From the Calcium Regulation Section, Laboratory of Signal
Transduction, NIEHS, National Institutes of Health, Research Triangle
Park, North Carolina 27709
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.
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INTRODUCTION |
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.
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EXPERIMENTAL PROCEDURES |
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 M
,
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).
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RESULTS |
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.
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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.
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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.
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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.
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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.
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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.
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DISCUSSION |
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.