CRAC Channel Activation Research Articles

Novel mutations in CRAC channel protein Orai1 cause immunodeficiency associated with non-immunological disease (89.23)

Abstract Orai1 has recently been identified as a pore subunit of the Ca2+ release activated Ca2+ (CRAC) channel essential for the function of T lymphocytes, mast cells and other cells types. A point mutation in Orai1 abrogates CRAC channel function and causes Severe Combined Immunodeficiency (SCID) in human infants. We here report that the immunodeficiency is accompanied by hypohidrotic ectodermal dysplasia (HED) and congenital myopathy, characterized by absent sweat production, dysplastic dental enamel, non-progressive muscle weakness and histologically a predominance of type I and atrophic type II mucle fibers. A major defect in the activation of NF-kB, known to cause HED accompanied by immune deficiency in patients with mutations in IkBα or IKKγ, was ruled out, although a partial, Ca2+ dependent impairment of NF-kB activation downstream of TCR crosslinking was observed. An novel nonsense mutation in Orai1 was observed in an unrelated SCID patient causing a premature truncation of Orai1 protein. The resulting lack of Ca2+ influx and CRAC channel activity could be rescued by ectopic expression of Orai1 in the patient’s cells. The immunodeficiency in this patient is accompanied by fatal progressive growth retardation and encephalopathy, but HED and myopathy are absent. These clinical phenotypes indicate that Orai1 may not only be necessary for regulating Ca2+ influx in the immune system but also several other tissues.

Physiological roles of STIM1 and Orai1 homologs and CRAC channels in the genetic model organism Caenorhabditis elegans

The nematode Caenorhabditis elegans provides numerous experimental advantages for developing an integrative molecular understanding of physiological processes and has proven to be a valuable model for characterizing Ca 2+ signaling mechanisms. This review will focus on the role of Ca 2+ release activated Ca 2+ (CRAC) channel activity in function of the worm gonad and intestine. Inositol 1,4,5-trisphosphate (IP 3)-dependent oscillatory Ca 2+ signaling regulates contractile activity of the gonad and rhythmic posterior body wall muscle contraction (pBoc) required for ovulation and defecation, respectively. The C. elegans genome contains a single homolog of both STIM1 and Orai1, proteins required for CRAC channel function in mammalian and Drosophila cells. C. elegans STIM-1 and ORAI-1 are coexpressed in the worm gonad and intestine and give rise to robust CRAC channel activity when coexpressed in HEK293 cells. STIM-1 or ORAI-1 knockdown causes complete sterility demonstrating that the genes are essential components of gonad Ca 2+ signaling. Knockdown of either protein dramatically inhibits intestinal cell CRAC channel activity, but surprisingly has no effect on pBoc, intestinal Ca 2+ oscillations or intestinal ER Ca 2+ store homeostasis. CRAC channels thus do not play obligate roles in all IP 3-dependent signaling processes in C. elegans. Instead, we suggest that CRAC channels carry out highly specialized and cell specific signaling roles and that they may function as a failsafe mechanism to prevent Ca 2+ store depletion under pathophysiological and stress conditions.

CRAC channel activity in C. elegans is mediated by Orai1 and STIM1 homologues and is essential for ovulation and fertility.

The Ca(2+) release-activated Ca(2+) (CRAC) channel is a plasma membrane Ca(2+) entry pathway activated by endoplasmic reticulum (ER) Ca(2+) store depletion. STIM1 proteins function as ER Ca(2+) sensors and regulate CRAC channel activation. Recent studies have demonstrated that CRAC channels are encoded by the human Orai1 gene and a homologous Drosophila gene. C. elegans intestinal cells express a store-operated Ca(2+) channel (SOCC) regulated by STIM-1. We cloned a full-length C. elegans cDNA that encodes a 293 amino acid protein, ORAI-1, homologous to human and Drosophila Orai1 proteins. ORAI-1 GFP reporters are co-expressed with STIM-1 in the gonad and intestine. Inositol 1,4,5-trisphosphate (IP(3))-dependent Ca(2+) signalling regulates C. elegans gonad function, fertility and rhythmic posterior body wall muscle contraction (pBoc) required for defecation. RNA interference (RNAi) silencing of orai-1 expression phenocopies stim-1 knockdown and causes sterility and prevents intestinal cell SOCC activation, but has no effect on pBoc or intestinal Ca(2+) signalling. Orai-1 RNAi suppresses pBoc defects induced by intestinal expression of a STIM-1 Ca(2+)-binding mutant, indicating that the proteins function in a common pathway. Co-expression of stim-1 and orai-1 cDNAs in HEK293 cells induces large inwardly rectifying cation currents activated by ER Ca(2+) depletion. The properties of this current recapitulate those of the native SOCC current. We conclude that C. elegans expresses bona fide CRAC channels that require the function of Orai1- and STIM1-related proteins. CRAC channels thus arose very early in animal evolution. In C. elegans, CRAC channels do not play obligate roles in all IP(3)-dependent signalling processes and ER Ca(2+) homeostasis. Instead, we suggest that CRAC channels carry out highly specialized and cell-specific signalling roles and that they may function as a failsafe mechanism to prevent Ca(2+) store depletion under pathophysiological and stress conditions.

Highlights From The Literature

Highlights From The Literature

ATP from Subplasmalemmal Mitochondria Controls Ca2+-dependent Inactivation of CRAC Channels

A sustained Ca2+ entry is the primary signal for T lymphocyte activation after antigen recognition. This Ca2+ entry mainly occurs through store-operated Ca2+ channels responsible for a highly selective Ca2+ current known as I(CRAC). Ca2+ ions act as negative feedback regulators of I(CRAC), promoting its inactivation. Mitochondria, which act as intracellular Ca2+ buffers, have been proposed to control all stages of CRAC current and, hence, intracellular Ca2+ signaling in several types of non-excitable cells. Using the whole-cell configuration of the patch clamp technique, which allows control of the intracellular environment, we report here that respiring mitochondria located close to CRAC channels can regulate slow Ca2+-dependent inactivation of I(CRAC) by increasing the Ca2+-buffering capacity beneath the plasma membrane, mainly through the release of ATP.

Ca2+ influx through CRAC channels activates cytosolic phospholipase A2, leukotriene C4secretion, and expression of c‐fos through ERK‐dependent and independent pathways in mast cells

Cytosolic phospholipase A2 (cPLA2) is a Ca2+-dependent enzyme that mediates agonist-dependent arachidonic acid release in most cell types. Arachidonic acid can then be metabolized by the 5-lipoxygenase enzyme to generate the proinflammatory signal leukotriene C4 (LTC4). Here we report that Ca2+ entry through store-operated CRAC (Ca2+ release-activated Ca2+) channels activates the extracellular signal-regulated kinases (ERKs), members of the mitogen-activated protein kinase family, within minutes and this is necessary for stimulation of cPLA2. Ca2+ entry activates ERK indirectly, via recruitment of Ca2+-dependent protein kinase C alpha and betaI. Ca2+ influx also promotes translocation of cytosolic 5-lipoxygenase to the nuclear membrane, a key step in the activation of this enzyme. Translocation is dependent on ERK activation. A role for gene activation is shown by the finding that CRAC channel opening results in increased transcription and translation of c-fos. Inhibition of ERK activation failed to prevent c-fos expression. Our results show that CRAC channel activation elicits short-term effects through the co-coordinated regulation of two metabolic pathways (cPLA2 and 5-lipoxygenase), which results in the generation of both intra- and intercellular messengers within minutes, as well as longer term changes involving gene activation. These short-term effects are mediated via ERK, whereas, paradoxically, c-fos expression is not. Ca2+ influx through CRAC channels can therefore activate different signaling pathways at the same time, culminating in a range of temporally diverse responses.

The elementary unit of store-operated Ca2+ entry: local activation of CRAC channels by STIM1 at ER–plasma membrane junctions

The activation of store-operated Ca2+ entry by Ca2+ store depletion has long been hypothesized to occur via local interactions of the endoplasmic reticulum (ER) and plasma membrane, but the structure involved has never been identified. Store depletion causes the ER Ca2+ sensor stromal interacting molecule 1 (STIM1) to form puncta by accumulating in junctional ER located 10–25 nm from the plasma membrane (see Wu et al. on p. 803 of this issue). We have combined total internal reflection fluorescence (TIRF) microscopy and patch-clamp recording to localize STIM1 and sites of Ca2+ influx through open Ca2+ release–activated Ca2+ (CRAC) channels in Jurkat T cells after store depletion. CRAC channels open only in the immediate vicinity of STIM1 puncta, restricting Ca2+ entry to discrete sites comprising a small fraction of the cell surface. Orai1, an essential component of the CRAC channel, colocalizes with STIM1 after store depletion, providing a physical basis for the local activation of Ca2+ influx. These studies reveal for the first time that STIM1 and Orai1 move in a coordinated fashion to form closely apposed clusters in the ER and plasma membranes, thereby creating the elementary unit of store-operated Ca2+ entry.

Regulation of CRAC Channel Activity by Recruitment of Silent Channels to a High Open-probability Gating Mode

CRAC (calcium release-activated Ca2+) channels attain an extremely high selectivity for Ca2+ from the blockade of monovalent cation permeation by Ca2+ within the pore. In this study we have exploited the blockade by Ca2+ to examine the size of the CRAC channel pore, its unitary conductance for monovalent cations, and channel gating properties. The permeation of a series of methylammonium compounds under divalent cation-free conditions indicates a minimum pore diameter of 3.9 Å. Extracellular Ca2+ blocks monovalent flux in a manner consistent with a single intrapore site having an effective Ki of 20 μM at −110 mV. Block increases with hyperpolarization, but declines below −100 mV, most likely due to permeation of Ca2+. Analysis of monovalent current noise induced by increasing levels of block by extracellular Ca2+ indicates an open probability (Po) of ∼0.8. By extrapolating the variance/mean current ratio to the condition of full blockade (Po = 0), we estimate a unitary conductance of ∼0.7 pS for Na+, or three to fourfold higher than previous estimates. Removal of extracellular Ca2+ causes the monovalent current to decline over tens of seconds, a process termed depotentiation. The declining current appears to result from a reduction in the number of active channels without a change in their high open probability. Similarly, low concentrations of 2-APB that enhance ICRAC increase the number of active channels while open probability remains constant. We conclude that the slow regulation of whole-cell CRAC current by store depletion, extracellular Ca2+, and 2-APB involves the stepwise recruitment of silent channels to a high open-probability gating mode.

Molecular identification of the CRAC channel by altered ion selectivity in a mutant of Orai

Recent RNA interference screens have identified several proteins that are essential for store-operated Ca2+ influx and Ca2+ release-activated Ca2+ (CRAC) channel activity in Drosophila and in mammals, including the transmembrane proteins Stim (stromal interaction molecule) and Orai. Stim probably functions as a sensor of luminal Ca2+ content and triggers activation of CRAC channels in the surface membrane after Ca2+ store depletion. Among three human homologues of Orai (also known as olf186-F), ORAI1 on chromosome 12 was found to be mutated in patients with severe combined immunodeficiency disease, and expression of wild-type Orai1 restored Ca2+ influx and CRAC channel activity in patient T cells. The overexpression of Stim and Orai together markedly increases CRAC current. However, it is not yet clear whether Stim or Orai actually forms the CRAC channel, or whether their expression simply limits CRAC channel activity mediated by a different channel-forming subunit. Here we show that interaction between wild-type Stim and Orai, assessed by co-immunoprecipitation, is greatly enhanced after treatment with thapsigargin to induce Ca2+ store depletion. By site-directed mutagenesis, we show that a point mutation from glutamate to aspartate at position 180 in the conserved S1-S2 loop of Orai transforms the ion selectivity properties of CRAC current from being Ca2+-selective with inward rectification to being selective for monovalent cations and outwardly rectifying. A charge-neutralizing mutation at the same position (glutamate to alanine) acts as a dominant-negative non-conducting subunit. Other charge-neutralizing mutants in the same loop express large inwardly rectifying CRAC current, and two of these exhibit reduced sensitivity to the channel blocker Gd3+. These results indicate that Orai itself forms the Ca2+-selectivity filter of the CRAC channel.

Genome-wide RNAi screen of Ca 2+ influx identifies genes that regulate Ca 2+ release-activated Ca 2+ channel activity

Recent studies by our group and others demonstrated a required and conserved role of Stim in store-operated Ca(2+) influx and Ca(2+) release-activated Ca(2+) (CRAC) channel activity. By using an unbiased genome-wide RNA interference screen in Drosophila S2 cells, we now identify 75 hits that strongly inhibited Ca(2+) influx upon store emptying by thapsigargin. Among these hits are 11 predicted transmembrane proteins, including Stim, and one, olf186-F, that upon RNA interference-mediated knockdown exhibited a profound reduction of thapsigargin-evoked Ca(2+) entry and CRAC current, and upon overexpression a 3-fold augmentation of CRAC current. CRAC currents were further increased to 8-fold higher than control and developed more rapidly when olf186-F was cotransfected with Stim. olf186-F is a member of a highly conserved family of four-transmembrane spanning proteins with homologs from Caenorhabditis elegans to human. The endoplasmic reticulum (ER) Ca(2+) pump sarco-/ER calcium ATPase (SERCA) and the single transmembrane-soluble N-ethylmaleimide-sensitive (NSF) attachment receptor (SNARE) protein Syntaxin5 also were required for CRAC channel activity, consistent with a signaling pathway in which Stim senses Ca(2+) depletion within the ER, translocates to the plasma membrane, and interacts with olf186-F to trigger CRAC channel activity.

Golli Protein Negatively Regulates Store Depletion-Induced Calcium Influx in T Cells

Calcium influx is crucial for T cell activation and differentiation. The detailed regulation of this process remains unclear. We report here that golli protein, an alternatively spliced product of the myelin basic protein gene, plays a critical role in regulating calcium influx in T cells. Golli-deficient T cells were hyperproliferative and showed enhanced calcium entry upon T cell receptor stimulation. We further found that golli regulates calcium influx in T cells through the inhibition of the store depletion-induced calcium influx. Mutation of the myristoylation site on golli disrupted its association with the plasma membrane and reversed its inhibitory action on Ca2+ influx, indicating that membrane association of golli was essential for its inhibitory action. These results indicate that golli functions in a unique way to regulate T cell activation through a mechanism involving the modulation of the calcium homeostasis.

A severe defect in CRAC Ca2+ channel activation and altered K+ channel gating in T cells from immunodeficient patients

Engagement of the TCR triggers sustained Ca2+ entry through Ca2+ release-activated Ca2+ (CRAC) channels, which helps drive gene expression underlying the T cell response to pathogens. The identity and activation mechanism of CRAC channels at a molecular level are unknown. We have analyzed ion channel expression and function in T cells from SCID patients which display 1–2% of the normal level of Ca2+ influx and severely impaired T cell activation. The lack of Ca2+ influx is not due to deficient regulation of Ca2+ stores or expression of several genes implicated in controlling Ca2+ entry in lymphocytes (kcna3/Kv1.3, kcnn4/IKCa1, trpc1, trpc3, trpv6, stim1). Instead, electrophysiologic measurements show that the influx defect is due to a nearly complete absence of functional CRAC channels. The lack of CRAC channel activity is correlated with diminished voltage sensitivity and slowed activation kinetics of the voltage-dependent Kv1.3 channel. These results demonstrate that CRAC channels provide the major, if not sole, pathway for Ca2+ entry activated by the TCR in human T cells. They also offer evidence for a functional link between CRAC and Kv1.3 channels, and establish a model system for molecular genetic studies of the CRAC channel.

STIM1, an essential and conserved component of store-operated Ca2+ channel function.

Store-operated Ca2+ (SOC) channels regulate many cellular processes, but the underlying molecular components are not well defined. Using an RNA interference (RNAi)-based screen to identify genes that alter thapsigargin (TG)-dependent Ca2+ entry, we discovered a required and conserved role of Stim in SOC influx. RNAi-mediated knockdown of Stim in Drosophila S2 cells significantly reduced TG-dependent Ca2+ entry. Patch-clamp recording revealed nearly complete suppression of the Drosophila Ca2+ release-activated Ca2+ (CRAC) current that has biophysical characteristics similar to CRAC current in human T cells. Similarly, knockdown of the human homologue STIM1 significantly reduced CRAC channel activity in Jurkat T cells. RNAi-mediated knockdown of STIM1 inhibited TG- or agonist-dependent Ca2+ entry in HEK293 or SH-SY5Y cells. Conversely, overexpression of STIM1 in HEK293 cells modestly enhanced TG-induced Ca2+ entry. We propose that STIM1, a ubiquitously expressed protein that is conserved from Drosophila to mammalian cells, plays an essential role in SOC influx and may be a common component of SOC and CRAC channels.

Ca2+-Calmodulin-dependent Facilitation and Ca2+ Inactivation of Ca2+ Release-activated Ca2+ Channels

In non-excitable cells, one major route for Ca2+ influx is through store-operated Ca2+ channels in the plasma membrane. These channels are activated by the emptying of intracellular Ca2+ stores, and in some cell types store-operated influx occurs through Ca2+ release-activated Ca2+ (CRAC) channels. Here, we report that intracellular Ca2+ modulates CRAC channel activity through both positive and negative feedback steps in RBL-1 cells. Under conditions in which cytoplasmic Ca2+ concentration can fluctuate freely, we find that store-operated Ca2+ entry is impaired either following overexpression of a dominant negative calmodulin mutant or following whole-cell dialysis with a calmodulin inhibitory peptide. The peptide had no inhibitory effect when intracellular Ca2+ was buffered strongly at low levels. Hence, Ca2+-calmodulin is not required for the activation of CRAC channels per se but is an important regulator under physiological conditions. We also find that the plasma membrane Ca2+ATPase is the dominant Ca2+ efflux pathway in these cells. Although the activity of the Ca2+ pump is regulated by calmodulin, the store-operated Ca2+ entry is more sensitive to inhibition by the calmodulin mutant than by Ca2+ extrusion. Hence, these two plasmalemmal Ca2+ transport systems may differ in their sensitivities to endogenous calmodulin. Following the activation of Ca2+ entry, the rise in intracellular Ca2+ subsequently feeds back to further inhibit Ca2+ influx. This slow inactivation can be activated by a relatively brief Ca2+ influx (30-60 s); it reverses slowly and is not altered by overexpression of the calmodulin mutant. Hence, the same messenger, intracellular Ca2+, can both facilitate and inactivate Ca2+ entry through store-operated CRAC channels and through different mechanisms.

Close Functional Coupling between Ca2+ Release-activated Ca2+ Channels, Arachidonic Acid Release, and Leukotriene C4 Secretion

In non-excitable cells, one major route for Ca2+ influx is through store-operated Ca2+ channels in the plasma membrane. These channels are activated by the emptying of intracellular Ca2+ stores, and in some cell types, particularly of hemopoietic origin, store-operated influx occurs through Ca2+ release-activated Ca2+ (CRAC) channels. However, little is known about the downstream consequences of CRAC channel activation. Here, we report that Ca2+ entry through CRAC channels stimulates arachidonic acid production, whereas Ca2+ release from the stores is ineffective even though the latter evokes a robust intracellular Ca2+ signal. We find that arachidonic acid released by Ca2+ entering through CRAC channels is used to synthesize the potent paracrine proinflammatory signal leukotriene C4 (LTC4). Both pharmacological inhibitors of CRAC channels and mitochondrial depolarization, which impairs CRAC channel activity, suppress arachidonic acid release and LTC4 secretion. Thus, arachidonic acid release is preferentially stimulated by elevated subplasmalemmal Ca2+ levels due to open CRAC channels, suggesting that the enzyme is located close to the CRAC channels. Our results also identify a novel role for CRAC channels, namely the activation of a downstream signal transduction pathway resulting in the secretion of LTC4. Finally, mitochondria are key determinants of the generation of both intracellular (arachidonic acid) and paracrine (LTC4) signals through their effects on CRAC channel activity.

Potent Inhibition of Ca2+ Release-activated Ca2+ Channels and T-lymphocyte Activation by the Pyrazole Derivative BTP2

Ca2+ entry through store-operated Ca2+release-activated Ca2+ (CRAC) channels is essential for T-cell activation and proliferation. Recently, it has been shown that 3,5-bistrifluoromethyl pyrazole (BTP) derivatives are specific inhibitors of Ca2+-dependent transcriptional activity in T-cells (Trevillyan, J. M., Chiou, X. G., Chen, Y. W., Ballaron, S. J., Sheets, M. P., Smith, M. L., Wiedeman, P. E., Warrior, U., Wilkins, J., Gubbins, E. J., Gagne, G. D., Fagerland, J., Carter, G. W., Luly, J. R., Mollison, K. W., and Djuric, S. W. (2001) J. Biol. Chem. 276, 48118-48126). Whereas inhibition of Ca2+ signals was reported for BTP2 (Ishikawa, J., Ohga, K., Yoshino, T., Takezawa, R., Ichikawa, A., Kubota, H., and Yamada, T. (2003) J. Immunol. 170, 4441-4449), it was not found for BTP3 (Chen, Y., Smith, M. L., Chiou, G. X., Ballaron, S., Sheets, M. P., Gubbins, E., Warrior, U., Wilkins, J., Surowy, C., Nakane, M., Carter, G. W., Trevillyan, J. M., Mollison, K., and Djuric, S. W. (2002) Cell. Immunol. 220, 134-142). We show that BTP2 specifically inhibits CRAC channels in T-cells with an IC(50) of approximately 10 nm. It does not interfere with other mechanisms important for Ca2+ signals in T-cells, including Ca2+ pumps, mitochondrial Ca2+ signaling, endoplasmic reticulum Ca2+ release, and K+ channels. BTP2 inhibits Ca2+ signals in peripheral blood T-lymphocytes (in particular in CD4+ T-cells) and in human Jurkat T-cells. Inhibition of Ca2+ signals is independent of the stimulation method as Ca2+ entry was blocked following stimulation with anti-CD3, which activates the T-cell receptor, and also following stimulation with thapsigargin or inositol 1,4,5-trisphosphate. BTP2 also inhibited Ca2+-dependent gene expression (interleukins 2 and 5 and interferon gamma) and proliferation of T-lymphocytes with similar IC(50) values. BTP2 is the first potent and specific inhibitor of CRAC channels in primary T-lymphocytes. The inhibition of CRAC channels as well as Ca2+-dependent signal transduction with similar IC(50) values in T-lymphocytes emphasizes the importance of CRAC channel activity during T-cell activation. Furthermore, BTP2 could prove to be a tool to finally unmask the molecular identity of CRAC channels.

Calcium entry and the control of calcium oscillations.

During oscillatory Ca(2+) signals, the agonist-induced enhanced entry of extracellular Ca(2+) plays a critical role in modulating the frequency of the oscillations. Although it was originally assumed that the entry of Ca(2+) under these conditions occurred via the well-known, and apparently ubiquitous, store-operated mechanism, subsequent studies suggested that this was unlikely. It is now known that, in many cell types, a novel non-capacitative Ca(2+)-selective pathway whose activation is dependent on arachidonic acid is responsible, and the channels involved [ARC channels (arachidonate-regulated Ca(2+) channels)] have been characterized. These ARC channels co-exist with the store-operated CRAC channels (Ca(2+)-release-activated Ca(2+) channel) in cells, but each plays a unique and non-overlapping role in Ca(2+) signalling. In particular, it is the ARC channels that are specifically activated at the low agonist concentrations that give rise to oscillatory Ca(2+) signals and provide the predominant mode of Ca(2+) entry under these conditions. The indications are that Ca(2+) entry through the ARC channels increases the likelihood that low concentrations of Ins(1,4,5)P(3) will trigger repetitive Ca(2+) release. At higher agonist concentrations, store-depletion is more complete and sustained resulting in the activation of CRAC channels. At the same time the ARC channels are turned off, resulting in what we have described as a reciprocal regulation of these two distinct Ca(2+) entry pathways.

Store-operated Ca2+ entry: dynamic interplay between endoplasmic reticulum, mitochondria and plasma membrane.

In eukaryotic cells, hormones and neurotransmitters that engage the phosphoinositide pathway evoke a biphasic increase in intracellular free Ca2+ concentration: an initial transient release of Ca2+ from intracellular stores is followed by a sustained phase of Ca2+ influx. This influx is generally store-dependent and is required for controlling a host of Ca2+-dependent processes ranging from exocytosis to cell growth and proliferation. In many cell types, store-operated Ca2+ entry is manifest as a non-voltage-gated Ca2+ current called ICRAC (Ca2+ release-activated Ca2+ current). Just how store emptying activates CRAC channels remains unclear, and some of our recent experiments that address this issue will be described. No less important from a physiological perspective is the weak Ca2+ buffer paradox: whereas macroscopic (whole cell) ICRAC can be measured routinely in the presence of strong intracellular Ca2+ buffer, the current is generally not detectable under physiological conditions of weak buffering following store emptying with the second messenger InsP3. In this review, I describe some of our experiments aimed at understanding just why InsP3 is ineffective under these conditions and which lead us to conclude that respiring mitochondria are essential for the activation of ICRAC in weak intracellular Ca2+ buffer. Mitochondrial Ca2+ uptake also increases the dynamic range over which InsP3 functions as the second messenger that controls Ca2+ influx. Finally, we find that Ca2+-dependent slow inactivation of Ca2+ influx, a widespread but poorly understood phenomenon that helps shape the profile of an intracellular Ca2+ signal, is regulated by mitochondrial Ca2+ buffering. Thus, by enabling macroscopic store-operated Ca2+ current to activate and then by controlling its extent and duration, mitochondria play a crucial role in all stages of store-operated Ca2+ influx. Store-operated Ca2+ entry reflects therefore a dynamic interplay between endoplasmic reticulum, mitochondria and plasma membrane.

Regulation of Ca2+ release-activated Ca2+ channels by INAD and Ca2+ influx factor.

The coupling mechanism between depletion of Ca(2+) stores in the endoplasmic reticulum and plasma membrane store-operated ion channels is fundamental to Ca(2+) signaling in many cell types and has yet to be completely elucidated. Using Ca(2+) release-activated Ca(2+) (CRAC) channels in RBL-2H3 cells as a model system, we have shown that CRAC channels are maintained in the closed state by an inhibitory factor rather than being opened by the inositol 1,4,5-trisphosphate receptor. This inhibitory role can be fulfilled by the Drosophila protein INAD (inactivation-no after potential D). The action of INAD requires Ca(2+) and can be reversed by a diffusible Ca(2+) influx factor. Thus the coupling between the depletion of Ca(2+) stores and the activation of CRAC channels may involve a mammalian homologue of INAD and a low-molecular-weight, diffusible store-depletion signal.

Potentiation and inhibition of Ca(2+) release-activated Ca(2+) channels by 2-aminoethyldiphenyl borate (2-APB) occurs independently of IP(3) receptors.

1. The effects of the IP(3)-receptor antagonist 2-aminoethyldiphenyl borate (2-APB) on the Ca(2+) release-activated Ca(2+) current (I(CRAC)) in Jurkat human T cells, DT40 chicken B cells and rat basophilic leukaemia (RBL) cells were examined. 2. 2-APB elicited both stimulatory and inhibitory effects on Ca(2+) influx through CRAC channels. At concentrations of 1-5 microM, 2-APB enhanced Ca(2+) entry in intact cells and increased I(CRAC) amplitude by up to fivefold. At levels > or = 10 microM, 2-APB caused a transient enhancement of I(CRAC) followed by inhibition. 3. 2-APB altered the kinetics of fast Ca(2+)-dependent inactivation of I(CRAC). At concentrations of 1-5 microM, 2-APB increased the rate of fast inactivation. In contrast, 2-APB at higher concentrations (> or = 10 microM) reduced or completely blocked inactivation. 4. 2-APB inhibited Ca(2+) efflux from mitochondria. 5. 2-APB inhibited I(CRAC) more potently when applied extracellularly than intracellularly. Furthermore, increased protonation of 2-APB at low pH did not affect potentiation or inhibition. Thus, 2-APB may have an extracellular site of action. 6. Neither I(CRAC) activation by passive store depletion nor the effects of 2-APB were altered by intracellular dialysis with 500 microg ml(-1) heparin. 7. I(CRAC) is present in wild-type as well as mutant DT40 B cells lacking all three IP(3) receptor isoforms. 2-APB also potentiates and inhibits I(CRAC) in both cell types, indicating that 2-APB exerts its effects independently of IP(3) receptors. 8. Our results show that CRAC channel activation does not require physical interaction with IP(3) receptors as proposed in the conformational coupling model. Potentiation of I(CRAC) by 2-APB may be a useful diagnostic feature for positive identification of putative CRAC channel genes, and provides a novel tool for exploring the physiological functions of store-operated channels.