Department of Physiology, University of Oxford, Oxford OX1 3PT, United Kingdom
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ABSTRACT |
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In many nonexcitable cells, depletion of the inositol 1,4,5-trisphosphate-sensitive store activates Ca2+ influx, a process termed store-operated Ca2+ entry. In rat basophilic leukemia cells, emptying of the stores activates a highly selective Ca2+ release-activated Ca2+ current (CRAC), ICRAC. We have recently found that ICRAC activates in an essentially all-or-none manner when the current is evoked by receptor stimulation, dialysis with inositol 1,4,5-trisphosphate via the patch pipette, or through the Ca2+ATPase inhibitor thapsigargin (Parekh, A. B., Fleig, A., and Penner, R. (1997) Cell 89, 973-980). Regulatory mechanisms must therefore operate to control the overall amount of Ca2+ that enters through CRAC channels. Such mechanisms include membrane potential and protein kinase C. In the present study, we have investigated additional inhibitory pathways that serve to determine just how much Ca2+ can enter through ICRAC. We have directly measured the current using the whole cell patch clamp technique. We report the presence of a slow Ca2+-dependent inactivation mechanism that curtails Ca2+ entry through CRAC channels. This inactivation mechanism is switched on by Ca2+ entering through CRAC channels, and therefore constitutes a slow negative feedback process. Although it requires a rise in intracellular Ca2+ for activation, it maintains CRAC channels inactive even under conditions that lower intracellular Ca2+ levels. The inactivation mechanism does not involve store refilling, protein phosphorylation, G proteins, nor Ca2+-dependent enzymes. It accounts for up to 70% of the total inactivation of ICRAC, and therefore appears to be a dominant inhibitory mechanism. It is likely to be an important factor that shapes the profile of the Ca2+ signal in these nonexcitable cells.
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INTRODUCTION |
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In many nonexcitable cells, depletion of the inositol 1,4,5-trisphosphate (InsP3)1-sensitive intracellular Ca2+ stores activates a Ca2+ influx pathway in the plasma membrane (1). This mechanism was originally proposed by Putney (2) and called capacitative Ca2+ influx. Patch-clamp experiments have identified a variety of Ca2+-permeable channels in the plasma membrane that seem to underlie capacitative Ca2+ influx (reviewed in Ref. 3). These channels differ in their biophysical properties and are generically referred to as store-operated Ca2+ channels (3, 4).
Of the store-operated Ca2+ currents, the best characterized is ICRAC, which was originally discovered in mast cells (5). ICRAC has subsequently been shown to exist in several different nonexcitable cells including basophils, T cells and megakaryocytes (3). CRAC channels are remarkably selective for Ca2+ ions and have a low single-channel conductance (3, 5).
Just how depletion of the stores activates CRAC channels is still unclear. Several potential mechanisms have been proposed but the signal has not been unequivocably identified (reviewed in Refs. 3 and 4). One interesting aspect of ICRAC is that the current activates in an essentially all-or-none manner, irrespective of whether activation is evoked by dialysis with inositol 1,4,5-trisphosphate, receptor stimulation, or thapsigargin (6). One consequence of this is that, if ICRAC activates in an all-or-none manner, mechanisms must exist that control the amount of Ca2+ entering the cell through CRAC channels. This is required in order to achieve graded activation of Ca2+-dependent processes like secretion that correlate with the level of cell stimulation by receptors. We have recently reported that Ca2+ entry through CRAC channels in rat basophilic leukemia (RBL) cells can be graded, despite all-or-none activation, because of several regulatory mechanisms that serve to control CRAC channel activity (6, 7). One way is by changing the membrane potential. Hyperpolarization increases the electrical gradient for Ca2+ entry, thus favoring further Ca2+ influx, whereas depolarization decreases the driving force and hence reduces Ca2+ entry (6).
ICRAC is also regulated by protein kinase C. Stimulation of this enzyme inactivates ICRAC. Since protein kinase C will be activated by diacylglycerol, which is produced following stimulation of receptors that engage the phosphoinositide pathway, it constitutes an important negative feedback mechanism on Ca2+ influx (7).
In mast cells and jurkat T lymphocytes, ICRAC is subjected to a fast inactivation process operating on a milliseconds time scale. This arises from Ca2+ ions entering the cell through CRAC channels and then binding to sites probably located on the channels themselves (5, 8).
Here we report an additional mechanism that serves to regulate CRAC channels. We find that Ca2+ influx through CRAC channels exerts a slow feedback inhibition that curtails further Ca2+ entry and which is dependent on a rise in intracellular Ca2+ levels. Slow inactivation accounts for up to 70% of the inhibition of ICRAC. Once activated, this slow inactivation mechanism can maintain CRAC channels in an inactivated state for several minutes, even after intracellular Ca2+ levels have been reduced. Hence slow inactivation appears to be a dominant inhibitory mechanism that determines the time course of Ca2+ influx following store depletion in RBL cells. It also provides an additional mechanism whereby the amount of Ca2+ entering through CRAC channels can be regulated despite all-or-none Ca2+ entry.
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EXPERIMENTAL PROCEDURES |
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Rat basophilic leukemia cells (RBL-2H3) cells were kindly
supplied by Michael Pilot, Max Planck Institute for Biophysical Chemistry, Goettingen, Germany, and were cultured essentially as
described previously (6, 7). Patch-clamp experiments were conducted in
the tight seal whole cell configuration at room temperature
(18-25 °C) as described previously (6, 7). Patch pipettes were
pulled from borosilicate glass (Hilgenberg), sylgard-coated, and
fire-polished. Pipettes had d.c. resistances of 2.5-4 megohms when
filled with standard internal solution that contained (in mM): cesium glutamate 145, NaCl 8, MgCl2 1, MgATP 1, InsP3 0.03, EGTA 1.4, HEPES 10, pH 7.2, with CsOH.
A correction of +10 mV was applied for the subsequent liquid junction
potential. In some experiments, the EGTA concentration was raised to 14 mM (indicated in text) or substituted with BAPTA. Drugs
were added to this internal solution as described in the text.
Extracellular solution contained (in mM): NaCl 145, KCl
2.8, CaCl2 10, MgCl2 2, CsCl 10, glucose 10, HEPES 10, pH 7.2 (NaOH). CsCl was present to block the activity of the
inwardly rectifying potassium channel. High resolution current
recordings were acquired by a computer-based patch-clamp amplifier
system (EPC-9, HEKA Electronics, Germany). Capacitative currents were
canceled before each voltage ramp using the automatic compensation of
the EPC-9. Series resistance was between 5 and 15 megohms. Currents
were filtered using an 8-pole Bessel filter at 2.5 kHz and digitized at
100 µs. ICRAC was measured using either voltage ramps (100 to +100 mV in 50 ms) or voltage steps (pulses to
80 mV for 200 ms) applied every 2 s using PULSE software (HEKA Electronics) on a 9500 PowerMac. Cells were held at 0 mV between pulses. All currents were leak subtracted by averaging the first two to
four ramps/steps after breaking in and then subtracting this from all
subsequent traces. Several parameters (capacitance, series resistance,
holding current) were displayed simultaneously on a second monitor at a
slower rate (2 Hz) using the X-Chart display (HEKA Electronics). Data
are presented as mean ± S.E., and statistical evaluation was
carried out using Student's unpaired t test.
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RESULTS |
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Slow Inactivation of ICRAC in the Presence of Moderate
intracellular calcium buffering--
Previous work has demonstrated
that ICRAC inactivates partially when RBL cells
are dialyzed with a patch pipette solution containing a high
concentration of the slow Ca2+ chelator EGTA (10 mM), and this is due to a kinase-mediated phosphorylation (7). In the present study, we have examined the effects of more
moderate calcium buffering on the properties of
ICRAC by including 1.4 mM EGTA in
the recording pipette. Fig.
1A(i) shows an
experiment in which the internal solution contained 30 µM
InsP3 (a supramaximal concentration) (6) and 1.4 mM EGTA. Cells were voltage clamped at 0 mV and
ICRAC was monitored using voltage ramps applied
every two seconds (shown in Fig. 1A(ii)). The
current was measured at 80 mV. Fig. 1A(i)
depicts the time course of the current during the experiment. Following
the onset of whole cell recording, ICRAC
activated as InsP3 diffused into the cell from the
recording pipette and depleted the stores. The time constant for
activation (
) was 15.2 ± 1.6 s, similar to our previous
measurements (19.4 ± 1.2 s) (9). The current peaked after
50-80 s and then inactivated substantially with time. For the cell
shown in Fig. 1A(i), the current had fallen by
70% (relative to the peak amplitude) after 300 s. Fig.
1A(ii) shows I-V relationships for the two time points indicated in Fig. 1A(i) (a and
b). The current measured in the ramps exhibited the classic
features of ICRAC: voltage-independent activation, inward rectification, and a reversal potential close to +60
mV.
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Variability in the Overall Extent of Slow Inactivation between Cell Preparations-- In some cells, slow inactivation was very prominent and contributed to an almost complete inactivation of ICRAC. Typical examples of this type of response are shown in Figs. 3B and 5A. In some other cells, slow inactivation contributed to around a 50% decrease in the amplitude of ICRAC (e.g. Fig. 5C). Overall, there was little difference between preparations, with the average decrease being 64.8 ± 7.9% (n = 35, measured using the step protocol after 300 s).
In experiments where drugs were used to interfere with slow inactivation, control responses were always obtained from the same preparation and the data were compared with these controls.Slow Inactivation Is Reduced by a Higher EGTA Concentration in the Recording Pipette-- To confirm that slow inactivation is dependent on a rise in intracellular Ca2+ levels, we dialyzed cells with a pipette solution in which the EGTA concentration had been increased 10-fold (to 14 mM). Pooled data from 10 cells in 14 mM EGTA and eight cells in 1.4 mM EGTA are summarized in Fig. 1D (paired recordings). Slow inactivation was reduced by almost 2-fold in the presence of the higher EGTA concentration and appears therefore to require a rise in intracellular free Ca2+ because it can be suppressed by increasing the concentration of a slow mobile Ca2+ buffer in the cytosol. The inset of Fig. 1D shows current traces taken from the voltage steps from two cells dialysed with 1.4 and 14 mM EGTA. Fast inactivation was the same in both cells, yet slow inactivation was less pronounced in the presence of higher EGTA. Fast and slow inactivation therefore reflect distinct processes.
Slow Inactivation Is Dependent on Ca2+ Entry into the
Cell--
To assess the contribution of Ca2+ influx
through CRAC channels to the slow
Ca2+-dependent inactivation, we carried out a
series of experiments in which the electrochemical gradient for
Ca2+ influx was altered. First, we modified the electrical
gradient by applying voltage pulses to potentials that either reduced
Ca2+ entry (40 mV) or enhanced it (
120 mV), relative to
the responses obtained on pulsing to
80 mV. Typical results are shown
in Fig. 2A, and the data are
summarized in Table I. Stepping the
voltage to
40 mV reduced the rate and extent of inactivation almost
2-fold (Fig. 2A(i) and Table I). The current at
40 mV in Fig. 2A(i) has been scaled so that it
has the same amplitude as that at
80 mV in order to clearly show the
slower inactivation. Voltage pulses to
120 mV, however, did not
increase the level of steady-state inactivation compared with that seen
on stepping to
80 mV (Fig. 2A(ii), where the
current at
80 mV has been scaled, see also Table I), which might
suggest that sufficient Ca2+ enters at
80 mV to maximally
activate the inhibitory process.
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Ca2+-dependent Slow Inactivation Is Not Affected by a Fast Ca2+ Chelator-- The preceding results demonstrate that ICRAC is subject to a negative feedback mechanism in which Ca2+ influx through CRAC channels induces a slow Ca2+-dependent inactivation. Fast negative feedback inactivation of CRAC channels operates on a milliseconds time-scale and can be substantially reduced by inclusion of the fast Ca2+ chelator BAPTA in the recording pipette, whereas it is not altered by increasing the concentration of the slower Ca2+ chelator EGTA (8). Replacing EGTA with the fast chelator BAPTA (both at 1.4 mM) did not significantly affect the rate or extent of inactivation (six cells, Fig. 2C and Table I). However, rapid inactivation of ICRAC was slightly slowed by this concentration of BAPTA (1.2-1.4-fold, data not shown), reinforcing the notion that the inactivation we observe is distinct from the fast inactivation process.
Once Initiated, Slow Inactivation Is Not Strongly Dependent on
Ca2+ Entry--
Although Ca2+ entry is
important for initiating slow inactivation, we set out to determine
whether slow inactivation still required Ca2+ entry even
after the inactivation process had started to develop. Fig.
3 describes two types of experiment which
were designed to address this. In Fig. 3A, hyperpolarizing
steps to 80 mV were repetitively applied every 2 s from a
holding potential of 0 mV. Once inactivation had clearly developed (at
200 s), the cell was held continuously at +20 mV for 30 s. At
this positive potential, very little Ca2+ enters through
CRAC channels (6). After 30 s, the cell was held again at 0 mV and
hyperpolarizing steps were resumed. The amplitude of
ICRAC increased only slightly relative to the
level it had reached prior to clamping the cell at +20 mV (four of nine cells). In the other five cells, no recovery was observed at all.
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Investigation into the Mechanism of Ca2+-dependent Slow Inactivation-- The following set of experiments were aimed at elucidating the molecular mechanism that gave rise to the slow inactivation of ICRAC.
Refilling of Intracellular Ca2+ Stores--
In the
presence of moderate concentrations of EGTA, it is conceivable that
Ca2+ entry through CRAC channels might enable the
intracellular Ca2+ stores to refill, a process that would
turn off ICRAC. If such a mechanism were
responsible for the inactivation of ICRAC, then one would predict that maneuvers directed toward reducing
Ca2+ uptake into the stores should prevent slow
inactivation from occurring. Thapsigargin is a specific inhibitor of
the Ca2+ATPase on the endoplasmic reticulum and prevents
store refilling (10). We therefore carried out paired recordings in
which six control cells were dialyzed with internal solution containing InsP3 and 1.4 mM EGTA, whereas six other cells
from the same preparations were dialyzed with this solution
supplemented with 2 µM thapsigargin. Fig.
4A shows the averaged results.
Slow inactivation was only slightly reduced by thapsigargin, but this
was not statistically significant. Thapsigargin is lipophilic, so it is
possible that the drug diffuses out of the cell into the bath solution.
Because we included a high concentration of thapsigargin in the pipette (2 µM), one would be surprised if the steady-state
cytoplasmic concentration was less than a few hundred nanomolar, a
concentration that is sufficient to reduce Ca2+ATPase
activity in a variety of cell types (10). In two cells, we applied
thapsigargin (1 µM) from the outside just after the onset
of ICRAC (thapsigargin was also included in the
recording pipette). The current still inactivated (by 53 and 61%).
Nevertheless, we sought additional ways to probe the effects of
compromised store refilling on slow inactivation. One method would be
to employ the Ca2+ ionophore ionomycin. Ionomycin increases
the permeability of the store membrane to Ca2+, and this
would enable any Ca2+ that had been pumped into the stores
to diffuse back into the cytosol thereby preventing stores from
refilling. We included 2 µM ionomycin in the recording
pipette together with InsP3 and 1.4 mM EGTA.
Fig. 4B shows the effects of ionomycin.
ICRAC activated slightly faster in
ionomycin-treated cells ( of 16.1 ± 1.7 s versus 22.7 ± 3.0 s in control paired cells),
which might indicate that ionomycin is diffusing into the cells rather
quickly and accelerating store depletion in combination with
InsP3. Slow inactivation of ICRAC
was still apparent in the presence of ionomycin, and the current
declined to a value similar to that seen in the absence of ionomycin in
paired recordings (six cells for ionomycin, five for controls).
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Protein Kinase-mediated Phosphorylation--
We tested the
possible involvement of a kinase-mediated phosphorylation reaction in
the Ca2+-dependent slow inactivation in RBL
cells in two independent ways. First, we examined the effects of
removing ATP from the pipette solution and then replacing it with
ATPS, and second, we tested the effects of broad kinase and
phosphatase inhibitors on the inactivation process.
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GTP-dependent Proteins and Ca2+-activated
Proteases--
To assess the role of G proteins, we included 300 µM GDPS in the recording pipette together with
InsP3. Slow inactivation still occurred (Fig. 5C
and Table II) and it was not significantly different from control
cells. Dialysis with 250 µM GTP
S did not affect slow
inactivation either (inactivation of 77 ± 9%). Later on in these
same cells (after a 5-10-min recording), the
GTP
S-dependent Na+ current activated (14),
demonstrating that the GTP
S was active under these conditions.
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DISCUSSION |
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Negative Feedback by Ca2+ Ions Entering through CRAC Channels Gives Rise to Slow Inactivation-- Our observation that slow inactivation could be significantly reduced by increasing the concentration of the Ca2+ chelator EGTA in the patch pipette solution (Fig. 1D) suggests that the inactivation mechanism requires a rise in cytoplasmic Ca2+ concentration. This rise in Ca2+ is accomplished by Ca2+ entry through CRAC channels because maneuvers that reduce the electrochemical gradient for Ca2+ influx result in less inactivation (Fig. 2).
It is likely that Ca2+ entry is the main source of Ca2+ for activating the slow inhibitory pathway with little contribution from Ca2+ release from the stores (e.g. Ca2+ entry evoked Ca2+ release) for the following reasons. First, dialysis with InsP3 and either ionomycin or thapsigargin, agents that would reduce the Ca2+ content of the stores, did not alter slow inactivation. Second, cells were dialyzed with a supramaximal concentration of InsP3 which is likely to maintain the stores in a Ca2+-depleted state (6). Although the InsP3 receptor might desensitize, thereby enabling stores to refill, we feel this process is not particularly active under our conditions. The InsP3 receptor that is involved in the activation of ICRAC in RBL cells does not seem to desensitize much because Ca2+ influx can still be activated more than 10 min after dialyzing with high InsP3 levels (6). Finally, slow inactivation still occurs in the presence of 2 mM ATPMechanism of Slow Inactivation-- Just how a rise in intracellular Ca2+ switches on the slow inactivation process is not clear. It is unlikely to reflect refilling of the stores because inclusion of thapsigargin or ionomycin in the pipette solution did not prevent the inactivation from occurring. Direct involvement of the Ca2+-binding protein calmodulin in the inactivation process is also unlikely because inclusion of the specific calmodulin inhibitory peptide fragment from Calbiochem failed to prevent slow inactivation (three cells, not shown).
One important clue to the mechanism of slow inactivation was the finding that, once activated, it did not require the continuous presence of elevated Ca2+ (Fig. 3). Such long-lasting effects are often mediated by protein phosphorylation reactions. However, several treatments designed to interfere with phosphorylation failed to affect slow inactivation of ICRAC. Involvement of Ca2+-activated proteases was also unlikely since a specific and potent inhibitor of this class of enzyme did not alter inactivation. It also seems unlikely that CRAC channels are being endocytosed slowly from the membrane in a Ca2+-dependent manner, because membrane capacitance did not change despite substantial inactivation of the Ca2+ current (data not shown). Finally, during whole cell recording small soluble components diffuse out of the cell and this washout may cause a general loss of CRAC channel activity. The fact that increasing the concentration of EGTA in the recording pipette reduced slow inactivation suggests that loss of cytoplasmic factors might not explain the inactivation. However, we cannot rule out the possibility that a rise in intracellular Ca2+ accelerates washout of an important component, perhaps by promoting its Ca2+-dependent dissociation from a bound to a diffusible form. A slow Ca2+-dependent inactivation of store-operated Ca2+ influx has been described in jurkat T cells (13) and NIH-3T3 fibroblasts (16). In T cells, it was reported that slow inactivation was inhibited by okadaic acid and by the kinase inhibitor H-7 (11, 13). It was concluded that recovery from slow inactivation occurred through the actions of a protein kinase. In NIH-3T3 cells, Ca2+ influx was measured using the fluorescent dye fura-2. Unlike T cells, slow inactivation was not affected by kinase or phosphatase inhibitors nor by Ca2+ chelators (loaded via the membrane-permeable acetoxymethyl ester form). Slow inactivation was suggested to arise from an action of Ca2+ not subject to Ca2+ chelation, perhaps by binding to an external part of the channel. The slow inactivation pathway described in this report exhibits some notable differences from these other inactivation ones. Unlike T cells, it was not sensitive to H-7 or okadaic acid, nor to conditions designed to facilitate protein phosphorylation. Slow inactivation was reduced by increasing the concentration of Ca2+ chelator in the recording pipette, demonstrating an intracellular action of Ca2+ rather than an extracellular site as proposed for the NIH-3T3 cells (16). Ca2+-dependent slow inactivation of ICRAC might therefore arise through different mechanisms in different nonexcitable cells.Comparison of Slow Inactivation with Other Inhibitory Mechanisms
That Regulate ICRAC--
Several inhibitory pathways have
been found to regulate CRAC channels in RBL cells, and it might be
instructive to ascertain their relative contributions to controlling
Ca2+ entry. ICRAC is subject to a
fast inactivation process (8). The peak current declines by up to 40%
following a hyperpolarization to 80 mV (Fig. 1B). Because
we have measured ICRAC at steady state
(i.e. once the rapid inactivation component was over, Fig. 1B), fast inactivation is not contributing to the
inactivation that we have measured and is therefore not relevant to the
discussion that follows.
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ACKNOWLEDGEMENTS |
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The critical comments of Professors Alison Brading, Erwin Neher, and Criss Hartzell are gratefully acknowledged.
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FOOTNOTES |
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* This work was supported by the Wellcome Trust.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.
Sir Edward Abraham Research Fellow at Keble College, Oxford. To
whom correspondence should be addressed: Dept. of Physiology, University of Oxford, Parks Road, Oxford OX1 3PT United Kingdom. Tel.:
44-1865-272439; Fax: 44-1865-272488; E-mail: anant.parekh{at}physiol.ox.ac.uk.
1
The abbreviations used are: InsP3,
inositol 1,4,5-trisphosphate; CRAC, calcium release-activated calcium
current; RBL, rat basophilic leukemia; BAPTA,
1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; ATPS, adenosine 5'-O-(thiotriphosphate); GDP
S,
guanosine 5'-O-2-(thiodiphosphate); GTP
S, guanosine
5'-3-O- (thiotriphosphate).
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REFERENCES |
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