J Biol Chem, Vol. 274, Issue 46, 32881-32888, November 12, 1999

Role of the Inositol 1,4,5-Trisphosphate Receptor in Ca²⁺ Feedback Inhibition of Calcium Release-activated Calcium Current (I_crac)^*

Lisa M. Broad, David L. Armstrong, and James W. Putney Jr.

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

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

We examined the activation and regulation of calcium release-activated calcium current (I_crac) in RBL-1 cells in response to various Ca²⁺ store-depleting agents. With [Ca²⁺]_i strongly buffered to 100 nM, I_crac was activated by ionomycin, thapsigargin, inositol 1,4,5-trisphosphate (IP₃), and two metabolically stable IP₃ receptor agonists, adenophostin A and L--glycerophospho-D-myoinositol-4,5-bisphosphate (GPIP₂). With minimal [Ca²⁺]_i buffering, with [Ca²⁺]_i free to fluctuate I_crac was activated by ionomycin, thapsigargin, and by the potent IP₃ receptor agonist, adenophostin A, but not by GPIP₂ or IP₃ itself. Likewise, when [Ca²⁺]_i was strongly buffered to 500 nM, ionomycin, thapsigargin, and adenophostin A did and GPIP₂ and IP₃ did not activate detectable I_crac. However, with minimal [Ca²⁺]_i buffering, or with [Ca²⁺]_i buffered to 500 nM, GPIP₂ was able to fully activate detectable I_crac if uptake of Ca²⁺ intracellular stores was first inhibited. Our findings suggest that when IP₃ activates the IP₃ receptor, the resulting influx of Ca²⁺ quickly inactivates the receptor, and Ca²⁺ is re-accumulated at sites that regulate I_crac. Adenophostin A, by virtue of its high receptor affinity, is resistant to this inactivation. Comparison of thapsigargin-releasable Ca²⁺ pools following activation by different IP₃ receptor agonists indicates that the critical regulatory pool of Ca²⁺ may be very small in comparison to the total IP₃-sensitive component of the endoplasmic reticulum. These findings reveal new and important roles for IP₃ receptors located on discrete IP₃-sensitive Ca²⁺ pools in calcium feedback regulation of I_crac and capacitative calcium entry.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Many extracellular stimuli act through cell surface receptors to promote generation of intracellular inositol 1,4,5-trisphosphate (IP₃)¹ and consequently release intracellular Ca²⁺ stores (1). The release of stored Ca²⁺ is commonly accompanied by influx of Ca²⁺ from the extracellular space, through the "capacitative Ca²⁺ entry" pathway (2-4). Although depletion of intracellular stores and activation of capacitative calcium entry are inextricably linked, the underlying mechanism remains poorly defined (4).

Store-operated currents, which are proposed to mediate capacitative calcium entry, have been measured in various cell types (5). To date, "Ca²⁺ release-activated Ca²⁺ current" (I_crac) is the best defined member of the store-operated current family (6). I_crac, measured in mast cells, T-lymphocytes, and rat basophilic leukemia (RBL-1) cells is both highly selective for Ca²⁺ as the permeant ion and strongly inhibited by Ca²⁺ feedback (5, 7-9). At least two forms of Ca²⁺-dependent inactivation of I_crac have been reported, termed fast and slow inactivation. Fast inactivation occurs on a sub-second time scale and is caused by increases in sub-plasmalemmal Ca²⁺ concentration in the vicinity of the Ca²⁺ channel (7, 9). Slow inactivation, on the time scale of tens to hundreds of seconds, is caused by increases in the bulk cytosolic [Ca²⁺] and is partly dependent on refilling of intracellular Ca²⁺ stores (8). In order to maximize, or indeed detect, the typically small whole-cell CRAC currents, it is necessary to minimize these forms of Ca²⁺ feedback. First, the [Ca²⁺]_i is tightly buffered to basal levels with high concentrations of Ca²⁺ chelators such as BAPTA or EGTA. Second, the cell membrane potential is held at depolarized levels to decrease the driving force for Ca²⁺ entry between current measurements. A major disadvantage of these conditions is the inability to measure simultaneous changes in [Ca²⁺]_i.

Recently, simultaneous measurements of [Ca²⁺]_i and I_crac have been reported in RBL-1 cells, an immortalized mast cell line (10). In this study, the degree of Ca²⁺ buffering was reduced from a level which effectively clamps [Ca²⁺]_i to basal concentrations, to a minimal level, which permitted fluctuations of [Ca²⁺]_i. Under these conditions, adenophostin A, an IP₃ receptor agonist with 100-fold higher affinity for the IP₃ receptor compared with the native ligand IP₃ (11) induced Ca²⁺ release and activation of I_crac (10). Several other agents that would be expected to deplete intracellular stores, including analogues of IP₃ ((2,4,5)IP₃, 3-deoxy-3-fluoro-IP₃), thapsigargin (a SERCA inhibitor), and ionomycin (a Ca²⁺ ionophore), did not activate I_crac under these conditions. It was suggested that the unique ability of adenophostin A to activate I_crac was perhaps due to action at the level of Ca²⁺-dependent inactivation of the CRAC channel.

In the present study we investigated activation of I_crac in RBL-1 cells under conditions of weak Ca²⁺ buffering, while simultaneously monitoring [Ca²⁺]_i. We compared the ability of various store depletion agents and combinations of these agents to induce a rise in [Ca²⁺]_i and to activate detectable I_crac. We now find that adenophostin A, thapsigargin, and ionomycin all activate I_crac under low buffering conditions; however, IP₃ and a stable analogue of IP₃, L--glycerophospho-D-myoinositol-4,5-bisphosphate (GPIP₂), do not. This failure was not the result of Ca²⁺ feedback on the CRAC channel. Rather, deactivation of the current appears due to inactivation of IP₃ receptors and rapid refilling of critical Ca²⁺ stores thereby diminishing the signal for activation of I_crac. This work reveals a new mechanism that involves the IP₃ receptor for Ca²⁺ feedback on I_crac. These IP₃ receptors are likely located in spatially restricted regions within the endoplasmic reticulum and are closely coupled to activation and regulation of I_crac.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Cell Culture-- Rat basophilic leukemia cells (RBL-1) were purchased from the ATCC (1378-CRL). Cells were cultured in Earle's minimal essential medium with Earle's salts, 10% fetal bovine serum, 2 mM L-glutamine, 50 units/ml penicillin, and 50 mg/ml streptomycin (37 °C, 5% CO₂). For experiments, cells were passaged onto glass coverslips (number 1_1/2) and used 12 -36 h after plating.

Fura-2 Loading and Fluorescence Measurements-- Coverslips with attached cells were mounted in a Teflon chamber and incubated at room temperature for 25 min in HEPES-buffered saline solution (HBSS; in mM, 140 NaCl, 4.7 KCl, 10 CsCl, 1.8 CaCl₂, 1.13 MgCl₂, 10 glucose, and 10 HEPES, pH 7.2) containing 1 µM Fura-2 AM (Molecular Probes). Cells were then washed and bathed in HBSS for at least 10 min before Ca²⁺ measurements were made.

Fluorescence was monitored by placing the Teflon chamber with the coverslip of Fura-2-loaded cells onto the stage of a Nikon Diaphot microscope (40× Neofluor objective). Cells were excited by light (340 and 380 nm) from a Deltascan D101 (Photon Technology International Ltd.) light source equipped with a light path chopper and dual excitation monochromators. Emitted fluorescence (510 nm) was collected by a photomultiplier tube (Omega). All experiments were conducted at room temperature (22 °C). Calibration of [Ca²⁺]_i was performed by reference to a look-up table created from Ca²⁺ standards supplied by Molecular Probes.

Electrophysiology-- Patch clamp experiments were conducted in the standard whole-cell recording configuration (12). Patch pipette (2-4 megaohm, Corning glass, 7052) solutions contained (in mM) 140 cesium aspartate, 2 MgCl₂, 10 HEPES, 1 MgATP, and either 10 BAPTA-Cs₄ (with free Ca²⁺ set to 100 or 500 nM, calculated using MaxChelator software (version 6.60) or 0.1 BAPTA-Cs₄ (with no Ca²⁺ added), pH 7.2. Fura-2 free acid (50 µM) was included in the pipette as indicated. Bath solution (HBSS) was as described above, except CaCl₂ was increased to 10 mM for Ca²⁺-HBSS or omitted for nominally Ca²⁺-free HBSS (10 mM MgCl₂ was included in nominally Ca²⁺-free HBSS). 0.2 mM EGTA was included where indicated.

In all experiments, upon forming the whole-cell configuration the cell membrane potential was held at +30 mV (to minimize Ca²⁺ entry and Ca²⁺-dependent inactivation of CRAC channels). Periodically (once every 5 s) the membrane potential was stepped to 100 mV (for 20 ms to assess I_crac), and then a voltage ramp to +60 mV, over a period of 160 ms, was applied. Currents are normalized to cell capacitance. All voltages are corrected for a 10 mV liquid-junction potential. Membrane currents were amplified with an Axopatch-1C amplifier (Axon Instruments, Burlingham, CA). Voltage clamp protocols were implemented and data acquisition performed with PCLAMP 6.1 software (Axon Instruments). Currents were filtered at 1 kHz and digitized at 200-µs intervals.

Materials-- Adenophostin A was a gift from Drs. M. Takahashi and K. Tanzawa (Sankyo Co., Ltd., Tokyo, Japan). IP₃ and ionomycin were from Calbiochem. Thapsigargin was from LC Laboratories, and GPIP₂ was from Roche Molecular Biochemicals. Cs₄BAPTA (1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid) and Fura-2 were from Molecular Probes (Eugene, OR).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Activation of I_crac Under Conditions of Strong and Weak [Ca²⁺]_i Buffering-- With pipette solutions strongly buffered (with 10 mM BAPTA) to 100 nM free [Ca²⁺], and following the voltage protocol described under "Experimental Procedures," I_crac was activated by a variety of store depletion agents in agreement with previous studies (6, 9, 10, 13) (Fig. 1). Intracellular delivery of IP₃ (20 or 40 µM), GPIP₂ (100 or 200 µM), a non-metabolizable analogue of IP₃, or adenophostin A (2 µM), or extracellular addition of thapsigargin (1 µM) or ionomycin (500 nM) activated an inward current with properties characteristic of I_crac. In each case, the current decreased rapidly upon removal of extracellular Ca²⁺ (Fig. 1B), showed inward rectification, and reversed direction at a potential positive of +30 mV (Fig. 1A). There were no significant differences among the maximum amplitudes of the currents activated by these agents (Fig. 1C). The development times were also similar, although slightly prolonged for thapsigargin (Fig. 1C; complete time course of I_crac activation with 10 mM BAPTA, 100 nM [Ca²⁺]_i is shown by the open circles in Fig. 3).

View larger version (23K): [in this window] [in a new window]   Fig. 1.   All store depletion agents activate Icrac to a similar extent when [Ca2+]i is strongly buffered to basal levels. All data are from RBL-1 cells patched in the whole-cell mode with 10 mM BAPTA in the pipette (free Ca2+ ~100 nM). A and B, cells were held at 30 mV, and Icrac was measured once every 5 s with voltage steps to 100 mV (20 ms) followed by voltage ramps to 60 mV (160 ms) (A, top panel). A, a and b show raw traces in response to these voltage ramps and correspond to the times labeled in B. A, lower panel, the leak current measured in Ca2+-free HBSS has been subtracted from the current measured in the presence of Ca2+-HBSS to give Icrac. B, activation of Icrac, measured at 100 mV, is plotted against time. Current is normalized against cell capacitance and the current density (pA/pF) is plotted. 2 µM adenophostin A was included in the patch pipette, and the whole-cell configuration was established at time 0. HBSS containing 10 mM Ca2+ was replaced with nominally Ca2+-free HBSS for the time shown. Traces are representative of 10 similar experiments. C, mean current density (filled bars) and mean time course of development (open bars) of whole-cell currents are plotted. 2 µM adenophostin A (AdA), 20 µM IP3, or 100 µM GPIP2 were included in pipettes where indicated. 500 nM ionomycin (IONO) or 1 µM thapsigargin (TG) were applied to the outside of the cell. Values are mean ± S.E. (n  6) in each case. A complete time course of Icrac activation with 10 mM BAPTA, 100 nM [Ca2+]i is shown by the open circles in Fig. 3.

With pipette solutions buffered with 0.1 mM BAPTA, [Ca²⁺]_i was weakly buffered and free to fluctuate. Under these conditions, each of the store-depleting agents caused an increase in [Ca²⁺]_i in Fura-2-loaded cells (Fig. 2, A-E upper traces). Adenophostin A (Fig. 2A), IP₃ (Fig. 2B), and GPIP₂ (Fig. 2C) each produced a biphasic increase in [Ca²⁺]_i as follows: first a peak then a lower plateau, sustained slightly but significantly above basal levels. Thapsigargin (Fig. 2D) and ionomycin (Fig. 2E), agents that bypass the IP₃ receptor, produced sustained and monophasic increases in [Ca²⁺]_i. Importantly, all the sustained increases in Ca²⁺ were reversed by removal of extracellular Ca²⁺ and therefore reflected Ca²⁺ entry. Ca²⁺ entry presumably occurs mostly during the brief hyperpolarizing pulses. In support of this idea, expansion of the Ca²⁺ traces revealed episodic Ca²⁺ peaks, with a 5-s periodicity, that correspond to membrane hyperpolarizations. These Ca²⁺ peaks were absent when extracellular Ca²⁺ was removed (data now shown).

View larger version (34K): [in this window] [in a new window]   Fig. 2.   Stimulation of Ca2+ release and activation of Icrac by adenophostin A, thapsigargin, and ionomycin but not IP3 or GPIP2 when [Ca2+]i is weakly buffered. A-E, single RBL-1 cells loaded with Fura-2 AM were patched with pipettes containing 50 µM Fura-2 free acid and a solution buffered with 0.1 mM BAPTA. A, 2 µM adenophostin A (AdA); B, 20 (in this figure) or 40 µM (not shown) IP3; C, 100 (in this figure) or 200 µM (not shown) GPIP2 was also included in the pipette solutions. D, 1 µM thapsigargin (TG); E, 500 nM ionomycin (IONO) was applied to the outside of the cell, for the period shown. HBSS containing 10 mM Ca2+ was replaced with nominally Ca2+-free HBSS as indicated. Establishment of whole-cell configuration is indicated by vertical arrows, at which time the voltage protocol described in Fig. 1 was initiated. The top panels show changes in [Ca2+]i recorded from single cells. The lower panels shows the simultaneous current densities from the same cell. F, mean current density (filled bars) and mean time course of development (open bars) of whole-cell currents are plotted. Data are from experiments similar to those shown in A-E. Values are mean ± S.E. (n  7).

Simultaneous measurements of membrane currents in the same Fura-2-loaded cells revealed that adenophostin A, thapsigargin, and ionomycin, but neither IP₃ nor GPIP₂, induced detectable activation of I_crac (Fig. 2, A-E, lower traces). The currents activated by adenophostin A (Fig. 2A), thapsigargin (Fig. 2D), and ionomycin (Fig. 2E) in weakly Ca²⁺-buffered cells displayed properties characteristic of I_crac. In each case, the current decreased rapidly upon removal of extracellular Ca²⁺. The currents also showed inward rectification and reversed direction at a potential positive of +30 mV (not shown).

Under these conditions adenophostin A (1.08 ± 0.12 pA/pF), thapsigargin (1.05 ± 0.14 pA/pF), and ionomycin (1.29 ± 0.13 pA/pF) caused similar increases in I_crac (Fig. 2F), but all of the values were significantly less than those measured with strong Ca²⁺ buffering (Fig. 1C), being 45 ± 5, 43 ± 6, and 46 ± 3%, respectively.

Following break in with pipettes containing adenophostin A, or following application of ionomycin, activation of I_crac occurred after short delays of 49 ± 11 and 88 ± 9 s, respectively (Fig. 2, A and E). However, there was a considerably longer delay between the delivery of thapsigargin and subsequent activation of I_crac (181 ± 19 s) (Fig. 2D). I_crac was activated by thapsigargin with a mean development time (time from the initial increase in current to development of current; see Ref. 13)) of 375 ± 35 s, considerably slower than that for adenophostin A (132 ± 25 s) or ionomycin (145 ± 22 s) (Fig. 2F, open bars). The initial rise in Ca²⁺ induced by thapsigargin indicates blockade of endoplasmic reticulum Ca²⁺-ATPases and the subsequent leak of Ca²⁺ from intracellular stores. The delay before detectable I_crac activation and the slow time course of development presumably reflect the time it takes to empty, to the necessary degree, the stores linked to I_crac activation. It is because of this delayed and slow activation of I_crac with thapsigargin that our earlier study failed to detect activation of I_crac by thapsigargin in RBL-1 cells (10). Also, in our earlier study (10) ionomycin failed to activate I_crac. However, a substantially higher concentration of ionomycin was used (5 µM, as opposed to 500 nM in the current study), and at this concentration ionomycin raises [Ca²⁺]_i to extremely high levels, in excess of 1 µM, which likely caused a strong Ca²⁺-dependent inactivation of I_crac.

Activation of I_crac at Elevated [Ca²⁺]_i-- We next considered how minimal Ca²⁺ buffering might prevent IP₃ and GPIP₂ from activating detectable I_crac. Direct effects of global [Ca²⁺]_i on CRAC channels is unlikely to be responsible, because the agents that activated I_crac actually raised steady-state [Ca²⁺]_i to somewhat higher levels than did IP₃ or GPIP₂. Thus, either IP₃ and GPIP₂ are capable of raising Ca²⁺ to higher levels than other agents in small, discrete regions close to the CRAC channels or, alternatively, I_crac activated by IP₃ and GPIP₂ is for some reason more sensitive to Ca²⁺ inhibition. Thus, we studied the effect of increased cytosolic Ca²⁺ on I_crac under conditions whereby both sub-plasmalemmal and global [Ca²⁺]_i were strongly buffered with 10 mM BAPTA. We buffered [Ca²⁺]_i to either a basal value ([Ca²⁺]_i ~100 nM) or a value ([Ca²⁺]_i ~500 nM) similar to that recorded in weakly buffered cells after activation with store depletion agents (Fig. 2E).

Under these conditions (10 mM BAPTA, [Ca²⁺]_i ~500 nM), adenophostin A (Fig. 3A), thapsigargin (Fig. 3D), and ionomycin (Fig. 3E) activated I_crac; IP₃ (Fig. 3B) and GPIP₂ (Fig. 3C) did not. Thus, IP₃ and GPIP₂ apparently fail to activate I_crac because with these agents the signaling mechanism is more sensitive to inhibition by elevated [Ca²⁺]_i.

View larger version (24K): [in this window] [in a new window]   Fig. 3.   Adenophostin A, thapsigargin, and ionomycin, but not IP3 and GPIP2, activate Icrac when [Ca2+]i is strongly buffered to activated levels (500 nM). A-E, cells were patched with pipette solutions containing 10 mM BAPTA and free Ca2+ set to either 500 nM (filled circles) or 100 nM (open circles). A, 2 µM adenophostin A (AdA); B, 20 (in this figure) or 40 µM (not shown) IP3; C, 100 (in this figure) or 200 µM (not shown) GPIP2 was also included in the pipette solution; D, 1 µM thapsigargin (TG); E, 500 nM ionomycin (IONO) was applied to the outside of the cell for the period shown. Cells were held at 30 mV, and the voltage protocol described in Fig. 1 was used. Traces are means ± S.E. (n  5).

Adenophostin A (2.18 ± 0.03 pA/pF), thapsigargin (2.57 ± 0.65 pA/pF), and ionomycin (2.95 ± 0.45 pA/pF) caused similar increases in I_crac. These values were also similar to measurements made with free Ca²⁺ buffered to 100 nM (Fig. 1C), being 90 ± 3, 104 ± 26, and 105 ± 20%, respectively. It is worth noting, however, that although the peak magnitude of I_crac measured with free Ca²⁺ set to 100 or 500 nM was similar, a slow inactivation of the current was prominent at the higher cytosolic [Ca²⁺]. I_crac activated by adenophostin A fell to 90 ± 12 and 56 ± 9% of the peak after 300 s, at 100 and 500 nM free Ca²⁺, respectively. Over the same period, I_crac activated by ionomycin also declined more at 500 nM, falling to 62 ± 9% of the peak compared with 87 ± 11% with 100 nM free Ca²⁺.

The mean development time of I_crac for thapsigargin at 500 nM [Ca²⁺]_i (415 ± 27 s) was again much slower than for adenophostin A (94 ± 8 s) and ionomycin (68 ± 5 s). There was also a considerable delay before thapsigargin induced detectable activation of I_crac (295 ± 34 s) compared to when free [Ca²⁺]_i was set to 100 nM (91 ± 14 s) (Fig. 3D). For both adenophostin A and ionomycin, activation of I_crac was seen after only a short delay (42 ± 12 and 88 ± 9 s, respectively).

Role of the IP₃ Receptor and SERCA Pumps-- Buffering sub-plasmalemmal [Ca²⁺] gradients (10 mM BAPTA), and raising bulk cytosolic [Ca²⁺] (500 nM), mimicked some of the effects of low Ca²⁺ buffering, namely failure of GPIP₂ and IP₃ to activate I_crac and slow activation of I_crac by thapsigargin. Given that adenophostin A, ionomycin, and thapsigargin all activate I_crac under conditions where IP₃ and GPIP₂ are ineffective, direct Ca²⁺-dependent inactivation of the CRAC channel seems unlikely to be the cause. Rather, an involvement of the IP₃ receptor itself would be indicated. Thus, I_crac could be inactivated if, when IP₃ and GPIP₂ are used, the IP₃ receptors were desensitized, and as a result the stores signaling I_crac activation were refilled. Increases in [Ca²⁺]_i are known to promote dissociation of IP₃ from the IP₃ receptor leading to faster IP₃ receptor inactivation (14). Adenophostin A, by virtue of its high affinity for the IP₃ receptor, may be less susceptible to this increased rate of dissociation. Adenophostin A would therefore prolong IP₃ receptor activation, relative to the lower affinity agonists (GPIP₂ and IP₃), and maintain depleted Ca²⁺ stores.

If indeed IP₃ receptor inactivation does lead to rapid store refilling and turns off detectable I_crac, then prevention of Ca²⁺ re-uptake (with thapsigargin) should prevent the re-uptake of Ca²⁺ that occurs with IP₃ and GPIP₂, and thus these agents would act more like adenophostin A. To test this hypothesis, thapsigargin was applied to cells loaded with Fura-2 and patch-clamped in the cell-attached mode (Fig. 4A). Patch pipettes contained 100 µM GPIP₂ and 0.1 mM BAPTA. After 50-70 s, a slight increase in [Ca²⁺]_i was detected indicating SERCA inhibition (Fig. 4A, upper trace). The whole-cell mode was then established in order to deliver GPIP₂ and measure I_crac (Fig. 4A, lower trace). GPIP₂ caused a further, more substantial increase in [Ca²⁺]_i which was sustained (compare with GPIP₂ alone, Fig. 2C). Importantly, the increase in [Ca²⁺]_i that occurred upon delivery of GPIP₂ was accompanied by rapid activation of I_crac. I_crac was activated after only a short delay (33 ± 8 s) and with a time course of 86 ± 14 s. Had the current been activated by thapsigargin alone, the delay from break-in would have been in excess of 3 min and the development time in excess of 5 min (inferred from Fig. 2D). Development of I_crac in the presence of GPIP₂ is therefore triggered by the rapid release of Ca²⁺ through activated IP₃ receptors and independent of the slow leak of Ca²⁺ induced by thapsigargin alone. The peak amplitude of I_crac was not significantly altered by the presence (1.53 ± 0.23 pA/pF) or absence of GPIP₂ (1.05 ± 0.14 pA/pF, Fig. 2F).² This is to be expected if GPIP₂ increases the rate of release, through opening IP₃ receptors, but not the overall extent.

View larger version (20K): [in this window] [in a new window]   Fig. 4.   GPIP2 activates Icrac when [Ca2+]i is raised if re-uptake of Ca2+ into stores is first eliminated. A and C, cells were loaded with Fura-2 AM and patched with pipettes containing 0.1 mM BAPTA, 50 µM Fura-2 free acid, and 100 µM GPIP2. B, cells were patched with pipettes containing 10 mM BAPTA (500 nM free Ca2+) and 100 µM GPIP2. A-C, establishment of whole-cell configuration is indicated by the vertical arrows, at which time the voltage protocol described in Fig. 1 was initiated. 1 µM thapsigargin (TG) was applied extracellularly 50-70 s before (A and B) or after (C) delivery of GPIP2. A and C, the top panels show changes in [Ca2+]i from single cells, and the lower panels show the simultaneous current densities measured from the same cell. Each trace is representative of similar responses from at least 3 similar experiments. B, traces represent mean current densities ± S.E. (n = 4).

The preceding data suggest that when Ca²⁺ is free to increase, re-uptake of Ca²⁺ prevents detectable activation of I_crac by GPIP₂. If this assumption is correct, then delivery of thapsigargin before GPIP₂, with Ca²⁺ clamped to 500 nM with 10 mM BAPTA, should also allow rapid activation of I_crac (Fig. 4B). As predicted, in cells pretreated with thapsigargin for 50-70 s, delivery of GPIP₂ led to a rapid activation of I_crac. Activation was detected after only a short delay (34 ± 11 s) and had a development time of 81 ± 13 s, compared with 415 ± 27 s for thapsigargin alone. The amplitude of the current induced by GPIP₂ and thapsigargin combined (3.16 ± 0.28 pA/pF) was once again not significantly greater than that that seen with thapsigargin alone (2.57 ± 0.65 pA/pF).

These results reveal that SERCA pumps need to be blocked if GPIP₂ is to activate detectable I_crac when [Ca²⁺]_i is weakly buffered. Refilling of stores by SERCA pumps must occur very quickly, and therefore, a decrease in IP₃ receptor activity must also occur quickly to explain the complete lack of detectable I_crac activation by GPIP₂ or IP₃. GPIP₂ is successful in activating I_crac after thapsigargin addition because despite rapid IP₃ receptor desensitization, Ca²⁺ cannot be re-accumulated into the critical stores. This predicts that if the addition of the SERCA inhibitor is delayed for even a short interval following addition of GPIP₂, I_crac activation will still fail. As shown in Fig. 4C, this is indeed the case. When thapsigargin was added 50-70 s after break-in with GPIP₂, no rapid activation of I_crac was observed; rather I_crac activation occurred slowly (311 ± 7 s) and after a latency of 222 ± 21 s, similar to activation of I_crac by thapsigargin alone (see Fig. 2D).

Collectively these results indicate that IP₃ and GPIP₂ activate IP₃ receptors and allow a rapid release of Ca²⁺ from intracellular stores, but this receptor activity and enhanced Ca²⁺ release are transient. Hence, when re-uptake of Ca²⁺ is blocked prior to GPIP₂ exposure, the release induced by GPIP₂ is sufficiently sustained to activate I_crac rapidly (Fig. 4A). However, if Ca²⁺ re-uptake is allowed to proceed for even a minute after GPIP₂ delivery, blockade of SERCA pumps at this stage does not rapidly activate I_crac, because the IP₃ receptors have already desensitized and Ca²⁺ has been re-accumulated (Fig. 4C).

The Size of the Critical Stores Regulating I_crac-- Finally, therefore, we attempted experiments designed to demonstrate more directly the re-uptake of Ca²⁺ into pools regulating I_crac and to determine their size relative to the presumably larger IP₃- and thapsigargin-sensitive stores. To this end, we activated signaling in cells with either GPIP₂ or adenophostin A, and we examined the size of the [Ca²⁺]_i signal on application of thapsigargin in Ca²⁺-free media (Fig. 5, lower panels). On delivery of adenophostin A or GPIP₂, cells were exposed to either Ca²⁺-free or Ca²⁺-containing media before assessing the thapsigargin-sensitive store content in order to determine the contribution of Ca²⁺ influx to the content of the stores. A residual, thapsigargin-sensitive, Ca²⁺ store was detected in cells after exposure to adenophostin A (Fig. 5A) or GPIP₂ (Fig. 5B) in the absence of Ca²⁺ influx (dotted lines). When Ca²⁺ influx was allowed before store assessment (dashed lines), some refilling of stores occurred after both agents, although slightly more refilling appeared to occur in the presence of GPIP₂. In each case, however, Ca²⁺ stores were not completely refilled, because control cells exposed only to thapsigargin showed the largest release of Ca²⁺ (solid lines).

View larger version (18K): [in this window] [in a new window]   Fig. 5.   Extent of intracellular Ca2+ pool depletion by adenophostin A and GPIP2. Cells were loaded with Fura-2 AM and patched with pipettes containing 0.1 mM BAPTA, 50 µM Fura-2 free acid, and either 2 µM adenophostin A (AdA) (A) or 100 µM GPIP2 (B). Establishment of the whole-cell configuration is indicated by the vertical arrows, at which time the voltage protocol described in Fig. 1 was initiated. Extracellular Ca2+ was either completely omitted (black dotted lines) or removed as indicated (black solid lines). In each trace 1 µM thapsigargin (TG) was applied to the outside of the cell as indicated. Thick black traces represent control cells exposed only to thapsigargin. The lower panels show mean changes in [Ca2+]i, collated from single cells, and the upper panels show the simultaneous current densities measured from those same cells. Traces are mean ± S.E. (n  4).

Assessment of the Ca²⁺ stores by inhibition of SERCA pumps revealed Ca²⁺ release kinetics for adenophostin A (Fig. 5A, dashed lines) which were distinct from that in control cells (Fig. 5, solid line) or GPIP₂-treated cells (Fig. 5B, dashed lines). For adenophostin A-treated cells, the rate of Ca²⁺ release was faster than in control cells, with [Ca²⁺]_i returning to base line within 514 ± 31 s, compared with 1058 ± 60 and 966 ± 92 s for control or GPIP₂-treated cells, respectively. This finding fits with our prediction that, in the presence of adenophostin A, IP₃ receptors remain more active and this maintains critical stores depleted and I_crac-detectable. GPIP₂ did not significantly increase the kinetics of Ca²⁺ release over that in control cells, suggesting minimal residual activation of IP₃ receptors.

Although the residual Ca²⁺ store was similar in cells treated with adenophostin A or GPIP₂, simultaneous current measurements revealed I_crac activation in response to adenophostin A only (Fig. 5, upper panels). This indicates that the pool of Ca²⁺ involved in controlling I_crac is very small in comparison to the total IP₃- or thapsigargin-sensitive Ca²⁺ pool. In support of this, thapsigargin, when added after adenophostin A, releases the remaining Ca²⁺ store but does not further increase I_crac amplitude (0.98 ± 0.26 versus 0.96 ± 0.12 pA/pF) (Fig. 6A). Also, adenophostin A and ionomycin activate I_crac to a similar amplitude (0.96 ± 0.12 pA/pF versus 1.16 ± 0.14 pA/pF), but only the latter entirely depletes thapsigargin-sensitive Ca²⁺ stores (Fig. 6B). Thus while neither adenophostin nor GPIP₂ release the entire thapsigargin-sensitive pool, adenophostin A releases and maintains empty, a small pool closely coupled to I_crac activation, which is not maintained empty by GPIP₂.

View larger version (23K): [in this window] [in a new window]   Fig. 6.   Adenophostin A activates Icrac to a similar extent as ionomycin and thapsigargin without depleting all thapsigargin-sensitive Ca2+ stores. Cells were loaded with Fura-2 AM and patched with pipettes containing 0.1 mM BAPTA, 50 µM Fura-2 free acid, with (A) or without (B) 2 µM AdA. Establishment of the whole-cell configuration is indicated by the vertical arrows, at which time the voltage protocol described in Fig. 1 was initiated. Extracellular Ca2+ was either completely omitted (dotted line in B) or removed where indicated (solid lines). Thapsigargin (TG, 1 µM) and ionomycin (IONO, 500 nM) were applied to the outside of the cell where indicated. The upper part of each panel shows the mean current densities measured simultaneously with [Ca2+]i in the same cells (lower traces). Traces are mean ± S.E. (n  3).

Our data strongly indicate that small specialized sub-compartments of the endoplasmic reticulum are coupled to activation of I_crac. These sub-compartments are drained of Ca²⁺ by adenophostin A and GPIP₂, but when [Ca²⁺]_i rises, only adenophostin A keeps them in a sufficiently depleted state to activate detectable I_crac, apparently because its high receptor affinity prevents Ca²⁺-dependent IP₃ receptor desensitization. Ionomycin and thapsigargin release Ca²⁺ from all compartments, but release of these additional compartments is not coupled to I_crac activation.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Our laboratory previously established a method for the simultaneous measurement of I_crac and [Ca²⁺]_i in RBL-1 cells (10). Prevention of Ca²⁺ entry between current measurements (by holding the cell membrane at a positive voltage) was the key to minimizing Ca²⁺-dependent inactivation of I_crac. In that study we showed that adenophostin A, but not IP₃ congeners, was an effective activator of I_crac when [Ca²⁺]_i was weakly buffered and free to rise. We now show that adenophostin A is effective because it keeps IP₃ receptors active, maintaining specific, critical stores sufficiently depleted of Ca²⁺ to signal I_crac activation. In contrast, other IP₃ receptor agonists, including IP₃ itself and GPIP₂, fail to maintain active IP₃ receptors, allowing refilling of the critical stores. Depletion of Ca²⁺ stores represents the sine qua non for capacitative calcium entry, and so the signal for I_crac is substantially diminished and the current is not activated to a detectable level. This is a novel mode of Ca²⁺-dependent inactivation of I_crac, in that the Ca²⁺-feedback occurs at the level of the IP₃ receptor, rather than the CRAC channel. The possible involvement of direct Ca²⁺-dependent inactivation of the CRAC channel being responsible for the failure of GPIP₂ or IP₃ to activate I_crac is ruled out by the ability of other store-depleting agents, including thapsigargin and ionomycin, to activate I_crac under similar conditions.

Our data reveal how IP₃ receptor activity can strongly influence the activity of CRAC channels. The IP₃ receptor participates "indirectly" in regulating CRAC channels in RBL-1 cells, because its activity determines the state of filling of a subset of Ca²⁺ stores intricately linked to I_crac activation. When the IP₃ receptor inactivates, these stores rapidly refill, through the activity of SERCA pumps, minimizing the signal for CRAC channel activation. Recently, the IP₃ receptor was also suggested to have a "direct" role in the regulation of Htrp3 channels stably expressed in HEK293 cells (15). The Htrp3 protein has previously been shown to form a channel activated by phospholipase C-linked agonists (16-18). Kiselyov and colleagues (20) now report that overexpressed Htrp3 channels can also be activated by store depletion but that the IP₃ receptor directly interacts with the Htrp3 channel and that occupation of the IP₃ receptor is a requirement for Htrp3 activation. Whether the IP₃ receptor works to modulate directly endogenous Ca²⁺ channels is unclear, although single channels that could be activated by either store depletion (in cell attached patches) or IP₃ (in excised patches) have been reported (19, 20).

In RBL-1 cells, when Ca²⁺ is free to fluctuate, adenophostin A, GPIP₂, and IP₃ each readily release Ca²⁺ from the specialized domains linked to I_crac activation. However, only adenophostin A keeps these stores sufficiently empty to signal detectable I_crac activation, because only it maintains IP₃ receptor activity. The distinct abilities of these agonists to maintain IP₃ receptor activity may reflect the very different affinities they display for the IP₃ receptor. Adenophostin A is ~100-fold more potent than 1,4,5-IP₃ (11), whereas GPIP₂ is ~10-fold weaker (21). Increases in Ca²⁺ are reported to increase the rate of dissociation of IP₃ from the IP₃ receptor (14). The lower affinity agonists would dissociate from the IP₃ receptor more quickly than adenophostin A, leading to a more rapid receptor inactivation. In the continued presence of adenophostin A some slow inactivation of I_crac is seen with higher cytosolic [Ca²⁺]_i (Fig. 3A). This may reflect either the slow dissociation of adenophostin A from the IP₃ receptor, reducing the signal for I_crac activation, or a slow Ca²⁺-dependent inactivation of the CRAC channel (8, 22).

RBL cells have been shown to express all three forms of the IP₃ receptor (23), with the predominating species being type 2. Because the number of IP₃ receptors involved in this subcompartment of the endoplasmic reticulum is likely small compared with the total number of receptors, any of the three types or possibly all three could be located there. There is evidence for specific involvement of the type 3 IP₃ receptor in regulating capacitative calcium entry (24). However, it is only the type-1 and type-2 receptors that appear to be regulated negatively by Ca²⁺ (25, 26), and thus type 1 and/or type 2 receptors would more likely be involved in the negative regulation seen here.

In addition to the findings discussed above, our results also suggest that the stores linked to I_crac activation in RBL-1 cells reside within a specialized subcompartment of the endoplasmic reticulum. Adenophostin A activates I_crac by releasing only a fraction of the thapsigargin- and ionomycin-releasable Ca²⁺ stores, yet further release of residual Ca²⁺ stores does not augment I_crac (Figs. 2F and 6, A and C). Hofer et al. (27) have also shown that in RBL-1 cells, full activation of I_crac occurs with only partial depletion of intracellular stores by ionomycin. We cannot determine if the failure of ionomycin and thapsigargin to induce a larger activation of I_crac than adenophostin A reflects subcompartments of the endoplasmic reticulum, some of which are and some of which are not linked to I_crac activation, or more simply a requirement for only partial depletion of all (or of critical) stores for maximal I_crac activation. However, we also observed that with minimal [Ca²⁺]_i buffering, GPIP₂ depletes nearly as much of the thapsigargin-sensitive store as does adenophostin A, yet GPIP₂ fails to activate detectable I_crac and adenophostin A activates it maximally. This striking distinction in the action of GPIP₂ and adenophostin A therefore must reflect the filling state of a pool that is very small in comparison to the total IP₃-sensitive Ca²⁺ stores.

There is already some evidence to suggest that specialized domains of the endoplasmic reticulum are coupled to activation of I_crac in RBL-1 cells (13, 28). Parekh et al. (28) observed differences in the concentration-effect relationships for Ca²⁺ release and I_crac activation which they interpreted as reflecting heterologous Ca²⁺ pools, only some of which were involved in I_crac regulation. Huang and Putney (13) observed different latencies for (2,4,5)IP₃, thapsigargin, and ionomycin activation of Ca²⁺ release and I_crac. A significant delay was observed between the initiation of Ca²⁺ stores depletion and the activation of I_crac for (2,4,5)IP₃ and thapsigargin. However, with ionomycin, little or no delay was observed. A small compartment of the endoplasmic reticulum was suggested to regulate Ca²⁺ entry, which was relatively resistant to store depletion by IP₃ and thapsigargin but not by ionomycin. This could result from a non-homogenous distribution of IP₃ receptors and leak channels. The more direct Ca²⁺-transporting action of ionomycin would not be affected by such distributions. There is evidence that sub-compartments of the endoplasmic reticulum regulate Ca²⁺ influx in other cell types. Treatment of NIH-3T3 cells with the phorbol ester phorbol 12-myristate 13-acetate caused a loss of Ca²⁺ (~70%) from thapsigargin- and IP₃-sensitive pools, but this depletion did not lead to activation of capacitative Ca²⁺ entry (29).

When Ca²⁺ stores are depleted by the SERCA inhibitor, thapsigargin, the release of Ca²⁺ depends on poorly understood "leak" channels in the endoplasmic reticulum. Interestingly, and consistent with our earlier conclusions (13), our data suggest that the specialized stores linked to I_crac activation are resistant to rapid emptying by the endogenous leak pathway. First, we observe a long delay between the initiation of Ca²⁺ store depletion and activation of I_crac by thapsigargin. Second, the current activated by thapsigargin in weakly buffered RBL-1 cells or in cells with [Ca²⁺]_i set to 500 nM has very slow development kinetics. These sub-compartments therefore either possess a low number of leak channels or lack them entirely or possess thapsigargin-insensitive pumps which act to slow the net Ca²⁺ leak (30, 31). The cause of the large increase in both the delay to onset and development kinetics of I_crac activation by thapsigargin after reduction of Ca²⁺ buffering from 10 to 0.1 mM BAPTA or on clamping [Ca²⁺]_i to 500 nM is unknown but presumably results from elevated [Ca²⁺]_i. There are at least three explanations. 1) The endogenous leak of Ca²⁺ from intracellular stores is slower at higher cytoplasmic [Ca²⁺], for example if basally active IP₃ receptors account for some of the endogenous leak. 2) In the presence of elevated [Ca²⁺]_i thapsigargin inhibition of SERCA pumps is slowed (32); therefore increased Ca²⁺ is protective. As a result, blockade of the SERCA pumps may be delayed, allowing the pumps to continue working for a longer period in the presence of thapsigargin. 3) RBL-1 cells may possess thapsigargin-resistant Ca²⁺ pumps, which are more active when cytoplasmic Ca²⁺ is allowed to rise and therefore slow the net loss of Ca²⁺ from intracellular stores (30).

This is the first study demonstrating that Ca²⁺ regulation of the IP₃ receptor plays a significant role in Ca²⁺-dependent negative feedback of I_crac. Furthermore, these findings provide a mechanism explaining the previously documented failure of IP₃ and its congeners to activate measurable I_crac in RBL-1 cells in the absence of maximal intracellular Ca²⁺ buffering. In Jurkat T-cells, with [Ca²⁺]_i moderately buffered (1.4 mM EGTA) and Ca²⁺ stores passively depleted (by the removal of extracellular Ca²⁺), a slow turn-off of I_crac, partially dependent on store refilling, accompanied the delayed rise in [Ca²⁺]_i induced by Ca²⁺ influx (8). In this setting, store refilling would be expected to occur as soon as extracellular Ca²⁺ is reapplied because nothing is actively keeping stores depleted. Another study in RBL cells used the low affinity Ca²⁺ chelator TPEN to rapidly chelate and reset stored [Ca²⁺] (27). I_crac was assessed after removal of TPEN, when stores were completely refilled. These authors observed a CRAC current, which turned off over a period of ~70 s, confirming that store refilling turns off I_crac but not immediately. Hence, both turn on and turn off of I_crac are rather slow (~1-2 min).

Because of these slow kinetics, in our experiments with IP₃ and GPIP₂ as activators, the stores linked to I_crac activation may never be depleted sufficiently or for a long enough period to activate I_crac to a detectable level. Importantly, in our experiments, despite some refilling of Ca²⁺ stores in the presence of GPIP₂, stores were not completely refilled (Fig. 5B), and although the currents underlying Ca²⁺ entry for GPIP₂ and IP₃ were beneath the level of detection, Ca²⁺ entry still occurred as evidenced by a sustained increase in [Ca²⁺]_i that reversed upon removal of extracellular Ca²⁺ (Fig. 2, B and C). Hence, the partially empty stores must signal some Ca²⁺ entry, but this is not detectable as a current. I_crac may be active under all conditions in which capacitative calcium entry occurs, but it may be below the threshold for detection. In the absence of specific pharmacological probes for these channels, it cannot be disproved that an as yet unidentified but distinct pathway contributes to entry under these conditions. Interestingly, Zhang and McCloskey (33) using the nystatin perforated patch technique and with no added intracellular calcium buffer were able to detect an inwardly rectifying Ca²⁺ current in RBL-2H3 cells in response to either immunoglobulin E (presumably acting through IP₃) or thapsigargin. This current was seen at 37 °C but not at room temperature. Hoth et al. (34) have demonstrated that in T-lymphocytes the ability to detect I_crac under conditions of low physiological calcium buffering in the patch pipette depends on endogenous calcium buffering by mitochondria. Thus, the ability to measure a calcium current associated with capacitative calcium entry may depend on a number of technical factors, including both experimentally applied as well as physiological calcium buffering. But it is clear from the data in this report and others in the literature that significant entry of calcium can occur through store-operated channels, whatever their nature, when the magnitude of the associated current is below the level of detection with presently available methodologies.

	ACKNOWLEDGEMENTS

We gratefully acknowledge the gift of adenophostin A by Drs. M. Takahashi and K. Tanzawa of Sankyo Co., Ltd. Helpful suggestions were provided by Drs. M. Ho, J. Yakel, G. Bird, and M. Carew.

	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.

To whom correspondence should addressed. Fax: 919-541-1898; E-mail: broad@niehs.nih.gov.

² Although the mean value for the current in the presence of GPIP₂ and thapsigargin was almost 50% larger than that for thapsigargin alone, the difference was not statistically significant by either t test or analysis of variance, owing to the variability in values of I_crac from one preparation to another. However, we note that the value for GPIP₂ plus thapsigargin (1.53 ± 0.23 pA/pF) was similar to the value for ionomycin under the same conditions (1.29 ± 0.13 pA/pF). This suggests that if GPIP₂ plus thapsigargin does induce a current larger than for thapsigargin alone, it is likely due to increased release of Ca²⁺ rather than a specific effect of GPIP₂ on the channel activation mechanism.

	ABBREVIATIONS

The abbreviations used are: IP₃, inositol 1,4,5-trisphosphate; I_crac, Ca²⁺ release-activated Ca²⁺ current; BAPTA, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; GPIP, L--glycerophospho-D-myo-inositol-4,5-bisphosphate; HBSS, HEPES-buffered saline solution.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES