Relationship between Intracellular Calcium Store Depletion and Calcium Release-activated Calcium Current in a Mast Cell Line (RBL-1)*

Yi Huang and J. W. Putney Jr.Dagger

From the Calcium Regulation Section, Laboratory of Signal Transduction, NIEHS, National Institutes of Health, Research Triangle Park, North Carolina 27709

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

The kinetic relationship between depletion of endoplasmic reticulum calcium stores and the activation of a calcium release-activated calcium current (Icrac) was investigated in the RBL-1 mast cell line. The inositol trisphosphate receptor activator, inositol 2,4,5-trisphosphate ((2,4,5)IP3), the sarcoplasmic-endoplasmic reticulum calcium ATPase inhibitor, thapsigargin, and the calcium ionophore, ionomycin, were used to deplete stored calcium. For (2,4,5)IP3 and thapsigargin, a significant delay was observed between the initiation of calcium store depletion and the activation of Icrac. However, for ionomycin, little or no delay was observed. This may indicate that a specialized subcompartment of the endoplasmic reticulum functions as a regulator of calcium entry and that this compartment is relatively resistant to depletion by (2,4,5)IP3 and thapsigargin but not to depletion by ionomycin. For all three calcium-depleting agents, the rate of development of Icrac, once initiated, was relatively constant, suggesting an all-or-none mechanism. However, there were also clear experimental situations in which submaximal, graded depletion of stored calcium resulted in submaximal activation of Icrac. This complex behavior could also result from the existence of a specific subcompartment of endoplasmic reticulum regulating Icrac. The kinetic behavior of this compartment may not be accurately reflected by the kinetics of calcium changes in the bulk of endoplasmic reticulum. These findings add to the growing body of evidence suggesting specialization of the endoplasmic reticulum calcium stores with regard to the control of capacitative calcium entry.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

Depletion of endoplasmic reticulum Ca2+ stores by (1,4,5)IP31 is generally accompanied by an increase in Ca2+ entry across the plasma membrane. In the majority of cases, this entry seems to be signaled by depletion of the intracellular stores, a process termed capacitative calcium entry (1) or store-operated calcium entry (2). Hoth and Penner (3) first described an inward Ca2+ current in RBL cells that seemed to underlie, or at least contribute to, this entry. This current they designated Icrac for Calcium Release-Activated Calcium current. Although other distinguishable currents have been described that may represent capacitative calcium entry currents in other cell types (4), to date, Icrac is the best characterized electrophysiological manifestation of capacitative calcium entry. Thus, its properties and modes of regulation have received considerable scrutiny by a number of laboratories. For example, Hoth and Penner (5) observed a variable latency for the activation of Icrac (4-14 s) when activated by external application of ionomycin or by break-in with IP3 in the patch pipette. These investigators assumed that release of intracellular Ca2+ by these two modes was essentially instantaneous and thus concluded that the latency observed reflected the time required for steps linking intracellular Ca2+ store depletion to plasma membrane channel activation. In a more recent report, Parekh et al. (6) described an all-or-none activation of Icrac by (1,4,5)IP3, as well as a dissociation of activation of Icrac by IP3 from the activation of Ca2+ release.

In the current studies, we have further investigated the latency for activation of Icrac utilizing IP3, the Ca2+-ATPase inhibitor, thapsigargin, and ionomycin to deplete intracellular stores and have attempted to relate these latencies to observed kinetics of intracellular Ca2+ store depletion by these same reagents. Surprisingly, our findings suggest that the latency between depletion of calcium stores and Icrac activation depends on the nature of the agent used to deplete the stores. We also find that the kinetics of activation are complex, with all-or-none behavior in some but not in all instances. Our results may suggest the existence of a specialized, kinetically distinct subcompartment of the endoplasmic reticulum that functions as a regulator of capacitative calcium entry.

    EXPERIMENTAL PROCEDURES
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Introduction
Procedures
Results
Discussion
References

Cell Culture and Materials-- Rat basophilic leukemia cells (RBL-1, ATCC 1378-CRL, batch F-13352) were cultured as recommended by ATCC. Briefly, cells were cultured in Earle's minimal essential medium with Earle's salt, 10% fetal bovine serum (heat-inactivated) and 50 units/ml penicillin and 50 µg/ml streptomycin. Ionomycin and (2,4,5)IP3 were obtained from Calbiochem. Thapsigargin was purchased from LC Laboratories.

Fura-2 Loading-- The cells were allowed to attach to cover slips, were mounted in a Teflon chamber, and were incubated with 3 µM fura-2/AM (Molecular Probes) for 25 min at room temperature. The cells were then washed and bathed in normal external saline solution (see below) at room temperature for at least 10 min before [Ca2+]i measurements were made.

Fluorescence Measurements-- The fluorescence of the fura-2 loaded cells was monitored with a photomultiplier-based system, mounted on a Nikon Diaphot microscope equipped with a Nikon 40× (1.3 N.A.) Neofluor objective. The fluorescence light source was provided by a PTI dual excitation light source equipped with a light path. The light path chopper enabled rapid interchange between two excitation wavelengths (340 and 380 nm), and a photomultiplier tube monitored the emission fluorescence at 510 nm, selected by a barrier filter (Omega). All experiments were carried out at 24 °C. Calibration and calculation of [Ca2+]i were carried out as described previously (7).

Electrophysiology-- The normal extracellular medium contained (in mM): 150 NaCl, 4.7 KCl, 1.8 CaCl2, 1.13 MgCl2, 10 glucose, and 10 HEPES (pH 7.2). Nominally Ca2+-free saline had the same composition, except no CaCl2 was added. The bath volume (0.4 ml) was rapidly exchanged with a gravity perfusion system. In the figures, exact times are indicated when new bath solution was introduced, without any correction for the dead time required for a new solution to reach the cell. When (2,4,5)IP3 was introduced into the cell for measurement of Ca2+ release, the patch pipette (2-4 MOmega , Corning glass, 7052) contained (in mM): 150 KCl, 10 NaCl, 2 MgCl2, 10 HEPES, 0.1 EGTA (or 0.1 BAPTA), 50 µM fura-2 free acid, and 1 MgATP (pH 7.2). For measurement of Icrac, the pipette solution was (in mM): 140 Cs-Asp, 2 MgCl2, 10 HEPES, 10 BAPTA-Cs4, and 1 MgATP (free Ca2+ ~100 nM, pH 7.2). The bath solution contained (in mM): 140 NaCl, 4.7 KCl, 10 CsCl, 10 CaCl2 (or 10 MgCl2 for Ca2+ free solution), 1.13 MgCl2, 10 glucose, and 10 HEPES (pH 7.2).

Ruptured-patch whole-cell voltage clamp was carried out as described previously (8, 9). The holding potential was 30 mV where little or no driving force for calcium entry exists. Icrac was measured from the current resulting from voltage ramps between -100 to +60 mV over a period of 160 ms executed every 5 s. The nonspecific current (the current before the induction of Icrac or the current remaining when external Ca2+ has been removed) was subtracted. All voltages were corrected for a 10-mV liquid-junction potential. Data acquisition and analysis were performed with Axopatch-1C amplifier and PCLAMP 6.1 software (Axon Instruments, Burlingame, CA). Currents were filtered at 1 kHz and digitized at 200-µs intervals. Intracellular application of (2,4,5)IP3 or external application of thapsigargin or ionomycin induced the appearance of an inward current presumed to represent Icrac because (i) the current was strongly inwardly rectifying with a magnitude and current-voltage relationship similar to that previously described for Icrac by Hoth and Penner (3) (not shown) and (ii) the current was seen with strong intracellular calcium buffering (10 mM BAPTA) but was lost when external calcium was removed (not shown).

Because the intracellular solutions used for Ca2+ release and Icrac generally differed in the major cationic species (Cs+ versus K+), some Ca2+ release experiments were carried out utilizing the Cs+-containing solutions from the Icrac protocol (but with BAPTA reduced to 0.1 mM). No statistically significant differences in the [Ca2+]i responses were noted. For 50 µM (2,4,5)IP3 with Cs+-containing solution, peak [Ca2+]i was 229.5 ± 19.9 nM and latency was 16.5 ± 8.1 s (n = 12); with K+-containing solution peak [Ca2+]i was 272.1 ± 19.3 and latency was 15.3 ± 1.9 s (n = 12). For 100 µM (2,4,5)IP3 with Cs+-containing solution, peak [Ca2+]i was 424.6 ± 50 nM and latency was 8.0 ± 1.2 s (n = 11); with K+-containing solution peak, [Ca2+]i was 385.2 ± 36.8 nM and latency was 10.6 ± 1.1 s (n = 12).

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

Icrac Is Activated after an Apparent Delay when Calcium Stores Are Emptied with (2,4,5)IP3-- The time course of activation of calcium release and inward calcium current (Icrac) was determined in single RBL-1 cells following the introduction of (2,4,5)IP3 into the cytoplasm via patch pipettes in the whole cell configuration. (2,4,5)IP3 was used to minimize effects of inositol phosphate metabolism (7). The cells were held at 30 mV to minimize Ca2+ entry (an approach similar to that employed by Parekh et al. (6)). After establishment of the whole cell configuration, cytosolic Ca2+ rose after a short latency as a consequence of Ca2+ being released from the intracellular stores by (2,4,5)IP3 (Fig. 1A). This initial short latency is apparently the result of the time required for IP3 to diffuse into the cell and reach a critical concentration in the vicinity of IP3 receptors. We measured Icrac activation in parallel experiments because the high levels of Ca2+ buffers required in the pipette in Icrac determinations (3) prevent observation of release of stored Ca2+. Icrac was also activated following introduction of IP3 after a significant latency following the establishment of whole cell configuration (Fig. 1B). However, at each of four different concentrations of (2,4,5)IP3 the latency for Icrac activation was greater than the latency for initiation of Ca2+ release (Fig. 1C). We assume that the filling state of the intracellular stores is initially the same in both the Ca2+ release and Icrac protocols because (2,4,5)IP3 was present in the pipette at the time of break-in, and in experiments without (2,4,5)IP3, Icrac was not activated after prolonged dialysis, and basal [Ca2+]i did not change appreciably. In the Icrac protocol, [Ca2+]i does not change because the intracellular solution contained ATP as well as sufficient added Ca2+ to keep the free [Ca2+]i in the physiological range (~100 nM). At (2,4,5)IP3 concentrations of 100 and 50 µM, the latency for Icrac activation exceeded the latency for initiation as well as for the peak of Ca2+ release (Fig. 1C), suggesting a significant delay between Ca2+ release and activation of Icrac. Both the latency for Ca2+ release and the latency for Icrac activation decreased with increasing concentration of (2,4,5)IP3, consistent with a causal relationship between store depletion and activation of Icrac (Fig. 1C). Intriguingly, the development time for Icrac (from the point where Icrac starts to be activated to the point where it is fully activated) remained relatively constant regardless of the concentration of IP3. In other words, the rate of store depletion only affects the initial latency for Icrac activation, not the time course of development of Icrac, at least over this (2,4,5)IP3 concentration range. A variable delay with similar development time for Icrac at different concentrations of (1,4,5)IP3 was also reported by Parekh et al. (6).


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Fig. 1.   Ca2+ release and Icrac activation with (2,4,5)IP3. A, a single RBL-1 cell, loaded with fura-2, was held at 30 mV under conventional whole cell configuration. At time 0, the whole cell configuration was established. After a short latency, (2,4,5)IP3 (50 µM in the pipette) caused a transient Ca2+ release from intracellular Ca2+ stores. The arrows indicate the intervals for estimation of latency for release and time to peak release. B, IP3 (50 µM in the pipette) activated Icrac with a latency longer than that for Ca2+ release. The current was measured at the potential of -100 mV from voltage ramps from -100 to +60 mV and plotted versus time. The nonspecific current (the current before the induction of Icrac or the current remaining when external Ca2+ was replaced) was subtracted. The arrows indicate the intervals for estimation of latency of Icrac and development time for Icrac. C, cumulative data for the four intervals illustrated in A and B determined with four different pipette concentrations of (2,4,5)IP3. Each column represents average measurements from 6 to 15 cells. The latency for Ca2+ release and Icrac decreases with increasing concentration of IP3. However, a significant discrepancy exists between the time for store depletion and activation of Icrac at all concentrations of IP3. The development time for Icrac was relatively constant.

The extent of intracellular release of Ca2+ was determined in parallel experiments in which the status of intracellular stores was assessed by application of the calcium ionophore, ionomycin, 5 min after breaking into the cells (by which time Icrac had reached its plateau level, Fig. 2A). Despite differing latencies for initiation of Ca2+ release and Icrac, at all but the lowest concentration of (2,4,5)IP3 (10 µM), the magnitude, both of intracellular release of Ca2+ and of Icrac, was relatively constant (Fig. 2, B and C). At 10 µM (2,4,5)IP3, however, both intracellular release and steady-state Icrac were clearly less than maximal (Fig. 2, B and C). In experiments with still lower concentrations of (2,4,5)IP3 (5 µM for example), a proportion of cells did not respond with release or with Icrac activation. Thus, over this range of concentrations of (2,4,5)IP3, Icrac activation seems all-or-none as described previously for (1,4,5)IP3 (6). However, this seems to result from the fact that most concentrations of IP3, which are sufficient to induce release of Ca2+, induce an all-or-none release of Ca2+. At 10 µM (2,4,5)IP3, where release is submaximal, steady-state Icrac activation is also submaximal (similar finding was reported for Icrac by Parekh et al. (6)). This indicates that at least over a narrow range of IP3 concentrations, activation of Icrac is likely a graded function of the extent of intracellular Ca2+ store depletion (see also data below with ionomycin, Fig. 4).


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Fig. 2.   Relationship between the extent of Ca2+ store depletion and activation of Icrac. A, protocol used. 300 s after breaking into fura-2-loaded cells with pipettes containing one of the four concentrations of (2,4,5)IP3, a high (5 µM) concentration of ionomycin was added to assess the content of intracellular stores. In this example, the dashed line indicates a control experiment (no (2,4,5)IP3 in the pipette), and the solid line indicates the result with 25 µM (2,4,5)IP3 in the pipette. B, average data from six to nine separate experiments (means ± S.E.). Baseline [Ca2+]i was not subtracted and is indicated by the arrow. C, the result from parallel experiments in which the extent of Icrac activation by the four (2,4,5)IP3 concentrations was determined (as in Fig. 1B). Means ± S.E. from 4 to 16 experiments.

A Delay for Icrac Activation Is Also Seen when the Intracellular Ca2+ Stores Are Depleted with Thapsigargin and Ionomycin-- In the above studies, fura-2 measurement of Ca2+ release is carried out under conditions of physiological low Ca2+ buffering (0.1 mM BAPTA or 0.1 mM EGTA, see Experimental Procedures). However, Icrac is necessarily measured under conditions of high Ca2+ buffering (10 mM BAPTA) to eliminate Ca2+-activated currents and Ca2+-dependent inactivation of Icrac (3). These differences in Ca2+ buffering may have significant effects on the binding of (2,4,5)IP3 to IP3 receptors and on the amplification of Ca2+ release owing to the Ca2+-induced Ca2+ release (CICR) behavior of the IP3 receptor (10). These factors could lead to a slower Ca2+ release by IP3 under the conditions for Icrac measurement, thus accounting for the longer latencies we observed. To minimize these potential problems, we investigated the time course for Icrac activation following depletion of stores with thapsigargin or ionomycin. Thapsigargin is a potent inhibitor of the endoplasmic reticulum Ca2+ pump (11) and depletes Ca2+ stores by blocking Ca2+ uptake and allowing Ca2+ to passively leak out. Ionomycin is a Ca2+ ionophore that depletes the stores by either directly transporting ions or functioning as an ion channel. In neither case is it expected that Ca2+ buffers would impede the rate of Ca2+ store depletion; if anything, intracellular depletion of Ca2+ would likely be augmented. We found that a delay between Ca2+ release and Icrac activation still seemed to be present when the store was depleted with thapsigargin (Fig. 3) or ionomycin (Fig. 4).2 As was seen for (2,4,5)IP3, the delay for activation of Icrac decreased with increasing concentration of these agents. In the case of ionomycin, the development time for Icrac was relatively constant for the three highest concentrations of ionomycin (58.5 ± 0.9, 62.6 ± 7.7, and 78.3 ± 2.4 s for 500, 50, and 5 nM ionomycin, respectively), and these times are comparable with the development time when Icrac was activated with (2,4,5)IP3. For thapsigargin, the development times for Icrac were slightly longer (75.9 ± 3.1, 105.2 ± 12.0, 115.0 ± 15.3 s for 1 µM, 100 nM and 10 nM thapsigargin, respectively). Furthermore, consistent with the (2,4,5)IP3 data, the extent of Icrac activation with different concentrations of thapsigargin (Fig. 3) or ionomycin (Fig. 4) was about the same except at the lowest concentration of ionomycin (0.5 nM, Fig. 4).


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Fig. 3.   The delay between Icrac activation and store depletion is also observed when the stores are depleted with thapsigargin. Above, time course of Icrac activation with thapsigargin. At time 0, the external solution containing different concentrations of thapsigargin was perfused into the chamber. Each trace represents the average current from five to seven cells. Below, time course for Ca2+ release with the indicated concentrations of thapsigargin. The external solution contained no added Ca2+. Each trace represents the average response of five to seven fura-2-loaded cells


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Fig. 4.   The relationship between Icrac activation and store depletion when the stores are depleted with ionomycin. Above, time course of Icrac activation with four concentrations of ionomycin. Each trace represents the average current from five to seven cells. Data for 1 µM thapsigargin are also included (from Fig. 3). Below, time course for Ca2+ release with ionomycin. The external solution contained no added Ca2+ and 1 EGTA with the indicated concentrations of ionomycin. Each trace represents the average response of five to seven fura-2-loaded cells.

Activation of Icrac Only Requires a Minimal Depletion of the Ca2+ Stores, and Full Activation of Icrac Does Not Require Full Depletion of the Store-- For IP3, thapsigargin, and ionomycin, there seems to be a significant delay between the release of intracellular Ca2+ and activation of Icrac. This could result either from an interval of time required to release and/or synthesize some signaling messenger or from the need to deplete intracellular stores below some critical level before the activation process begins. From inspection of the data in Figs. 3 and 4, it seems that for both agents release of Ca2+ is well under way prior to the activation of Icrac. However, the amount of Ca2+ needed to increase [Ca2+]i into the 100-300 nM range is potentially very small in comparison with the total Ca2+ content of intracellular stores. Thus, we attempted to determine more quantitatively the extent of depletion required to activate Icrac by estimating the Ca2+ store content at the time when Icrac is initially activated. The latency and time course of activation of Icrac for 1 µM thapsigargin and 5 nM ionomycin are very similar (Fig. 4). In parallel experiments with intact RBL-1 cells, we added a high dose of ionomycin (5 µM) 50 s after treatment with either 1 µM thapsigargin or 5 nM ionomycin, corresponding to the time of initiation of Icrac (see Fig. 3), to assess the Ca2+ content of the stores and compared it with that of control cells that had not been exposed to either agent. As shown in Fig. 5, at the time when Icrac was initiated by 1 µM thapsigargin, significant release of Ca2+ had already occurred. 100 s later, when Icrac activation was maximal, a small but significant amount of stored Ca2+ remained in the cells. Thus, for thapsigargin, it seems that either a significant amount of Ca2+ must be released before the initiation of Icrac or a significant amount of time is required for steps linking Ca2+ store depletion to Icrac activation.


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Fig. 5.   Status of intracellular Ca2+ stores 50 and 150 s after addition of 1 µM thapsigargin. The status of intracellular stores was assessed by addition of 5 µM ionomycin 50 s (above) after addition of 1 µM thapsigargin (dashed line), at which time Icrac is just beginning, and 150 s (below), at which time Icrac activation is maximal (Fig. 3). Comparison of the responses to ionomycin in control cells (no addition of thapsigargin) indicates that at 50 s, substantial depletion of stores has occurred, and by 150 s, near maximal depletion has occurred. Each trace represents an average of data obtained from 5 to 10 cells analyzed in separate experiments.

Surprisingly, when ionomycin was used to deplete intracellular stores, a different result was obtained. 50 s after application of 5 nM ionomycin, despite the fact that a discernible elevation in [Ca2+]i had occurred, the Ca2+ content of the stores seemed to be about the same in ionomycin-treated and control cells (Fig. 6). Thus, the stores are apparently only slightly depleted at 50 s with 5 nM ionomycin. This result suggests that when ionomycin is used to deplete stored Ca2+ only a very small reduction is required to initiate activation of Icrac. It also indicates that for ionomycin, the delay in activating of Icrac may not reflect a longer delay than that required to significantly reduce the Ca2+ content of intracellular Ca2+ stores.


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Fig. 6.   Status of intracellular Ca2+ stores 50 and 150 s after addition of 5 nM ionomycin. The status of intracellular stores was assessed by addition of 5 µM ionomycin 50 s (above) after addition of 5 nM ionomycin (dashed line), at which time Icrac is just beginning, and 150 s (below), at which time Icrac activation is maximal (Fig. 4). Comparison of the responses to high ionomycin in control cells (no addition of 5 nM ionomycin) indicates that at 50 s, very little depletion of stores has occurred, and at 150 s, only about 50% of maximal depletion has occurred. Each trace represents an average of data obtained from 5 to 10 cells analyzed in separate experiments.

At 150 s following addition of 5 nM ionomycin, Icrac was fully activated (Fig. 4). When we used the same strategy to assess the store content at this time, we found that the Ca2+ stores were depleted by about 50% (Fig. 6). This result suggests that Icrac can be fully activated with only partial depletion of intracellular Ca2+ stores.

    DISCUSSION
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Abstract
Introduction
Procedures
Results
Discussion
References

The temporal relationship between the discharge of intracellular stores of Ca2+ and the activation of capacitative calcium entry is key to understanding the nature of the signaling process. However, it is difficult to determine these two parameters under similar conditions because the very high [Ca2+]i buffering required to detect Icrac prevents detection of Ca2+ release to the cytoplasm. To minimize this problem, we have relied on comparisons among three agents that cause depletion of endoplasmic reticulum Ca2+ stores by clearly distinct mechanisms: IP3 by activating a membrane receptor/ion channel; thapsigargin, which blocks the SERCA pumps that accumulate Ca2+ in the endoplasmic reticulum; and ionomycin, which passively transports Ca2+ down its concentration gradient. Although we expect experimental conditions such as Ca2+ buffering to affect release of Ca2+ by IP3, we expect this to be much less of a factor with thapsigargin and ionomycin. Nonetheless, we cannot know with absolute certainty that this is so. Ideally, one would like to be able to assess the Ca2+ content of intracellular stores in the experiments in which Icrac is measured. Recently described technologies may permit such a determination in the near future (12).

In experiments utilizing (2,4,5)IP3 as an activator of intracellular IP3 receptors, we observed a clear distinction between the time required for detectable release of stored Ca2+ and that required for activation of Icrac. That is, significantly shorter intervals were required for mobilization of intracellular Ca2+ stores than for activation of Icrac. As discussed above, because different Ca2+ buffering conditions were necessarily used for these two determinations, it is not clear whether the latency for release of Ca2+ was identical in the two experimental conditions. In fact, one might expect augmentation of the Ca2+ release process with minimal Ca2+ buffering through calcium-induced calcium release. However, if that were the case, one might also expect that concentrations of (2,4,5)IP3 inducing only partial depletion of stores with high concentrations of intracellular buffers might cause complete depletion with lower buffer concentrations. From the data in Fig. 2, this does not seem to be the case; 25-100 µM (2,4,5)IP3 induced maximal responses in the high and low buffer conditions (Icrac in the former, depletion in the latter), whereas 10 µM (2,4,5)IP3 induced a partial activation with both buffer conditions.

Differences in Ca2+ buffering should be less of an issue for experiments utilizing thapsigargin or ionomycin. These agents deplete Ca2+ stores by distinct and passive mechanisms, such that cytoplasmic Ca2+ buffering should affect the kinetics of depletion minimally if at all. From inspection of the time courses of Icrac activation and of [Ca2+]i increase in Figs. 3 and 4, it seems that Ca2+ release from the endoplasmic reticulum does indeed precede Icrac by some tens of seconds. However, the data in Fig. 6 show that for low concentrations of ionomycin at least, the level of cytoplasmic Ca2+ can be a poor indicator of the extent of depletion of Ca2+ stores. Despite a significant rise in cytoplasmic Ca2+ 50 s after addition of 5 nM ionomycin, the total stored Ca2+ content of the endoplasmic reticulum was changed minimally. These results indicate that especially with low concentrations of Ca2+-depleting agents, changes in [Ca2+]i can misrepresent the extent of changes in intracellular Ca2+ stores. Thus, for this concentration of ionomycin, and in contrast to the findings with (2,4,5)IP3 and thapsigargin, there may be very little delay between the fall in Ca2+ content of the endoplasmic reticulum and the initiation of Icrac. In an earlier report, McDonald et al. (13) reported minimal latency for Icrac activation when (1,4,5)IP3 was rapidly released within Jurkat T cells by flash photolysis.

What then is the meaning of the delay between Ca2+ store depletion and Icrac activation seen with (2,4,5)IP3 and thapsigargin? As discussed above, for (2,4,5)IP3, the difference may reflect differences in rates of Ca2+ discharge with the two different intracellular Ca2+ buffering systems used. But perhaps a more fundamental difference among these three modes of Ca2+ release is that only ionomycin can be assumed to release Ca2+ in a spatially nonspecific manner throughout the cell. In other words, because of its presumed mechanism of action, we expect ionomycin to release Ca2+ from all components or regions of the endoplasmic reticulum with similar facility. However, for (2,4,5)IP3 and thapsigargin, this may not be the case. (2,4,5)IP3 will cause activation of IP3 receptor channels and cause discharge of Ca2+ only at the specific sites where these receptors are located. Thapsigargin will lead to depletion of stores by passive leak of Ca2+ following inhibition of SERCA pumps. Virtually nothing is known about the channels mediating this presumably IP3-insensitive or basal movement of Ca2+, but such sites could also be localized in a non-homogeneous manner in specific regions of the endoplasmic reticulum. Thus, the current findings may indicate that a specific subfraction of the endoplasmic reticulum regulates the Ca2+ channels underlying Icrac and that as Ca2+ is depleted from the endoplasmic reticulum through IP3 receptors, or through the leak pathway involved with thapsigargin action, this specific subcompartment is more slowly depleted than the majority of the endoplasmic reticulum. To our knowledge, this is the first evidence for differential effects of thapsigargin in subcompartments of the endoplasmic reticulum. The suggestion of a specific subfraction of endoplasmic reticulum involved in the regulation of Icrac is consistent with the finding that for both ionomycin and thapsigargin, full depletion of the endoplasmic reticulum store is not required for maximal activation of Icrac. In at least one earlier report, specialization of the endoplasmic reticulum Ca2+ stores with respect to regulation of capacitative Ca2+ entry has been suggested (14). It was suggested that the subfraction of endoplasmic reticulum coupled to capacitative calcium entry was at most 30% of the total thapsigargin-sensitive Ca2+ stores.

The current findings indicate that the kinetics of activation of Icrac are complex. Over certain concentration ranges with all three modes of activation, the time course of Icrac activation is relatively constant. This may simply result from the fact that in most experimental situations, release of Ca2+ occurs more rapidly than the steps involved in signaling Icrac. This may also be suggestive of an all-or-none mechanism of activation, as proposed by Parekh et al. (6). However, graded activation of Icrac was observed with 10 µM (2,4,5)IP3, as well as in an study earlier utilizing cyclopiazonic acid to deplete endoplasmic reticulum calcium (14). As in the current study, Parekh et al. (6) concluded that a small component of the total intracellular Ca2+ stores regulates Icrac, but this conclusion was based on findings that low concentrations of (1,4,5)IP3 seemed to activate Ca2+ release without activating Ca2+ entry. In our study, we have exclusively utilized (2,4,5)IP3, a poorly metabolizable analog of (1,4,5)IP3, to avoid possible complications of differential metabolism under different experimental conditions. With (2,4,5)IP3 as the mobilizing ligand, no dissociation between the concentrations required for release and those required for Icrac activation were observed (Fig. 2). However, it is the differential latencies observed for ionomycin, thapsigargin, and (2,4,5)IP3 that lead us to propose a specialized, quantitatively minor component of the endoplasmic reticulum as the site of control of capacitative calcium entry and Icrac. If in fact a small compartment of the endoplasmic reticulum is responsible for regulation of Icrac, it is possible that the kinetics of Icrac activation reflect the kinetic behavior of this small pool of Ca2+ and that these kinetics are not accurately reflected by the average time course of changes in cytoplasmic and stored Ca2+ in the cell. Future work must concentrate on experimental dissection of these functionally distinguishable subcompartments of endoplasmic reticulum Ca2+ stores if we are to fully understand how the plasma membrane Ca2+ movements underlying Icrac and capacitative calcium entry are regulated.

    FOOTNOTES

* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed: NIEHS, National Institutes of Health, P.O. Box 12233, Research Triangle Park, NC 27709. Tel.: 919-541-1420; Fax: 919-541-1898; E-mail: putney{at}niehs.nih.gov.

1 The abbreviations used are: (1,4,5)IP3, inositol 1,4,5-triphosphate; Icrac, calcium release-activated calcium current; BAPTA, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid.

2 For experiments measuring Ca2+ release owing to ionomycin or thapsigargin, we used intact rather than dialyzed cells. This is because at low concentrations of ionomycin, when release is gradual, the rise in [Ca2+]i associated with release is difficult to detect presumably owing to constant exchange of the Ca2+-associated dye through the pipette. With maximal concentrations of ionomycin where this is less of a problem, the magnitude and extent of the response to ionomycin are comparable (compare Fig. 5 with Fig. 2). Because ionomycin and thapsigargin deplete Ca2+ stores by passive means, we presume that the rates of depletion will be similar under the different conditions, but the data in intact cells give a more accurate reflection of the extent of depletion.

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Abstract
Introduction
Procedures
Results
Discussion
References

  1. Putney, J. W., Jr. (1986) Cell Calcium 7, 1-12[Medline] [Order article via Infotrieve]
  2. Clapham, D. E. (1995) Nature 375, 634-635[CrossRef][Medline] [Order article via Infotrieve]
  3. Hoth, M., and Penner, R. (1992) Nature 355, 353-355[CrossRef][Medline] [Order article via Infotrieve]
  4. Putney, J. W., Jr. (1997) Cell Calcium 21, 257-261[Medline] [Order article via Infotrieve]
  5. Hoth, M., and Penner, R. (1993) J. Physiol. (Lond.) 465, 359-386[Abstract]
  6. Parekh, A. B., Fleig, A., and Penner, R. (1997) Cell 89, 973-980[Medline] [Order article via Infotrieve]
  7. Bird, G., St, J., Rossier, M. F., Hughes, A. R., Shears, S. B., Armstrong, D. L., and Putney, J. W., Jr. (1991) Nature 352, 162-165[CrossRef][Medline] [Order article via Infotrieve]
  8. Hamill, O. P., Marty, A., Neher, E., Sakmann, B., and Sigworth, F. J. (1981) Pflügers Arch. 391, 85-100[Medline] [Order article via Infotrieve]
  9. White, R. E., Schonbrunn, A., and Armstrong, D. L. (1991) Nature 351, 570-573[CrossRef][Medline] [Order article via Infotrieve]
  10. Finch, E. A., Turner, T. J., and Goldin, S. M. (1991) Science 252, 443-446[Medline] [Order article via Infotrieve]
  11. Thastrup, O., Dawson, A. P., Scharff, O., Foder, B., Cullen, P. J., Drobak, B. K., Bjerrum, P. J., Christensen, S. B., and Hanley, M. R. (1994) Agents Actions 43, 187-193[Medline] [Order article via Infotrieve]
  12. Miyawaki, A., Llopis, J., Heim, R., McCaffery, J. M., Adams, J. A., Ikura, M., and Tsien, R. Y. (1997) Nature 388, 882-887[CrossRef][Medline] [Order article via Infotrieve]
  13. McDonald, T. V., Premack, B. A., and Gardner, P. (1993) J. Biol. Chem. 268, 3889-3896[Abstract/Free Full Text]
  14. Ribeiro, C. M. P., and Putney, J. W., Jr. (1996) J. Biol. Chem. 271, 21522-21528[Abstract/Free Full Text]


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