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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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The TG-insensitive store senses Ca2+ near CRAC channels. Experimental conditions and analysis were identical to Fig. 2 with  the exception that conditions during the plateau phase were varied. (A and B) 2 mM Ca2+ (dotted trace, ○) or 20 mM Ca2+ with 8 mM  Ni2+ (solid trace) or 5–10 mM Ni2+ (•) was added to create plateaus of comparable magnitude. (C and D) 2 mM Ca2+ was applied to parental Jurkat cells (dotted trace, ○), and 20 mM Ca2+ was applied to CJ-4 cells (solid trace, •), a Jurkat mutant with reduced CRAC  channel function. (E and F) 20 mM Ca2+ was added (solid trace, •) and compared to the response to 2 mM Ca2+ (○, data from Fig. 2 B).  Graphs show mean values ± SD: parent cells, 2 mM Ca2+ (964 cells/11 experiments), 20 mM Ca2+ + Ni2+ (604/6), 20 mM Ca2+ (439/3);  CJ-4 cells, 20 mM Ca2+ (900/5). Each of the experiments in A, C, and E represents the average response of ∼300 cells.
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Figure 3: The TG-insensitive store senses Ca2+ near CRAC channels. Experimental conditions and analysis were identical to Fig. 2 with the exception that conditions during the plateau phase were varied. (A and B) 2 mM Ca2+ (dotted trace, ○) or 20 mM Ca2+ with 8 mM Ni2+ (solid trace) or 5–10 mM Ni2+ (•) was added to create plateaus of comparable magnitude. (C and D) 2 mM Ca2+ was applied to parental Jurkat cells (dotted trace, ○), and 20 mM Ca2+ was applied to CJ-4 cells (solid trace, •), a Jurkat mutant with reduced CRAC channel function. (E and F) 20 mM Ca2+ was added (solid trace, •) and compared to the response to 2 mM Ca2+ (○, data from Fig. 2 B). Graphs show mean values ± SD: parent cells, 2 mM Ca2+ (964 cells/11 experiments), 20 mM Ca2+ + Ni2+ (604/6), 20 mM Ca2+ (439/3); CJ-4 cells, 20 mM Ca2+ (900/5). Each of the experiments in A, C, and E represents the average response of ∼300 cells.

Mentions: To quantify loading of the TG-insensitive store (see Figs. 2 and 3), cells were chosen for analysis if they satisfied two criteria: (a) [Ca2+]i was approximately constant (Δ[Ca2+]i/Δt between −0.1 and 0.1 nM/s) over the final 800 s of the Ca2+ readdition period; and (b) [Ca2+]i did not oscillate or undergo spontaneous transitions (e.g., see Fig. 9). 33–53% of the cells met these two criteria. The relative store content was estimated in these cells from the amount of Ca2+ released by 4 μM ionomycin in three different ways: (a) from the maximum rate of [Ca2+]i rise, (b) from the peak [Ca2+]i as fitted by a polynomial function, and (c) by the integral of [Ca2+]i during the ionomycin release transient after correction for the ongoing clearance rate (Bergling, S., R. Dolmetsch, R.S. Lewis, and J. Keizer, manuscript in preparation). Upon addition of ionomycin, the rate at which [Ca2+]i changes is proportional to the rate of Ca2+ release from the TG-insensitive store minus the rate of Ca2+ clearance across the plasma membrane. The clearance rate in Jurkat cells is an approximately linear function of [Ca2+]i up to 1.5 μM (rate constant = 0.057 s−1); thus the clearance rate as a function of time can be estimated by scaling [Ca2+]i by this rate constant. By adding the clearance rate at each time point to the corresponding time derivative of [Ca2+]i, we obtained the rate of release from the store. This curve was then integrated to give a measure of total mitochondrial Ca2+ released. This method is equivalent to a summation of the amount of Ca2+ irreversibly pumped across the plasma membrane, which reflects the total Ca2+ released from mitochondria under the given conditions.


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

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

The TG-insensitive store senses Ca2+ near CRAC channels. Experimental conditions and analysis were identical to Fig. 2 with  the exception that conditions during the plateau phase were varied. (A and B) 2 mM Ca2+ (dotted trace, ○) or 20 mM Ca2+ with 8 mM  Ni2+ (solid trace) or 5–10 mM Ni2+ (•) was added to create plateaus of comparable magnitude. (C and D) 2 mM Ca2+ was applied to parental Jurkat cells (dotted trace, ○), and 20 mM Ca2+ was applied to CJ-4 cells (solid trace, •), a Jurkat mutant with reduced CRAC  channel function. (E and F) 20 mM Ca2+ was added (solid trace, •) and compared to the response to 2 mM Ca2+ (○, data from Fig. 2 B).  Graphs show mean values ± SD: parent cells, 2 mM Ca2+ (964 cells/11 experiments), 20 mM Ca2+ + Ni2+ (604/6), 20 mM Ca2+ (439/3);  CJ-4 cells, 20 mM Ca2+ (900/5). Each of the experiments in A, C, and E represents the average response of ∼300 cells.
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Related In: Results  -  Collection

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Figure 3: The TG-insensitive store senses Ca2+ near CRAC channels. Experimental conditions and analysis were identical to Fig. 2 with the exception that conditions during the plateau phase were varied. (A and B) 2 mM Ca2+ (dotted trace, ○) or 20 mM Ca2+ with 8 mM Ni2+ (solid trace) or 5–10 mM Ni2+ (•) was added to create plateaus of comparable magnitude. (C and D) 2 mM Ca2+ was applied to parental Jurkat cells (dotted trace, ○), and 20 mM Ca2+ was applied to CJ-4 cells (solid trace, •), a Jurkat mutant with reduced CRAC channel function. (E and F) 20 mM Ca2+ was added (solid trace, •) and compared to the response to 2 mM Ca2+ (○, data from Fig. 2 B). Graphs show mean values ± SD: parent cells, 2 mM Ca2+ (964 cells/11 experiments), 20 mM Ca2+ + Ni2+ (604/6), 20 mM Ca2+ (439/3); CJ-4 cells, 20 mM Ca2+ (900/5). Each of the experiments in A, C, and E represents the average response of ∼300 cells.
Mentions: To quantify loading of the TG-insensitive store (see Figs. 2 and 3), cells were chosen for analysis if they satisfied two criteria: (a) [Ca2+]i was approximately constant (Δ[Ca2+]i/Δt between −0.1 and 0.1 nM/s) over the final 800 s of the Ca2+ readdition period; and (b) [Ca2+]i did not oscillate or undergo spontaneous transitions (e.g., see Fig. 9). 33–53% of the cells met these two criteria. The relative store content was estimated in these cells from the amount of Ca2+ released by 4 μM ionomycin in three different ways: (a) from the maximum rate of [Ca2+]i rise, (b) from the peak [Ca2+]i as fitted by a polynomial function, and (c) by the integral of [Ca2+]i during the ionomycin release transient after correction for the ongoing clearance rate (Bergling, S., R. Dolmetsch, R.S. Lewis, and J. Keizer, manuscript in preparation). Upon addition of ionomycin, the rate at which [Ca2+]i changes is proportional to the rate of Ca2+ release from the TG-insensitive store minus the rate of Ca2+ clearance across the plasma membrane. The clearance rate in Jurkat cells is an approximately linear function of [Ca2+]i up to 1.5 μM (rate constant = 0.057 s−1); thus the clearance rate as a function of time can be estimated by scaling [Ca2+]i by this rate constant. By adding the clearance rate at each time point to the corresponding time derivative of [Ca2+]i, we obtained the rate of release from the store. This curve was then integrated to give a measure of total mitochondrial Ca2+ released. This method is equivalent to a summation of the amount of Ca2+ irreversibly pumped across the plasma membrane, which reflects the total Ca2+ released from mitochondria under the given conditions.

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