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Single channel properties and regulated expression of Ca(2+) release-activated Ca(2+) (CRAC) channels in human T cells.

Fomina AF, Fanger CM, Kozak JA, Cahalan MD - J. Cell Biol. (2000)

Bottom Line: Passive Ca(2+) store depletion resulted in the opening of 41-pS CRAC channels characterized by high open probabilities, voltage-dependent block by extracellular Ca(2+) in the micromolar range, selective Ca(2+) permeation in the millimolar range, and inactivation that depended upon intracellular Mg(2+) ions.Capacitative Ca(2+) influx induced by thapsigargin was also significantly enhanced in activated T cells.We conclude that a surprisingly low number of CRAC channels are sufficient to mediate Ca(2+) influx in human resting T cells, and that the expression of CRAC channels increases approximately 10-fold during activation, resulting in enhanced Ca(2+) signaling.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology and Biophysics, University of California Irvine, Irvine, California 92697-4561, USA.

ABSTRACT
Although the crucial role of Ca(2+) influx in lymphocyte activation has been well documented, little is known about the properties or expression levels of Ca(2+) channels in normal human T lymphocytes. The use of Na(+) as the permeant ion in divalent-free solution permitted Ca(2+) release-activated Ca(2+) (CRAC) channel activation, kinetic properties, and functional expression levels to be investigated with single channel resolution in resting and phytohemagglutinin (PHA)-activated human T cells. Passive Ca(2+) store depletion resulted in the opening of 41-pS CRAC channels characterized by high open probabilities, voltage-dependent block by extracellular Ca(2+) in the micromolar range, selective Ca(2+) permeation in the millimolar range, and inactivation that depended upon intracellular Mg(2+) ions. The number of CRAC channels per cell increased greatly from approximately 15 in resting T cells to approximately 140 in activated T cells. Treatment with the phorbol ester PMA also increased CRAC channel expression to approximately 60 channels per cell, whereas the immunosuppressive drug cyclosporin A (1 microM) suppressed the PHA-induced increase in functional channel expression. Capacitative Ca(2+) influx induced by thapsigargin was also significantly enhanced in activated T cells. We conclude that a surprisingly low number of CRAC channels are sufficient to mediate Ca(2+) influx in human resting T cells, and that the expression of CRAC channels increases approximately 10-fold during activation, resulting in enhanced Ca(2+) signaling.

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Na+ current through CRAC channels in a resting T cell. All currents recorded in divalent-free solution using a Cs+-aspartate pipette solution containing BAPTA with no Mg2+. (A) Sequence of channel activation displaying currents at −120 mV at the indicated times following break in. Horizontal lines are multiples of 4.8 pA, with the number of open channels shown on the left. Please note baseline shifts in channel numbers for the middle and right columns. (B) All-points amplitude histogram (bin size = 0.2 pA) accumulated during nine consecutive current traces recorded at −120 mV, beginning 135 s after break in. Note that the histogram peaks are equally spaced with intervals of 4.8 pA, corresponding to the sequential and sustained opening of eight channels. (C) Time course of whole-cell current recorded from the same resting T cell. The current amplitude at each time point was measured off-line by averaging 5-ms intervals randomly selected within square-pulse traces. (D) Open probability (Po) obtained from current traces with one channel active at the beginning of current activation (a) and after currents ran down to a single remaining active channel (b). a and b correspond to time intervals marked on C. Po values were measured by integrating current traces containing one active channel. Missing points within the horizontal scale break represent skipped traces with more than one channel active. (E) Activation by passive store dialysis, IP3, and Tg. Average time course of CRAC channel currents from representative resting T cells dialyzed with either normal BAPTA-containing pipette solution (Control, •, n = 4), with 30 μM IP3 added to the normal pipette solution (IP3, ▪, n = 3), or after preincubation with 2 μM Tg for 15–45 min (Tg, ▵, n = 3). All current traces were normalized to maximal current amplitude and then averaged.
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Figure 1: Na+ current through CRAC channels in a resting T cell. All currents recorded in divalent-free solution using a Cs+-aspartate pipette solution containing BAPTA with no Mg2+. (A) Sequence of channel activation displaying currents at −120 mV at the indicated times following break in. Horizontal lines are multiples of 4.8 pA, with the number of open channels shown on the left. Please note baseline shifts in channel numbers for the middle and right columns. (B) All-points amplitude histogram (bin size = 0.2 pA) accumulated during nine consecutive current traces recorded at −120 mV, beginning 135 s after break in. Note that the histogram peaks are equally spaced with intervals of 4.8 pA, corresponding to the sequential and sustained opening of eight channels. (C) Time course of whole-cell current recorded from the same resting T cell. The current amplitude at each time point was measured off-line by averaging 5-ms intervals randomly selected within square-pulse traces. (D) Open probability (Po) obtained from current traces with one channel active at the beginning of current activation (a) and after currents ran down to a single remaining active channel (b). a and b correspond to time intervals marked on C. Po values were measured by integrating current traces containing one active channel. Missing points within the horizontal scale break represent skipped traces with more than one channel active. (E) Activation by passive store dialysis, IP3, and Tg. Average time course of CRAC channel currents from representative resting T cells dialyzed with either normal BAPTA-containing pipette solution (Control, •, n = 4), with 30 μM IP3 added to the normal pipette solution (IP3, ▪, n = 3), or after preincubation with 2 μM Tg for 15–45 min (Tg, ▵, n = 3). All current traces were normalized to maximal current amplitude and then averaged.

Mentions: Fig. 1 shows inward Na+ current through single CRAC channels in a resting T cell. After patch rupture (break in) to initiate whole-cell recording, the first indication of channel activity was the appearance of very brief opening events, <1 ms in duration, within an average of 68 ± 12 s after break in (n = 18 cells; Fig. 1 A, top left trace). Brief openings were typically observed for ∼1 min, and then the first open channel stabilized abruptly to a state characterized by long openings, short closed events, and high open probability (Po = 0.95 at –120 mV). The average latency between break in and stable openings of the first channel was 116.4 ± 11.3 s (n = 33). Additional single channels opened sequentially, with an average time constant of activation (τact) of 85 ± 15 s (n = 12). Amplitude histograms during the activation phase showed equally spaced peaks of 4.8 ± 0.03 pA at −120 mV (n = 23), corresponding to a single channel chord conductance of 41 pS (Fig. 1 B). Fig. 1 C shows the time course of whole-cell current illustrating activation of a total of 16 channels in a resting T cell. The number of open channels gradually declined with a run-down time constant (τr) of 940 ± 430 s (n = 12). Single channel activity at the beginning of activation and at the end of run-down exhibited the same high Po values and open and closed channel lifetimes (Fig. 1A and Fig. D), indicating that changes in the number of open channels underlie both activation and run-down processes. As expected for a store-operated channel, IP3 (30 μM in the pipette) significantly decreased the latency between break in and the onset of channel activation (29.8 ± 15.3 s, n = 13), without affecting the average total number of open channels (Fig. 1 E). In cells pretreated with Tg (2 μM in Ringer for 15–45 min), many channels were already open and current was observed immediately after break in, with an additional increase during prolonged recording (Fig. 1 E). These results indicate that single CRAC channels can be detected in human T cells using Na+ as the permeant ion, permitting channel activation, kinetic properties, and functional expression levels to be investigated with single channel resolution. In resting T cells, the number of CRAC channels simultaneously open during whole cell recording ranged from 2 to 50 per cell.


Single channel properties and regulated expression of Ca(2+) release-activated Ca(2+) (CRAC) channels in human T cells.

Fomina AF, Fanger CM, Kozak JA, Cahalan MD - J. Cell Biol. (2000)

Na+ current through CRAC channels in a resting T cell. All currents recorded in divalent-free solution using a Cs+-aspartate pipette solution containing BAPTA with no Mg2+. (A) Sequence of channel activation displaying currents at −120 mV at the indicated times following break in. Horizontal lines are multiples of 4.8 pA, with the number of open channels shown on the left. Please note baseline shifts in channel numbers for the middle and right columns. (B) All-points amplitude histogram (bin size = 0.2 pA) accumulated during nine consecutive current traces recorded at −120 mV, beginning 135 s after break in. Note that the histogram peaks are equally spaced with intervals of 4.8 pA, corresponding to the sequential and sustained opening of eight channels. (C) Time course of whole-cell current recorded from the same resting T cell. The current amplitude at each time point was measured off-line by averaging 5-ms intervals randomly selected within square-pulse traces. (D) Open probability (Po) obtained from current traces with one channel active at the beginning of current activation (a) and after currents ran down to a single remaining active channel (b). a and b correspond to time intervals marked on C. Po values were measured by integrating current traces containing one active channel. Missing points within the horizontal scale break represent skipped traces with more than one channel active. (E) Activation by passive store dialysis, IP3, and Tg. Average time course of CRAC channel currents from representative resting T cells dialyzed with either normal BAPTA-containing pipette solution (Control, •, n = 4), with 30 μM IP3 added to the normal pipette solution (IP3, ▪, n = 3), or after preincubation with 2 μM Tg for 15–45 min (Tg, ▵, n = 3). All current traces were normalized to maximal current amplitude and then averaged.
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Related In: Results  -  Collection

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Figure 1: Na+ current through CRAC channels in a resting T cell. All currents recorded in divalent-free solution using a Cs+-aspartate pipette solution containing BAPTA with no Mg2+. (A) Sequence of channel activation displaying currents at −120 mV at the indicated times following break in. Horizontal lines are multiples of 4.8 pA, with the number of open channels shown on the left. Please note baseline shifts in channel numbers for the middle and right columns. (B) All-points amplitude histogram (bin size = 0.2 pA) accumulated during nine consecutive current traces recorded at −120 mV, beginning 135 s after break in. Note that the histogram peaks are equally spaced with intervals of 4.8 pA, corresponding to the sequential and sustained opening of eight channels. (C) Time course of whole-cell current recorded from the same resting T cell. The current amplitude at each time point was measured off-line by averaging 5-ms intervals randomly selected within square-pulse traces. (D) Open probability (Po) obtained from current traces with one channel active at the beginning of current activation (a) and after currents ran down to a single remaining active channel (b). a and b correspond to time intervals marked on C. Po values were measured by integrating current traces containing one active channel. Missing points within the horizontal scale break represent skipped traces with more than one channel active. (E) Activation by passive store dialysis, IP3, and Tg. Average time course of CRAC channel currents from representative resting T cells dialyzed with either normal BAPTA-containing pipette solution (Control, •, n = 4), with 30 μM IP3 added to the normal pipette solution (IP3, ▪, n = 3), or after preincubation with 2 μM Tg for 15–45 min (Tg, ▵, n = 3). All current traces were normalized to maximal current amplitude and then averaged.
Mentions: Fig. 1 shows inward Na+ current through single CRAC channels in a resting T cell. After patch rupture (break in) to initiate whole-cell recording, the first indication of channel activity was the appearance of very brief opening events, <1 ms in duration, within an average of 68 ± 12 s after break in (n = 18 cells; Fig. 1 A, top left trace). Brief openings were typically observed for ∼1 min, and then the first open channel stabilized abruptly to a state characterized by long openings, short closed events, and high open probability (Po = 0.95 at –120 mV). The average latency between break in and stable openings of the first channel was 116.4 ± 11.3 s (n = 33). Additional single channels opened sequentially, with an average time constant of activation (τact) of 85 ± 15 s (n = 12). Amplitude histograms during the activation phase showed equally spaced peaks of 4.8 ± 0.03 pA at −120 mV (n = 23), corresponding to a single channel chord conductance of 41 pS (Fig. 1 B). Fig. 1 C shows the time course of whole-cell current illustrating activation of a total of 16 channels in a resting T cell. The number of open channels gradually declined with a run-down time constant (τr) of 940 ± 430 s (n = 12). Single channel activity at the beginning of activation and at the end of run-down exhibited the same high Po values and open and closed channel lifetimes (Fig. 1A and Fig. D), indicating that changes in the number of open channels underlie both activation and run-down processes. As expected for a store-operated channel, IP3 (30 μM in the pipette) significantly decreased the latency between break in and the onset of channel activation (29.8 ± 15.3 s, n = 13), without affecting the average total number of open channels (Fig. 1 E). In cells pretreated with Tg (2 μM in Ringer for 15–45 min), many channels were already open and current was observed immediately after break in, with an additional increase during prolonged recording (Fig. 1 E). These results indicate that single CRAC channels can be detected in human T cells using Na+ as the permeant ion, permitting channel activation, kinetic properties, and functional expression levels to be investigated with single channel resolution. In resting T cells, the number of CRAC channels simultaneously open during whole cell recording ranged from 2 to 50 per cell.

Bottom Line: Passive Ca(2+) store depletion resulted in the opening of 41-pS CRAC channels characterized by high open probabilities, voltage-dependent block by extracellular Ca(2+) in the micromolar range, selective Ca(2+) permeation in the millimolar range, and inactivation that depended upon intracellular Mg(2+) ions.Capacitative Ca(2+) influx induced by thapsigargin was also significantly enhanced in activated T cells.We conclude that a surprisingly low number of CRAC channels are sufficient to mediate Ca(2+) influx in human resting T cells, and that the expression of CRAC channels increases approximately 10-fold during activation, resulting in enhanced Ca(2+) signaling.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology and Biophysics, University of California Irvine, Irvine, California 92697-4561, USA.

ABSTRACT
Although the crucial role of Ca(2+) influx in lymphocyte activation has been well documented, little is known about the properties or expression levels of Ca(2+) channels in normal human T lymphocytes. The use of Na(+) as the permeant ion in divalent-free solution permitted Ca(2+) release-activated Ca(2+) (CRAC) channel activation, kinetic properties, and functional expression levels to be investigated with single channel resolution in resting and phytohemagglutinin (PHA)-activated human T cells. Passive Ca(2+) store depletion resulted in the opening of 41-pS CRAC channels characterized by high open probabilities, voltage-dependent block by extracellular Ca(2+) in the micromolar range, selective Ca(2+) permeation in the millimolar range, and inactivation that depended upon intracellular Mg(2+) ions. The number of CRAC channels per cell increased greatly from approximately 15 in resting T cells to approximately 140 in activated T cells. Treatment with the phorbol ester PMA also increased CRAC channel expression to approximately 60 channels per cell, whereas the immunosuppressive drug cyclosporin A (1 microM) suppressed the PHA-induced increase in functional channel expression. Capacitative Ca(2+) influx induced by thapsigargin was also significantly enhanced in activated T cells. We conclude that a surprisingly low number of CRAC channels are sufficient to mediate Ca(2+) influx in human resting T cells, and that the expression of CRAC channels increases approximately 10-fold during activation, resulting in enhanced Ca(2+) signaling.

Show MeSH
Related in: MedlinePlus