Morphological changes of T cells following formation of the immunological synapse modulate intracellular calcium signals

Sustained Ca(2+) influx through plasma membrane Ca(2+) released-activated Ca(2+) (CRAC) channels is essential for T cell activation. Since inflowing Ca(2+) inactivates CRAC channels, T cell activation is only possible if Ca(2+)-dependent inactivation is prevented. We have previously reported that sustained Ca(2+) influx through CRAC channels requires both mitochondrial Ca(2+) uptake and mitochondrial translocation towards the plasma membrane in order to prevent Ca(2+)-dependent channel inactivation. Here, we show that morphological changes following formation of the immunological synapse (IS) modulate Ca(2+) influx through CRAC channels. Cell shape changes were dependent on the actin cytoskeleton, and they sustained Ca(2+) entry by bringing mitochondria and the plasma membrane in closer proximity. The increased percentage of mitochondria beneath the plasma membrane following shape changes occurred in all 3 dimensions and correlated with an increase in the amplitude of Ca(2+) signals. The shape change-dependent mitochondrial localization close to the plasma membrane prevented CRAC channel inactivation even in T cells in which dynein motor protein-dependent mitochondria movements towards the plasma membrane were completely abolished, highlighting the importance of the shape change-dependent control of Ca(2+) influx. Our results suggest that morphological changes do not only facilitate an efficient contact with antigen presenting cells but also strongly modulate Ca(2+) dependent T cell activation.     

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

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

Potentiation of Fcepsilon receptor I-activated Ca(2+) current (I(CRAC)) by cholera toxin: possible mediation by ADP ribosylation factor

Antigen-evoked influx of extracellular Ca(2+) into mast cells may occur via store-operated Ca(2+) channels called calcium release-activated calcium (CRAC) channels. In mast cells of the rat basophilic leukemia cell line (RBL-2H3), cholera toxin (CT) potentiates antigen-driven uptake of (45)Ca(2+) through cAMP-independent means. Here, we have used perforated patch clamp recording at physiological temperature to test whether cholera toxin or its substrate, Gs, directly modulates the activity of CRAC channels. Cholera toxin dramatically amplified (two- to fourfold) the Ca(2)+ release-activated Ca(2+) current (I(CRAC)) elicited by suboptimal concentrations of antigen, without itself inducing I(CRAC), and this enhancement was not mimicked by cAMP elevation. In contrast, cholera toxin did not affect the induction of I(CRAC) by thapsigargin, an inhibitor of organelle Ca(2+) pumps, or by intracellular dialysis with low Ca(2+) pipette solutions. Thus, the activity of CRAC channels is not directly controlled by cholera toxin or Gsalpha. Nor was the potentiation of I(CRAC) due to enhancement of phosphoinositide hydrolysis or calcium release. Because Gs and the A subunit of cholera toxin bind to ADP ribosylation factor (ARF) and could modulate its activity, we tested the sensitivity of antigen-evoked I(CRAC) to brefeldin A, an inhibitor of ARF-dependent functions, including vesicle transport. Brefeldin A blocked the enhancement of antigen-evoked I(CRAC) without inhibiting ADP ribosylation of Gsalpha, but it did not affect I(CRAC) induced by suboptimal antigen or by thapsigargin. These data provide new evidence that CRAC channels are a major route for Fcin receptor I-triggered Ca(2+) influx, and they suggest that ARF may modulate the induction of I(CRAC) by antigen.     

Mitochondrial regulation of store-operated CRAC channels

In eukaryotic cells, one major route for Ca(2+) influx is through store-operated CRAC channels, which are activated following a fall in Ca(2+) content within the endoplasmic reticulum. Mitochondria are key regulators of this ubiquitous Ca(2+) influx pathway. Respiring mitochondria rapidly take up some of the Ca(2+) released from the stores, resulting in more extensive store depletion and thus robust activation of CRAC channels. As CRAC channels open, the ensuing rise in cytoplasmic Ca(2+) feeds back to inactivate the channels. By buffering some of the incoming Ca(2+) mitochondria reduce Ca(2+)-dependent inactivation of the CRAC channels, resulting in more prolonged Ca(2+) influx. However, mitochondria can release Ca(2+) close to the endoplasmic reticulum, accelerating store refilling and thus promoting deactivation of the CRAC channels. Mitochondria thus regulate all major transitions in CRAC channel gating, revealing remarkable versatility in how this organelle impacts upon Ca(2+) influx. Recent evidence suggests that mitochondria also control CRAC channels through mechanisms that are independent of their Ca(2+)-buffering actions and ability to generate ATP. Furthermore, pyruvic acid, a key intermediary metabolite and precursor substrate for the Krebs cycle, reduces the extent of Ca(2+)-dependent inactivation of CRAC channels. Hence mitochondrial metabolism impacts upon Ca(2+) influx through CRAC channels and thus on a range of key downstream cellular responses.     

Fundamental Ca2+ signaling mechanisms in mouse dendritic cells: CRAC is the major Ca2+ entry pathway

Although Ca(2+)-signaling processes are thought to underlie many dendritic cell (DC) functions, the Ca(2+) entry pathways are unknown. Therefore, we investigated Ca(2+)-signaling in mouse myeloid DC using Ca(2+) imaging and electrophysiological techniques. Neither Ca(2+) currents nor changes in intracellular Ca(2+) were detected following membrane depolarization, ruling out the presence of functional voltage-dependent Ca(2+) channels. ATP, a purinergic receptor ligand, and 1-4 dihydropyridines, previously suggested to activate a plasma membrane Ca(2+) channel in human myeloid DC, both elicited Ca(2+) rises in murine DC. However, in this study these responses were found to be due to mobilization from intracellular stores rather than by Ca(2+) entry. In contrast, Ca(2+) influx was activated by depletion of intracellular Ca(2+) stores with thapsigargin, or inositol trisphosphate. This Ca(2+) influx was enhanced by membrane hyperpolarization, inhibited by SKF 96365, and exhibited a cation permeability similar to the Ca(2+) release-activated Ca(2+) channel (CRAC) found in T lymphocytes. Furthermore, ATP, a putative DC chemotactic and maturation factor, induced a delayed Ca(2+) entry with a voltage dependence similar to CRAC. Moreover, the level of phenotypic DC maturation was correlated with the extracellular Ca(2+) concentration and enhanced by thapsigargin treatment. These results suggest that CRAC is a major pathway for Ca(2+) entry in mouse myeloid DC and support the proposal that CRAC participates in DC maturation and migration.     

Low cytoplasmic [Ca(2+)] activates I(CRAC) independently of global Ca(2+) store depletion in RBL-1 cells

Release of Ca(2+) from inositol (1,4,5)-trisphosphate-sensitive Ca(2+) stores causes "capacitative calcium entry," which is mediated by the so-called "Ca(2+) release-activated Ca(2+) current" (I(CRAC)) in RBL-1 cells. Refilling of the Ca(2+) stores or high cytoplasmic [Ca(2+)] ([Ca(2+)](cyt)) inactivate I(CRAC). Here we address the question if also [Ca(2+)](cyt) lower than the resting [Ca(2+)](cyt) influences store-operated channels. We therefore combined patch clamp and mag fura-2 fluorescence methods to determine simultaneously both I(CRAC) and [Ca(2+)] within Ca(2+) stores of RBL-1 cells ([Ca(2+)](store)). We found that low [Ca(2+)](cyt) in the range of 30-50 nM activates I(CRAC) and Ca(2+) influx spontaneously and independently of global Ca(2+) store depletion, while elevation of [Ca(2+)](cyt) to the resting [Ca(2+)](cyt) (100 nM) resulted in store dependence of I(CRAC) activation. We conclude that spontaneous activation of I(CRAC) by low [Ca(2+)](cyt) could serve as a feedback mechanism keeping the resting [Ca(2+)](cyt) constant.     

Calumin, a Ca²⁺-binding protein on the endoplasmic reticulum, alters the ion permeability of Ca²⁺ release-activated Ca²⁺ (CRAC) channels

Store-operated channels (SOC) are Ca(2+)-permeable channels that are activated by IP(3)-receptor-mediated Ca(2+) depletion of the endoplasmic reticulum (ER). Recent studies identify a membrane pore subunits, Orai1 and a Ca(2+) sensor on ER, STIM1 as components of Ca(2+) release-activated Ca(2+) (CRAC) channels, which are well-characterized SOCs. On the other hand, proteins that act as modulators of SOC activity remain to be identified. Calumin is a Ca(2+)-binding protein that resides on the ER and functional experiments using calumin-null mice demonstrate that it is involved in SOC function, although its role is unknown. This study used electrophysiological analysis to explore whether calumin modulates CRAC channel activity. CRAC channel currents were absent in HEK293 cells co-expressing calumin with the CRAC channel components, Orai1 or STIM1. Meanwhile, HEK cells that co-expressed calumin with CRAC channels exhibited larger currents with slower inactivation than cells expressing CRAC channels alone. The current-voltage relationship showed an inwardly rectifying current, but a negative shift in the reversal potential of greater than 60mV was observed in HEK cells co-expressing calumin with CRAC channels. In addition, the permeability coefficient ratio of Ca(2+) over monovalent cations was much lower than that of cells expressing CRAC channels alone. Replacement of Na(+) with N-methyl-d-glucamine(+) in the external solution noticeably diminished the CRAC current in HEK cells co-expressing calumin and CRAC channels. In a Cs(+)-based external solution, CRAC current was not observed in either cell-type. In addition, Ca(2+) imaging analysis revealed that co-transfection of calumin reduced extracellular Ca(2+) influx via CRAC channels. Further, calumin was shown to be directly associated with CRAC channels. These results reveal a novel mechanism for the regulation of CRAC channels by calumin.     

Calcium inhibition and calcium potentiation of Orai1, Orai2, and Orai3 calcium release-activated calcium channels

The recent discoveries of Stim1 and Orai proteins have shed light on the molecular makeup of both the endoplasmic reticulum Ca(2+) sensor and the calcium release-activated calcium (CRAC) channel, respectively. In this study, we investigated the regulation of CRAC channel function by extracellular Ca(2+) for channels composed primarily of Orai1, Orai2, and Orai3, by co-expressing these proteins together with Stim1, as well as the endogenous channels in HEK293 cells. As reported previously, Orai1 or Orai2 resulted in a substantial increase in CRAC current (I(crac)), but Orai3 failed to produce any detectable Ca(2+)-selective currents. However, sodium currents measured in the Orai3-expressing HEK293 cells were significantly larger in current density than Stim1-expressing cells. Moreover, upon switching to divalent free external solutions, Orai3 currents were considerably more stable than Orai1 or Orai2, indicating that Orai3 channels undergo a lesser degree of depotentiation. Additionally, the difference between depotentiation from Ca(2+) and Ba(2+) or Mg(2+) solutions was significantly less for Orai3 than for Orai1 or -2. Nonetheless, the Na(+) currents through Orai1, Orai2, and Orai3, as well as the endogenous store-operated Na(+) currents in HEK293 cells, were all inhibited by extracellular Ca(2+) with a half-maximal concentration of approximately 20 mum. We conclude that Orai1, -2, and -3 channels are similarly inhibited by extracellular Ca(2+), indicating similar affinities for Ca(2+) within the selectivity filter. Orai3 channels appeared to differ from Orai1 and -2 in being somewhat resistant to the process of Ca(2+) depotentiation.     

Dissecting ICRAC, a store-operated calcium current

The use of Ca(2+) for intracellular signalling necessitates tight local and global control of cytoplasmic Ca(2+) concentration, and mechanisms for maintaining the net Ca(2+) balance. It has long been recognized that intracellular Ca(2+) stores exert control over Ca(2+) influx at the plasma membrane through a process of store-operated Ca(2+) entry (SOCE). The Ca(2+) current I(CRAC) is the best characterized instance of SOCE, but the elements of the pathway leading to I(CRAC) have eluded biochemical definition for more than a decade. However, the recent identification of key proteins underlying I(CRAC)--STIM1 and Orai1--has led to several insights into this ER-to-plasma membrane signalling system and to the recognition that it is an ancient and conserved mechanism in multicellular organisms.     

Microdomains of high calcium are not required for exocytosis in RBL-2H3 mucosal mast cells

We have previously shown that store-associated microdomains of high Ca(2+) are not essential for exocytosis in RBL-2H3 mucosal mast cells. We have now examined whether Ca(2+) microdomains near the plasma membrane are required, by comparing the secretory responses seen when Ca(2+) influx was elicited by two very different mechanisms. In the first, antigen was used to activate the Ca(2+) release-activated Ca(2+) (CRAC) current (I(CRAC)) through CRAC channels. In the second, a Ca(2+) ionophore was used to transport Ca(2+) randomly across the plasma membrane. Since store depletion by Ca(2+) ionophore will also activate I(CRAC), different means of inhibiting I(CRAC) before ionophore addition were used. Ca(2+) responses and secretion in individual cells were compared using simultaneous indo-1 microfluorometry and constant potential amperometry. Secretion still takes place when the increase in intracellular Ca(2+) occurs diffusely via the Ca(2+) ionophore, and at an average intracellular Ca(2)+ concentration that is no greater than that observed when Ca(2+) entry via CRAC channels triggers secretion. Our results suggest that microdomains of high Ca(2+) near the plasma membrane, or associated with mitochondria or Ca(2+) stores, are not required for secretion. Therefore, we conclude that modest global increases in intracellular Ca(2+) are sufficient for exocytosis in these nonexcitable cells.     

Functional properties of endogenous receptor- and store-operated calcium influx channels in HEK293 cells

Activation of phospholipase C (PLC)-mediated signaling pathways in non-excitable cells causes the release of calcium (Ca2+) from inositol 1,4,5-trisphosphate (InsP3)-sensitive intracellular Ca2+ stores and activation of Ca2+ influx via plasma membrane Ca2+ channels. The properties and molecular identity of plasma membrane Ca2+ influx channels in non-excitable cells is a focus of intense investigation. In the previous studies we used patch clamp electrophysiology to describe the properties of Ca2+ influx channels in human carcinoma A431 cell lines. Now we extend our studies to human embryonic kidney HEK293 cells. By using a combination of Ca2+ imaging and whole cell and single channel patch clamp recordings we discovered that: 1) HEK293 cells contain four types of plasma membrane Ca2+ influx channels: I(CRAC), Imin, Imax, and I(NS); 2) I(CRAC) channels are highly Ca2+-selective (P(Ca/Cs)>1000) and I(CRAC) single channel conductance is too small for single channel analysis; 3) Imin channels in HEK293 cells display functional properties identical to Imin channels in A431 cells, with single channel conductance of 1.2 pS for divalent cations, 10 pS for monovalent cations, and divalent cation selectivity P(Ba/K)=20; 4) Imin channels in HEK293 cells are activated by InsP3 and inhibited by phosphatidylinositol 4,5-bisphosphate, but store-independent; 5) when compared with Imin, Imax channels have higher conductance for divalent (17 pS) and monovalent (33 pS) cations, but less selective for divalent cations (P(Ba/K)=4), 6) Imax channels in HEK293 cells can be activated by InsP3 or by Ca2+ store depletion; 7) I(NS) channels are non-selective (P(Ba/K)=0.4) and display a single channel conductance of 5 pS; and 8) I(NS) channels are not gated by InsP3 but activated by depletion of intracellular Ca2+ stores. Our findings provide novel information about endogenous Ca2+ channels supporting receptor-operated and store-operated Ca2+ influx pathways in HEK293 cells.     

Ion channels modulating mouse dendritic cell functions

Ca(2+)-mediated signal transduction pathways play a central regulatory role in dendritic cell (DC) responses to diverse Ags. However, the mechanisms leading to increased [Ca(2+)](i) upon DC activation remained ill-defined. In the present study, LPS treatment (100 ng/ml) of mouse DCs resulted in a rapid increase in [Ca(2+)](i), which was due to Ca(2+) release from intracellular stores and influx of extracellular Ca(2+) across the cell membrane. In whole-cell voltage-clamp experiments, LPS-induced currents exhibited properties similar to the currents through the Ca(2+) release-activated Ca(2+) channels (CRAC). These currents were highly selective for Ca(2+), exhibited a prominent inward rectification of the current-voltage relationship, and showed an anomalous mole fraction and a fast Ca(2+)-dependent inactivation. In addition, the LPS-induced increase of [Ca(2+)](i) was sensitive to margatoxin and ICAGEN-4, both inhibitors of voltage-gated K(+) (Kv) channels Kv1.3 and Kv1.5, respectively. MHC class II expression, CCL21-dependent migration, and TNF-alpha and IL-6 production decreased, whereas phagocytic capacity increased in LPS-stimulated DCs in the presence of both Kv channel inhibitors as well as the I(CRAC) inhibitor SKF-96365. Taken together, our results demonstrate that Ca(2+) influx in LPS-stimulated DCs occurs via Ca(2+) release-activated Ca(2+) channels, is sensitive to Kv channel activity, and is in turn critically important for DC maturation and functions.     

A store-operated nonselective cation channel in lymphocytes is activated directly by Ca(2+) influx factor and diacylglycerol

Agonist-receptor interactions at the plasma membrane often lead to activation of store-operated channels (SOCs) in the plasma membrane, allowing for sustained Ca(2+) influx. While Ca(2+) influx is important for many biological processes, little is known about the types of SOCs, the nature of the depletion signal, or how the SOCs are activated. We recently showed that in addition to the Ca(2+) release-activated Ca(2+) (CRAC) channel, both Jurkat T cells and human peripheral blood mononuclear cells express novel store-operated nonselective cation channels that we termed Ca(2+) release-activated nonselective cation (CRANC) channels. Here we demonstrate that activation of both CRAC and CRANC channels is accelerated by a soluble Ca(2+) influx factor (CIF). In addition, CRANC channels in inside-out plasma membrane patches are directly activated upon exposure of their cytoplasmic side to highly purified CIF preparations. Furthermore, CRANC channels are also directly activated by diacylglycerol. These results strongly suggest that the Ca(2+) store-depletion signal is a diffusible molecule and that at least some SOCs may have dual activation mechanisms.     

Molecular basis of the CRAC channel

Ca(2+) release-activated Ca(2+) (CRAC) channels, located in the plasma membrane, are opened upon release of Ca(2+) from intracellular stores, permitting Ca(2+) entry and sustained [Ca(2+)](i) signaling that replenishes the store in numerous cell types. This mechanism is particularly important in T lymphocytes of the immune system, providing the missing link in the signal transduction cascade that is initiated by T cell receptor engagement and leads to altered expression of genes that results ultimately in the production of cytokines and cell proliferation. In the past three years, RNA interference screens together with over-expression and site-directed mutagenesis have identified the triggering molecule (Stim) that links store depletion to CRAC channel-mediated Ca(2+) influx and the pore subunit (Orai) of the CRAC channel that allows highly selective entry of Ca(2+) ions into cells.     

Plasma membrane calcium channels in human carcinoma A431 cells are functionally coupled to inositol 1,4,5-trisphosphate receptor-phosphatidylinositol 4,5-bisphosphate complexes

In most nonexcitable cells, calcium (Ca(2+)) release from inositol 1,4,5-trisphosphate (InsP(3))-sensitive intracellular Ca(2+) stores is coupled to Ca(2+) influx (calcium release-activated channels (I(CRAC))) pathway. Despite intense investigation, the molecular identity of I(CRAC) and the mechanism of its activation remain poorly understood. InsP(3)-dependent miniature calcium channels (I(min)) display functional properties characteristic for I(CRAC). Here we used patch clamp recordings of I(min) channels in human carcinoma A431 cells to demonstrate that I(min) activity was greatly enchanced in the presence of anti-phosphatidylinositol 4, 5-bisphosphate antibody (PIP(2)Ab) and diminished in the presence of PIP(2). Anti-PIP(2) antibody induced a greater than 6-fold increase in I(min) sensitivity for InsP(3) activation and an almost 4-fold change in I(min) maximal open probability. The addition of exogenous PIP(2) vesicles to the cytosolic surface of inside-out patches inhibited I(min) activity. These results lead us to propose an existence of a Ca(2+) influx pathway in nonexcitable cells activated via direct conformational coupling with a selected population of InsP(3) receptors, located just underneath the plasma membrane and coupled to PIP(2). The described pathway provides for a highly compartmentalized Ca(2+) influx and intracellular Ca(2+) store refilling mechanism.     

All-or-none activation of CRAC channels by agonist elicits graded responses in populations of mast cells

In nonexcitable cells, receptor stimulation evokes Ca(2+) release from the endoplasmic reticulum stores followed by Ca(2+) influx through store-operated Ca(2+) channels in the plasma membrane. In mast cells, store-operated entry is mediated via Ca(2+) release-activated Ca(2+) (CRAC) channels. In this study, we find that stimulation of muscarinic receptors in cultured mast cells results in Ca(2+)-dependent activation of protein kinase Calpha and the mitogen activated protein kinases ERK1/2 and this is required for the subsequent stimulation of the enzymes Ca(2+)-dependent phospholipase A(2) and 5-lipoxygenase, generating the intracellular messenger arachidonic acid and the proinflammatory intercellular messenger leukotriene C(4). In cell population studies, ERK activation, arachidonic acid release, and leukotriene C(4) secretion were all graded with stimulus intensity. However, at a single cell level, Ca(2+) influx was related to agonist concentration in an essentially all-or-none manner. This paradox of all-or-none CRAC channel activation in single cells with graded responses in cell populations was resolved by the finding that increasing agonist concentration recruited more mast cells but each cell responded by generating all-or-none Ca(2+) influx. These findings were extended to acutely isolated rat peritoneal mast cells where muscarinic or P2Y receptor stimulation evoked all-or-none activation of Ca(2+)entry but graded responses in cell populations. Our results identify a novel way for grading responses to agonists in immune cells and highlight the importance of CRAC channels as a key pharmacological target to control mast cell activation.     

Dynamic simulation of the effect of calcium-release activated calcium channel on cytoplasmic Ca2+ oscillation

A mathematical model is proposed to illustrate the activation of STIM1 (stromal interaction molecule 1) protein, the assembly and activation of calcium-release activated calcium (CRAC) channels in T cells. In combination with De Young-Keizer-Li-Rinzel model, we successfully reproduce a sustained Ca(2+) oscillation in cytoplasm. Our results reveal that Ca(2+) oscillation dynamics in cytoplasm can be significantly affected by the way how the Orai1 CRAC channel are assembled and activated. A low sustained Ca(2+) influx is observed through the CRAC channels across the plasma membrane. In particular, our model shows that a tetrameric channel complex can effectively regulate the total quantity of the channels and the ratio of the active channels to the total channels, and a period of Ca(2+) oscillation about 29 s is in agreement with published experimental data. The bifurcation analyses illustrate the different dynamic properties between our mixed Ca(2+) feedback model and the single positive or negative feedback models.     

Role of the inositol 1,4,5-trisphosphate receptor in Ca(2+) feedback inhibition of calcium release-activated calcium current (I(crac)).

We examined the activation and regulation of calcium release-activated calcium current (I(crac)) in RBL-1 cells in response to various Ca(2+) store-depleting agents. With [Ca(2+)](i) strongly buffered to 100 nM, I(crac) was activated by ionomycin, thapsigargin, inositol 1,4,5-trisphosphate (IP(3)), and two metabolically stable IP(3) receptor agonists, adenophostin A and L-alpha-glycerophospho-D-myoinositol-4,5-bisphosphate (GPIP(2)). With minimal [Ca(2+)](i) buffering, with [Ca(2+)](i) free to fluctuate I(crac) was activated by ionomycin, thapsigargin, and by the potent IP(3) receptor agonist, adenophostin A, but not by GPIP(2) or IP(3) itself. Likewise, when [Ca(2+)](i) was strongly buffered to 500 nM, ionomycin, thapsigargin, and adenophostin A did and GPIP(2) and IP(3) did not activate detectable I(crac). However, with minimal [Ca(2+)](i) buffering, or with [Ca(2+)](i) buffered to 500 nM, GPIP(2) was able to fully activate detectable I(crac) if uptake of Ca(2+) intracellular stores was first inhibited. Our findings suggest that when IP(3) activates the IP(3) receptor, the resulting influx of Ca(2+) quickly inactivates the receptor, and Ca(2+) 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 Ca(2+) pools following activation by different IP(3) receptor agonists indicates that the critical regulatory pool of Ca(2+) may be very small in comparison to the total IP(3)-sensitive component of the endoplasmic reticulum. These findings reveal new and important roles for IP(3) receptors located on discrete IP(3)-sensitive Ca(2+) pools in calcium feedback regulation of I(crac) and capacitative calcium entry.