake  and release of Ca2+, mitochondria redistribute the Ca2+ derived from enhanced ICRAC to distant sites where it can elevate global [Ca2+]i.
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Related In: Results  -  Collection

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Figure 10: A hypothesis for mitochondrial modulation of capacitative Ca2+ entry. (A) When Ca2+ uptake by mitochondria is blocked, Ca2+ accumulates near CRAC channels and inhibits them (bold dashed line). A steady-state [Ca2+]i near the channels is reached as a result of this negative feedback. (B) Sequestration of Ca2+ by mitochondria in the absence of export reduces the local [Ca2+]i, thereby partially relieving inactivation of ICRAC (thin dashed line). At steady state, ICRAC is larger than in A, but much of the Ca2+ from this increased influx is sequestered and hence is not available to enhance global [Ca2+]i. (C) In the presence of normal mitochondrial uptake and release of Ca2+, mitochondria redistribute the Ca2+ derived from enhanced ICRAC to distant sites where it can elevate global [Ca2+]i.
Mentions: Inhibition of either mitochondrial Ca2+ uptake (by CCCP or antimycin A1) or export (by Na+ depletion) attenuates the high Ca2+ plateau, suggesting that both uptake and export of Ca2+ are necessary to maintain a high rate of capacitative Ca2+ entry. A model based on Ca2+-dependent inhibition of CRAC channels (Hoth and Penner, 1993; Zweifach and Lewis, 1995a,b) may provide a way to reconcile these results. As shown in Fig. 10 A, in the absence of functional mitochondria (e.g., with CCCP or antimycin + oligomycin present), Ca2+ entering the cell through CRAC channels accumulates near the plasma membrane and inhibits Ca2+ entry, leading eventually to a steadystate level of ICRAC and [Ca2+]i. Intracellular Ca2+ inhibits ICRAC in several ways. Fast inactivation is thought to involve binding of Ca2+ to sites located within several nanometers of the intracellular mouth of the channels (Zweifach and Lewis, 1995a), while slow inactivation occurs via store refilling as well as by a store-independent process that persists in the presence of TG (Zweifach and Lewis, 1995b). Slow inactivation can be prevented by intracellular EGTA, implying that the Ca2+ binding sites underlying this process are located further than ∼100 nm from the channels (Zweifach and Lewis, 1995a,b). Considering the evidence that mitochondria can sense [Ca2+]i gradients near CRAC channels (Fig. 3), it seems reasonable that they may reduce [Ca2+]i at locations that influence slow inactivation. Effects on fast inactivation are less likely given the extreme constraints that the inactivation site would place on mitochondrial location and uptake rates. Thus, when mitochondria are allowed to take up but not export Ca2+ (e.g., in Na+-depleted cells; Fig. 10 B), they may act as a sink to reduce the local [Ca2+]i. The reduction of local [Ca2+]i would lessen CRAC channel inactivation until a new steady state is reached, in which ICRAC is somewhat greater than it was before and the local [Ca2+]i is somewhat less than it was before. Thus, the paradoxical result would be that, without export, mitochondrial uptake would enhance ICRAC but not necessarily global [Ca2+]i. Only if Ca2+ export from mitochondria is also permitted (e.g., in untreated cells; Fig. 10 C) can the increased Ca2+ influx contribute effectively to the global [Ca2+]i. Thus, mitochondria may serve two functions; as Ca2+ buffers, they remove Ca2+ from sites where it inhibits CRAC channels, and through Ca2+ export, they redistribute it to distant sites where it does not inhibit ICRAC. To explain why transitions are uncommon in normal cells, we would speculate that the redistribution of Ca2+ by mitochondria helps to prevent transitions. As discussed above, further study will be required to test this hypothesis.

Bottom Line: Ca2+ uptake by the mitochondrial store is sensitive (threshold is <400 nM cytosolic Ca2+), rapid (detectable within 8 s), and does not readily saturate.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cellular Physiology, Stanford University School of Medicine, California 94305-5426, USA. mhoth@leland.stanford.edu

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus