Cell Calcium 42 (2007) 145–156

Signalling to transcription: Store-operated Ca2+ entry and NFAT activation in lymphocytes
Yousang Gwack, Stefan Feske, Sonal Srikanth, Patrick G. Hogan, Anjana Rao ∗
Department of Pathology, Harvard Medical School, The CBR Institute for Biomedical Research, 200 Longwood Avenue, Boston, MA 02115, USA Received 3 March 2007; received in revised form 20 March 2007; accepted 21 March 2007 Available online 18 June 2007

Abstract In cells of the immune system that are stimulated by antigen or antigen–antibody complexes, Ca2+ entry from the extracellular medium is driven by depletion of endoplasmic reticulum Ca2+ stores and occurs through specialized store-operated Ca2+ channels known as Ca2+ release-activated Ca2+ (CRAC) channels. The process of store-operated Ca2+ influx is essential for short-term as well as long-term responses by immune-system cells. Short-term responses include mast cell degranulation and killing of target cells by effector cytolytic T cells, whereas long-term responses typically involve changes in gene transcription and include T and B cell proliferation and differentiation. Transcription downstream of Ca2+ influx is in large part funneled through the transcription factor nuclear factor of activated T cells (NFAT), a heavily phosphorylated protein that is cytoplasmic in resting cells, but that enters the nucleus when dephosphorylated by the calmodulin-dependent serine/threonine phosphatase calcineurin. The importance of the Ca2+ /calcineurin/NFAT signalling pathway for lymphocyte activation is underscored by the finding that the underlying defect in a family with a hereditary severe combined immune deficiency (SCID) syndrome is a defect in CRAC channel function, store-operated Ca2+ entry, NFAT activation and transcription of cytokines, chemokines and many other NFAT target genes whose transcription is essential for productive immune defence. We recently used a two-pronged genetic approach to identify Orai1 as the pore subunit of the CRAC channel. On the one hand, we initiated a positional cloning approach in which we utilised genome-wide single nucleotide polymorphism (SNP) mapping to identify the genomic region linked to the mutant gene in the SCID family described above. In parallel, we used a genome-wide RNAi screen in Drosophila to identify critical regulators of NFAT nuclear translocation and store-operated Ca2+ entry. These approaches, together with subsequent mutational and electrophysiological analyses, converged to identify human Orai1 as a pore subunit of the CRAC channel and as the gene product mutated in the SCID patients. © 2007 Elsevier Ltd. All rights reserved.
Keywords: Nuclear factor of activated T cells; CRAC channels; Calcineurin; T cell activation; Cytokine expression

Ca2+ is a universal regulator of intracellular signalling [1–4]. As described in other reviews in this volume, Ca2+ is utilised as a second messenger by essentially all cells in multicellular organisms, where it regulates diverse aspects of cellular function. Increases in intracellular free Ca2+ ([Ca2+ ]i ) levels modulate many intracellular processes by activating ubiquitous Ca2+ sensors such as calmodulin (CaM); in
∗ Corresponding author at: Department of Pathology, Harvard Medical School, The CBR Institute for Biomedical Research, Rm 152, Warren Alpert Bldg, 200 Longwood Avenue, Boston, MA 02115, USA. Tel.: +1 617 278 3260; fax: +1 617 278 3280. E-mail address: arao@cbr.med.harvard.edu (A. Rao).

turn, calmodulin activates a large number of calmodulindependent proteins – including the kinases CaMKII and CaMKIV and the phosphatase calcineurin – which together shape both the early and late phases of the subsequent cellular response [5]. The importance of Ca2+ as an intracellular second messenger is emphasised by the many different mechanisms, which work together to maintain Ca2+ homeostasis within intracellular compartments (reviewed in [1,2]). The endoplasmic reticulum (ER) is a substantial reservoir of stored Ca2+ , and is the principal Ca2+ store mobilised for signalling. The free Ca2+ concentration in the ER is estimated to be in the 100–700 M range, and about an order of magnitude

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showing a preference for Ca2+ over Na+ in physiological solutions that is estimated at ∼1000:1 [3. macrophages and natural killer cells. InsP3 generation – that lead to store depletion can be bypassed and store depletion can be achieved directly by treating cells with a variety of agents: thapsigargin.9. which are often members of the TRP (transient receptor potential) family. ∼1–2 mM.10].3 channels cause membrane depolarisation.4. Of these.146 Y. The gating mechanism for these channels involves depletion of Ca2+ stores per se. thus reducing the driving force on Ca2+ . or calcium ionophores such as ionomycin. 1 illustrates one of our own experiments. Another hallmark of the CRAC channel is its susceptibilty to potentiation by low concentrations (3–5 M) of 2-aminoethoxydiphenyl borate (2-APB) [4.5-trisphosphate (InsP3 ) and diacylglycerol. etc. 1.5-bisphosphate (PIP2). The ratio of the permeability coefficients for Ca2+ and Na+ has been estimated at ∼1000:1. performed in collaboration with Drs. whereas extracellular Ca2+ levels ([Ca2+ ]o ) are 104 -fold higher.9]. / Cell Calcium 42 (2007) 145–156 more Ca2+ is bound to low-affinity sites in the ER lumen and is therefore available for eventual mobilisation. Ca2+ enters the cytoplasm through channels located in ER and plasma membranes. The basic biophysical and electrophysiological features of CRAC channels have been well established through studies in many laboratories (reviewed in [3. 1). Store-operated Ca2+ channels open upon stimulation of receptors coupled to phospholipase C (reviewed in [1–4]).11]). the volume of the ER is estimated at only ∼1% of cytoplasmic volume in T lymphocytes and ∼3% in the RBL (rat basophilic leukemia) mast cell line (reviewed in [11]). voltage-gated Ca2+ channels. CRAC channels and the regulation of Ca2+ entry in lymphocytes and mast cells In this review. thus leading to a sustained increase in [Ca2+ ]i that lasts until the antigenic stimulus dies away. intracellular application of Ca2+ chelators such as BAPTA.9. Four general classes of Ca2+ entry channels have been described: voltagegated Ca2+ channels (Cav) such as the L-type Ca2+ channel (LTCC).9] (Fig. Fig.4. The receptor-proximal events – PLC activation. which promote passive store depletion by acting as a cytoplasmic Ca2+ sink. and opens store-operated Ca2+ channels through a process whose molecular mechanism is not yet fully understood (see below). at the concentrations commonly used to activate immune cells. and store-operated Ca2+ channels gated by depletion of intracellular Ca2+ stores (reviewed in [1–4. Gwack et al. BCR).9.9].4. The resulting depletion of ER Ca2+ stores promotes a transient elevation of [Ca2+ ]i . a degree of selectivity otherwise documented only in voltage-gated (Cav) Ca2+ channels. in which the functional consequences of Ca2+ signalling have been exhaustively studied [3. Since cytoplasmic Ca2+ is extruded by PMCA pumps.6–9].4. However. and is extruded across the plasma membrane and into ER Ca2+ stores by Ca2+ ATPases localised in the ER and the plasma membrane (SERCA and PMCA pumps.4. rather than a response to Ca2+ released into the cytoplasm or to second messengers such as diacylglycerol (reviewed in [3. Rather. respectively). Kv1. Toxins and compounds that block Kv1. Under normal physiological conditions. InsP3 releases Ca2+ from the ER by binding to InsP3 receptors (InsP3 R).11]). the CRAC channels of mast cells and lymphocytes have been unambiguously established as store-operated Ca2+ channels [3. as mentioned above. results in a transient rise in intracellular free Ca2+ ([Ca2+ ]i ) levels as a result of the release of Ca2+ from ER stores triggered by InsP3 . channels gated by ligand/receptor interaction. operate primarily by providing an additional pathway for Ca2+ efflux from stores (reviewed in [3. Cells have several mechanisms for regulated Ca2+ entry. are markedly Ca2+ -selective. mechanical forces. T cells also express voltage-gated and Ca2+ -activated + channels in a pattern that depends on the activaK tion/differentiation status of the cells [13].4. release of Ca2+ from ER stores cannot by itself support a sustained elevation of [Ca2+ ]i . which blocks the SERCA pump responsible for maintaining the stores.3 channels maintain membrane potential and therefore the driving force on Ca2+ : in turn Ca2+ entry and the resulting increased [Ca2+ ]i levels are thought to activate the opening of Ca2+ -activated IKCa1 channels.4. channels gated by physical parameters (temperature. we focus on cells of the immune system. Unlike TRP channels [7]. CRAC channels remain open as long as antigen is present and stores remain depleted. Na+ /Ca2+ exchangers in the plasma membrane (NCX) contribute to maintaining the low resting levels of [Ca2+ ]i . and binding of antigen–antibody complexes to Fc receptors on mast cells. the predominant mechanism utilised depends on the cell type and manner of stimulation involved [1–4].11]). Activated PLC hydrolyses phosphatidylinositol-4. atten- . Murali Prakriya and Richard Lewis at Stanford [12]. thereby generating the second messengers inositol-1. Binding of antigen to T and B cell antigen receptors (TCR. Cytoplasmic Ca2+ ([Ca2+ ]i ) levels are typically 70–100 nM. while certain G protein-coupled receptors (GPCR) activate PLC . These K+ channels modulate the rate of Ca2+ entry through CRAC channels by altering membrane potential: hyperpolarisation increases the driving force for Ca2+ influx while depolarisation decreases it. Receptor tyrosine kinases (RTKs) and immunoreceptors (antigen and Fc receptors on immune cells) activate phospholipase C gamma (PLC ). CRAC channels show a characteristic I–V relationship with pronounced inward rectification and a very high selectivity for Ca2+ over monovalent cations. store depletion triggers the opening of a specific class of store-operated Ca2+ channels known as CRAC channels [3.9].). some of which are also TRP family members. which. and the subtype of store-operated Ca2+ channels found in immune cells and termed Ca2+ -release-activated Ca2+ (CRAC) channels. monocytes. In some cells.

osteoporosis. respectively. among others. while the two conserved motifs flanking the NLS (SP-2 and SP-3) control DNA-binding affinity. 1. The cell was pretreated with thapsigargin to activate CRAC channels.14–17]. dephosphorylation results in a conformational change that masks the NES and exposes a nuclear localization sequence (NLS) which binds importins [18]. (A) Ca2+ and Na+ currents through CRAC channels in a control T cell (wholecell configuration). 2). when cells are stimulated. Gwack et al. controls NLS exposure and accessibility of the remaining phosphorylated residues to calcineurin [18]. / Cell Calcium 42 (2007) 145–156 147 Fig. CK1 and Fig. The phosphorylated serine residues of NFAT are located primarily within four conserved sequence motifs in a conserved regulatory domain [10. they are dephosphorylated by calcineurin. a calmodulin-dependent serine/threonine phosphatase. NFAT regulates gene transcription during T cell activation and differentiation. Electrophysiological characteristics of CRAC channels in human T cells. which binds the exportin Crm1. Reproduced with permission from ref. the reader is referred to the web version of this article).14–17]) (Fig. [12] (For interpretation of the references to colour in this figure legend. NFAT proteins are heavily phosphorylated and reside in the cytoplasm. 2. and translocate to the nucleus [10. uating Ca2+ influx and [Ca2+ ]i increase.Y. a family of four transcription factors (NFAT14.14–17]. 2. it is also implicated in many pathological processes. The most N-terminal phosphorylated motif. also known as NFATc1-c4) (reviewed in [10. Schematic view of the NFAT activation cycle. Phosphorylated NFAT exposes a nuclear export sequence (NES). The kinases can be classified as “maintenance” kinases which act in the cytoplasm of resting cells to keep NFAT in its phosphorylated state. NES exposure and nuclear export (reviewed in [10]). myocardial hypertrophy. and inhibiting T cell activation [13]. (C) 2-APB (5 M) strongly potentiates ICRAC . Peak currents during steps to −100 mV are shown. SRR1.18]. Note the pronounced inward rectification characteristic of ICRAC . Three families of constitutively-active kinases – DYRK. osteoclast differentiation. cardiac valve development and differentiation of slow-twitch skeletal muscle fibers. and export” kinases which re-phosphorylate NFAT in the nucleus. . then exposed sequentially to 20 mM Ca2+ or to a divalent-free solution (DVF) extracellularly to record Ca2+ and Na+ currents. The different serine-rich motifs of NFAT proteins are targeted by different kinases. (B) Current–voltage relationship of the Ca2+ current recorded in A (average of 10 traces recorded at the time points indicated by the blue bar). NFAT is a major target of Ca2+ signalling in many cell types Ca2+ signalling activates nuclear factor of activated T cells (NFAT). allergy and autoimmune disease [10. allowing for fine-tuned regulation of NFAT activation [18–20]. among them transplant rejection.

Gwack et al. as well as heterodimers of Fos and Jun [25. leukotrienes.5-phosphate (PIP3 ) in individual T cells for many hours [32]. the activity of GSK3 is suppressed by Akt. thereby priming for GSK3-mediated phosphorylation at the SP-2 motif. Activation of NFAT and NF B was also sensitive to the frequency of [Ca2+ ]i oscillations: low frequency oscillations activated NF B. which resides in the nucleus. which nevertheless causes low-level activation of multiple signalling pathways. which were insufficient to activate JNK or NF B. phosphatidylinositol (PI) 3-kinase. and natural killer (NK) cells which bind antigen-IgG complexes through Fc receptors – activate similar downstream signalling pathways and transcription factors (reviewed in [21–24]). In addition.and heterodimers of Jun family proteins. and consequent generation of the second messengers InsP3 and diacylglycerol. serves as the maintenance kinase while DYRK1A.15. (iii) A third and very interesting Ca2+ -dependent programme is activated in B cells exposed to self-antigens while circulating in a healthy host: these cells show only a modest basal elevation of [Ca2+ ]i (∼150–200 nM).30]. NFAT. NFAT. InsP3 -mediated depletion of ER Ca2+ stores results in CRAC channel opening and Ca2+ influx across the plasma membrane. as shown by adding inhibitors (e. Transient high Ca2+ spikes evoked sustained activation of JNK and NF B. cytolytic T cells attack and lyse infected. as shown by monitoring [Ca2+ ]i elevation and production of PI-3. which consists of homo. DYRK-family kinases phosphorylate the SP-3 motif of NFAT1. oscillations enhanced signalling efficiency specifically at low levels of stimulation [29. sustained activation is required to maintain ongoing transcriptional responses. (i) The rapid responses are independent of new gene transcription: they include degranulation of allergen-exposed mast cells. Diacylglycerol binds to two distinct classes of signalling enzymes. Biological consequences of Ca2+ entry in immune-system cells [Ca2+ ]i increases in lymphocytes and mast cells are coupled to a variety of antigen-dependent responses. thus activating MAP kinase and IKK (I B kinase) pathways. whereas prolonged low increases in [Ca2+ ]i . NFAT and other signalling pathways. DYRK2. whose activation results in tyrosine phosphorylation and activation of PLC . AP-1 and NF B were shown to be optimally activated in response to different patterns of Ca2+ signalling in Jurkat T cells [28–30]. the PI 3-kinase inhibitor wortmannin or the calcineurin inhibitors cyclosporin A (CsA) or FK506) to T cells after initiation of T cell activation [32–34]. 3. CK1family kinases are both maintenance and export kinases. is the export kinase [20].25–27]). similarly. the long-term responses involve transcriptional programmes initiated by sustained Ca2+ signalling (reviewed in [10. 4.148 Y. which lead to activation of the AP-1 (Fos-Jun) and NF B transcription factors respectively [25–27]. Continuous occupancy of the BCR activates potent negative feedback mechanisms that cause the B cells to enter an “anergic” or unresponsive state.26].g. conversely. cells of the immune system – B cells which bind antigen through the BCR. anergic B cells are very short-lived . and sends a substantial fraction of the total NFAT in the cell (∼30%) to the nucleus [35–37]. and target cell killing by cytolytic T cells. MAP kinases facilitate the nuclear export of NFAT proteins by potentiating the ability of CK1 to phosphorylate the SRR1 motif. AP-1 and NF B act in concert with secondary transcription factors to drive the transcription of a large number of genes that regulate lymphocyte proliferation and differentiation (reviewed in [10. sufficed to activate NFAT [28]. T cells which bind MHC/peptide complexes through the TCR. Thus the level of active nuclear NFAT depends both on the parameters of Ca2+ influx as described below. Mast cells that are coated with immunoglobulin E (IgE) degranulate and release proteases. whereas high frequencies activated both NFAT and NF B [29. Moreover. For instance. Each of these immunoreceptors is coupled to tyrosine kinases of the Src and ZAP70/Syk families. such that they become unable to respond to strong antigen stimulation with an elevation of [Ca2+ ]i . which occurs in minutes. both rapid and long-term.30]. In response to stimulation through immunoreceptors. for instance. histamine and many other mediators when exposed to appropriate allergens [23].25]): they include proliferation. Together. Moreover. differentiation and acquisition of effector function by “na¨ve” T and B lymphoı cytes following their first encounter with antigen. (ii) In contrast. which is complete within a few hours. as well as transcription of cytokine. and on which inducible kinases are active under the particular stimulation conditions imposed. prostaglandins. thus driving activation of the transcription factor NFAT. Jun is modified by members of the family of Jun N-terminal kinases (JNK) [26]. In vivo. Fos and Jun proteins are synthesised and also activated posttranslationally by site-specific phosphorylation. malignant or transplanted cells by secreting the pore-forming protein perforin and specialised proteases known as granzymes [31]. which phosphorylate the SRR-1 region of NFAT1 and other NFAT proteins [19]. RasGRP and protein kinase C. Ca2+ signalling and transcriptional activation in cells of the immune system Upon stimulation through “immunoreceptors”.4. chemokine and other activationassociated genes by differentiated “effector” T cells upon secondary exposure to antigen. other intracellular signalling pathways influence the phosphorylation state of NFAT. MAP kinases are responsible for activation of the AP-1 transcription factor. / Cell Calcium 42 (2007) 145–156 GSK3 – act concertedly to phosphorylate NFAT [20]. The productive interaction of T cells with antigen-presenting cells is accompanied by sustained activation of Ca2+ . but not NFAT. a kinase activated in response to diverse signalling pathways in different cell types (reviewed in [10]). which is cytoplasmic. mast cells which bind antigen-IgE complexes through the Fc receptor.

Surprisingly. The first was a positional cloning approach aimed at identifying the gene defective in the SCID patients mentioned above (Fig.42]. Orai1 ([54. 4). was rescued by administration of recombinant IL-2 and bone marrow transplantation. this transient activation permits almost normal induction of a small number of NFAT-dependent genes [52]. Cbl-b and Grail [46. The second was a genome-wide Drosophila RNAi screen (Fig. and leads to transient NFAT dephosphorylation and nuclear import [12. Anergy is a transcriptional state that is strongly dependent on NFAT. Even brief nuclear entry of NFAT can have significant biological consequences.. as shown by analysis of B cell anergy in mice lacking NFAT1 [39]. The anergic state decays rapidly upon dissociation of bound antigen from the B cell surface [36]. (ii) the transcriptional regulators Egr2 and Ikaros [44. One patient. and many of the relevant target genes (“anergy-associated genes”) have been identified [41. The existence of a similar negative regulatory programme is well established in T cells [40]. unpublished). In addition to identifying CRAC channel components and regulators. and later shown to be completely deficient in store-operated Ca2+ entry as measured by Ca2+ imaging in single cells. there is clearly some lymphocyte selectivity that could be exploited therapeutically.47]. it . Use of the calcineurin inhibitor CsA showed that a majority of Ca2+ -dependent gene expression in T cells is funneled through the calcineurin (and presumably the NFAT) signalling pathway [53]. Also. The children with SCID were originally identified by their susceptibility to recurrent infections. as shown by using T cells from the SCID patients mentioned above. calcineurin and NFAT include: (i) the diacylglycerol kinases DGK and DGK [43]. implying that calcium signalling activates a complex transcriptional programme in which nearly as many genes are repressed (∼40%) as activated (∼60%).7]). At the time that we initiated our efforts. 6.19].54]. Despite the fact that they have almost imperceptible store-operated Ca2+ influx and CRAC channel activation. Severe combined immunodeficiencies resulting from defects in CRAC channel function The importance of Ca2+ influx through CRAC channels for normal immune defence against pathogens is highlighted by the existence of at least three families of patients with severe combined immunodeficiency (SCID) secondary to a lack of Ca2+ influx and ICRAC [49–51]. the molecular identity of the CRAC channel was not known. The cells are completely deficient in CRAC channel function as judged by electrophysiological measurements [12]. since it had eluded biochemical identification for many years [18.52. 3). However. As a result.F. nor did we find any mutations in coding and junctional sequences of the TRP genes that we analysed (S.. made possible (i) by the fact that Drosophila cells have a store-operated Ca2+ channel with electrophysiological characteristics very reminiscent of CRAC channels [56]. we did not observe altered expression of TRP mRNA and/or protein. cDNA microarrays were used to compare the transcriptional profiles of normal T cells and SCID T cells under both resting and activated conditions [53]. release of Ca2+ from ER stores is normal in these cells.45]. see below).53]. thereby facilitating T cell signalling [48]. whose older sibling succumbed to a severe infection.55]. Gwack et al. This finding emphasises that although CRAC currents are broadly apparent in non-excitable cells (including Drosophila S2 cells. with no obvious defect in activation of the transcription factors AP-1 and NF B [49. Surprisingly. the T cells are defective in transcription of multiple cytokine and chemokine genes. We have shown that T cells from the two affected patients in one of these families show marked attenuation of NFAT dephosphorylation and nuclear translocation in response to TCR stimulation or treatment with pharmacological agents such as ionomycin or thapsigargin. and he is now essentially normal except for a slight degree of non-progressive muscle hypotonia and a mild case of a syndrome termed ectodermal dysplasia with anhydrosis [52]. fully explaining the patients’ severe immune deficiency [52. almost half of the genes whose expression was altered upon activation showed increased expression in the SCID patients’ cells. we were interested in identifying the kinase that phosphorylated the SP3 motif of NFAT1.Y. and (iv) an unidentified palmitoyltransferase that anchors the transmembrane adaptor LAT in cholesterol-rich lipid microdomains in the plasma membrane.53]. Identification of CRAC channel components and regulators: a two-pronged genetic approach We used two different but complementary genetic screens to identify upstream regulators of NFAT [20.53].R. A. [56]). and sequenced the relevant TRP genes. 5. We therefore examined the SCID patients’ T cells and fibroblasts for TRP family members known to be expressed in these cell types. loss of CRAC channel function was linked to pronounced changes in the expression of activation-associated genes.52. The molecular defect in these patients has been identified as a point mutation in the CRAC channel pore subunit. Established negative regulators that are transcriptional targets of the anergy programme mediated by Ca2+ . A substantial number of these are known signalling proteins or transcription factors that function in negative feedback loops to attenuate T cell responses. and (ii) by the establishment of the Drosophila RNAi Screening Centre by Norbert Perrimon and Bernard Mathey-Prevot at Harvard Medical School [57–60]. but there was much discussion of the possibility that the CRAC channel was formed by one or more TRP family members (reviewed in [4. To make a more quantitative assessment of the number of genes controlled by Ca2+ signalling. / Cell Calcium 42 (2007) 145–156 149 and thus are unable to provoke autoimmune destruction by producing antibodies that react to self [36–38]. As expected. (iii) the E3 ligases Itch.

two fluorescence-based screens for candidates whose depletion abolished store-operated Ca2+ influx. bringing the total number of family members analysed to 23. Because our objective was to find all potential regulators of NFAT. Identification by genome-wide SNP analysis of the region linked to the mutation in the SCID patients’ cells. the sec- . We had access to T cells and DNA from two parents who were first cousins and so were presumed to be heterozygous for the mutant gene (assuming an autosomal recessive mode of inheritance) and their two affected children. SNP data were evaluated using two independent linkage analyses. and evaluated SNP data just from the two patients. their parents. Adapted with permission from ref. respectively [61. [54] (For interpretation of the references to colour in this figure legend. This choice turned out to be ideal – in our screen of 21. 3. Gwack et al. (b) Map of the genomic region linked to the SCID mutation. we obtained only 16 robust candidates whose depletion prevented NFAT nuclear translocation.63]. when Ca2+ influx was monitored at lower concentrations of [Ca2+ ]o (0. of whom 13 appeared to be heterozygous carriers of the mutant gene (Fig.800 gene products. yielded ∼1500 candidates and 75 “filtered hits”. The first (standard homozygosity mapping) assumed an autosomal recessive mode of inheritance based on the clinical phenotype. This modification of the assay allowed us to test a panel of additional family members. monitored by use of a Ca2+ indicator dye. but identifying other components obviously called for a more global approach. and initial testing of their T cells showed no obvious defect in store-operated Ca2+ influx when 2 mM extracellular Ca2+ was used.5 mM). 4).62]. was clear that the positional cloning approach would yield one component of the pathway leading from store depletion to CRAC channel opening. and – as expected – the candidates included nuclear transport proteins and calcineurin subunits [20. unaffected and presumed heterozygous carriers of the disease gene based on experimentally measured Ca2+ influx. their unaffected brother and their grandparents (grey shaded area in Fig. (a) Pedigree of the SCID family. including the grandparents and an unaffected sibling. with lower peak [Ca2+ ]i as well as a lower apparent rate of [Ca2+ ]i increase observed in the parents’ cells [54].62].150 Y. The ORAI1 gene is indicated. Positional cloning of the mutation in the SCID patients’ cells The SCID patient family that we were investigating was initially very small. rather than thapsigarginevoked changes in [Ca2+ ]i as preferred by other groups that performed similar screens [61. we decided to use NFAT nuclear translocation as the readout of the global RNAi screen (Fig. Black squares. a significant deficit became apparent.000 SNPs in each individual’s genome [54]. affected patients. 3a). However. The parents were clinically normal. 3a). / Cell Calcium 42 (2007) 145–156 Fig.2 to 0. 7. Yellow and double-coloured symbols. DNA samples from all family members were then used for genome-wide SNP (single nucleotide polymorphism) mapping using microarrays. the reader is referred to the web version of this article). In contrast. and not amenable to standard positional cloning approaches. permitting simultaneous genotyping of more than 10.

this protein is more closely related to mammalian NFAT5 (also termed TonEBP or OREBP) than to the calcium-regulated vertebrate NFATs [65–67].8 Mb candidate region that was overwhelmingly likely to contain the true gene (Fig. Genomic sequencing of six known genes in this region with a potential role in Ca2+ signalling or Ca2+ binding (shown in blue in Fig. for instance. Drosophila does possess one protein termed NFAT (Drosophila NFAT.8. Our decision to use NFAT in the Drosophila RNAi screen was unusual because the four calcium-regulated NFAT proteins emerged only in vertebrates and are not represented in Drosophila. Long double-stranded nucleic acids cannot be used in mammalian cells since they elicit an interferon response. / Cell Calcium 42 (2007) 145–156 151 Fig. 8. We chose to perform the RNAi screen in Drosophila rather than in mammalian cells. 3b) did not reveal any mutations in exons or immediately adjacent genomic regions. LOD scores 1. partly because the mammalian RNAi screens that were being set up at the same time at Harvard Medical School did not cover the entire human genome. (Left) Candidates whose RNAi-mediated depletion ( ) would be expected to prevent nuclear translocation of a GFP-NFAT fusion protein in thapsigargin-treated cells. The dominant analysis identified a unique region on chromosome 12q24 with a LOD score of ∼3. but did allow us to narrow down the candidate homozygous region from ∼9.63]. (Right) Candidates whose RNAi-mediated depletion ( ) would be expected to cause aberrant nuclear localisation of NFAT in unstimulated cells. This is primarily because long double-stranded RNAs (300–700 basepairs) are used. Gwack et al. Only 2 of the 16 candidates. it was possible to add the parametric LOD scores to obtain a statistically robust combined LOD score of 5. were unambiguously identified as regulators of storeoperated Ca2+ influx in a secondary flow cytometry-based screen [54. Stromal interaction molecule (STIM) had been previously identified as an essential regulator of . thereby reducing redundancy. clearly overlapping with one of six regions (on six different chromosomes. 3b).000: one in favour of linkage).5–1. b). Drosophila has one member each of the Stim and Orai protein families. Genome-wide Drosophila RNAi screen to identify upstream regulators of NFAT. because RNAi in Drosophila cell cultures is much more efficient than in mammalian cells [64]. Genome-wide Drosophila RNAi screen to identify upstream regulators of NFAT In parallel with genome-wide SNP mapping. defining an ∼9. respectively.7 (odds of ∼500. 4. which yield multiple 21-nt siRNAs after processing by Dicer. 3a). and assumed an autosomal dominant mode of inheritance based on our identification of heterozygous carriers of the mutant gene by phenotypic analysis of store-operated Ca2+ influx.8 to an ∼6.Y.5 Mb interval which contained ∼74 genes [54].9) identified in the homozygosity mapping analysis. 5a. dOrai and dStim (Fig. ond utilized the remainder of the pedigree (green box in Fig. and more importantly. dNFAT). Another advantage of Drosophila RNAi screens is that there are typically fewer members of each protein family in Drosophila than in mammals. however. whereas mammals have two and three members. Because the two analyses were run on different sets of individuals and were fully independent. we conducted a genome-wide RNAi screen for NFAT regulators in Drosophila.

The approximate positions of the N-linked carbohydrate moiety and the HA epitope tag inserted into the TM3-TM4 loop are shown.69]. that ER STIM1 is sufficient for activation of store-operated Ca2+ influx. TMEM142B and TMEM142C (HUGO Gene Nomenclature Committee). 4). and a multiude of other kinases. The cytoplasmic region of STIM1 is long enough to span the distance between the ER and a closely apposed plasma membrane.G. The protein product of the Drosophila gene olf186-F was named Drosophila Orai by one of us (Y. however. The same screen yielded diverse regulators of calcium homeostasis. bound Ca2+ dissociates from the EF-hand (red). The ER Ca2+ sensor STIM1 is distributed throughout the ER in resting cells. At sites of ER-plasma membrane apposition. this topic is also exhaustively reviewed elsewhere in this volume and will not be discussed here. glutamate residues with a key role in ion selectivity. especially those bearing conditional alleles of the Stim1 and Stim2 genes. signals from STIM1 (red arrows) activate Ca2+ influx through the CRAC channel. STIM proteins possess conserved N-terminal Ca2+ -binding EF hands . (b) Human Orai1 is an intrinsic plasma membrane protein with four transmembrane segments. (a) The luminal portion of human STIM1 contains an EF-hand paired with a second vestigial EF-hand sequence that does not bind Ca2+ (not shown). The red rectangle shows the location of the R91W mutation in SCID patients [54]. / Cell Calcium 42 (2007) 145–156 Fig. STIM1 is a single-pass transmembrane protein (Fig. and will not be addressed here. were identified in a screen for proteins that sent NFAT-GFP to the nucleus even under resting conditions—the converse of the screen used to identify Stim and Orai (Fig. There is considerable controversy about the role of plasma membrane STIM1. a subunit of which is Orai 1. 5.70]. followed by a SAM domain. 5a) localized both in the ER and in the plasma membrane. led to a marked decrease in store-operated Ca2+ influx [68. NFAT kinases. or depletion of either of its two human homologues. The genes encoding the human proteins have been assigned the symbols TMEM142A. M2 and M3 [61. 2 and 3 [54] with an alternate proposed nomenclature being CRACM1. store-operated Ca2+ influx in RNAi screens performed independently in Drosophila and in mammalian cells [68. the reader is referred to the web version of this article). in Jurkat cells STIM1 depletion suppressed thapsigargin-mediated Ca2+ influx whereas STIM2 depletion had little or no effect [68]. Adapted with permission from ref. Na+ /Ca2+ exchangers. The question of the relative roles of STIM1 and STIM2 is discussed in many of the other reviews in this volume. In contrast. with its EF-hand occupied by Ca2+ (grey).72]. and various cation/Ca2+ channels [20]. The black rectangles in TM1 and TM3 show the approximate locations of E106 and E190. Orai1. Analysis of the mammalian homologues of several of these kinases showed unambiguously that the SP-3 motif of NFAT1 is a target for phosphorylation by members of the DYRK family of kinases [20]. The cytoplasmic segment contains coiled-coil regions and other regions whose structure is not known (shown as blobs). eliciting a conformational change in STIM1 and causing it to localise into puncta. 9. STIM1 and STIM2.152 Y. [11] (For interpretation of the references to colour in this figure legend. and CRAC channel activation. (c) The current model of STIM1-Orai1 signalling. the corresponding names for the human proteins are Orai1. at least in the initial activation of Ca2+ influx [71. in HeLa cells.69]. It is clear. will likely resolve the controversies. Analysis of gene-disrupted mice. A successful byproduct of the RNAi screen was the identification of the elusive SP-3 kinase for NFAT1 [20]. STIM1. and that there is no absolute requirement for STIM1 in the plasma membrane. The ER Ca2+ sensor STIM RNAi-mediated depletion of Drosophila Stim in S2R cells. On depletion of luminal Ca2+ . including SERCA pumps. Gwack et al.). K+ channels.

In Jurkat T cells treated with cytochalasin D. large STIM1 aggregates were shown to correspond to sites of localised Ca2+ influx. close enough for a potential direct interaction between STIM1 in the ER membrane and Orai1 in the plasma membrane [71]. treatment of cells with a cell-permeant crosslinking agent showed that the protein existed as dimers and possibly as higher-order multimers (tetramers?) in cells. or Orai1 and STIM1 in HEK293 cells or RBL cells. Further experiments are needed to confirm these latter associations and determine whether mixed dimers or multimers of Orai proteins exist in cells. and there is evidence that overexpressed Orai1 coaggregates with overexpressed STIM1 after store depletion [76. The minimum mean distance between the ER and the plasma membrane at sites of punctum formation has been estimated by electron microscopy as 17 ± 10 nm. Orai1 could co-immunoprecipitate with Orai2 and Orai3 under the same conditions [63]. The EF hand of STIM1 has a Kd for Ca2+ binding that is estimated at 200–600 M [73].70]. but mutation of this residue does not impair either surface localisation or function [63] (Fig. As an ER Ca2+ sensor. Although the splicing factor Noi emerged as a potential candidate in two of three screens (our own screen and one of the Ca2+ based screens [61]). proteins that recruit STIM1 to sites of ERplasma membrane apposition.Y. Orai2 and Orai3 All three Orai proteins can localise to the plasma membrane. Biochemical properties of Orai1. a SNARE protein involved in vesicle fusion. was detected by surface staining of unpermeabilised cells [54. suggesting that these two proteins are the only limiting components of the pathway. also showed that Orai1 molecules bearing different epitope tags could associate with one another in detergent solution [63.77]. Stim and Orai are clearly key components of the pathway leading from from ER store depletion to CRAC channel opening. 11. and roughly. it is likely that STIM1 has roles in a variety of pathways unrelated to CRAC.61–63]. Moreover.79]. unpublished).75]. a technique that provides no information about stoichiometry. 5c). Orai1 is a stable dimer in non-ionic detergent solutions. The mechanism by which STIM1 couples ER store depletion to CRAC channel opening is not clear at present. Indeed there have been several reports that STIM1 couples to TRP channels and is involved in their gating. which are visible in the light microscope as small clusters or aggregates that have been termed “puncta” (Fig. Co-immunoprecipitation. or within the ER lumen. Syntaxin 5. to relocalise into regions of ER-plasma membrane apposition. closely matched to the Ca2+ concentration in the ER lumen (100–700 M) [74]. in CRAC currents [62.72. 10. indeed Stim served as a positive control based on previous reports [68. which are essential for Ca2+ permeation through all Ca2+ channels that have been analysed so far [6]. to sites of Orai1 aggregation as well [76]. Stim and Orai were the only ones found in common in the three Drosophila genome-wide RNAi screens performed by three independent groups [54. in which the HA epitope tag was inserted into the predicted TM3-TM4 loop. These presumed additional players may be abundant and stable proteins that are difficult to deplete by RNAi. and proteins involved in organising Orai1 within this putative larger complex.63]. and the adjacent SAM domain (sterile. In our own experiments [55]. suggesting a causal role [76]. / Cell Calcium 42 (2007) 145–156 153 localised either extracellularly. EF hand mutants of STIM1 that do not bind Ca2+ effectively are constitutively localised in puncta [69. an adjacent vestigial EF hand (P. was identified in only one of the screens. we first showed – by RNAi-mediated knockdown of endogenous Orai in Drosophila cells and reconstitution with RNAi-resistant mutants in which the conserved glutamates had been substituted with the sterically similar glutamine – that two conserved glutamates in transmembrane segments 1 and 3 were essential for store-operated Ca2+ entry. Other components may yet be discovered: potential players include proteins involved in Stim trafficking. as shown by glycerol gradient centrifugation [63]. but was suggested to have a role in the pathway based on the fact that its depletion inhibited store-operated Ca2+ influx by 2–3-fold [62]. 5b). which is normally diffusely distributed in the ER.H. both before and after treatment with thapsigargin. We then replaced the corresponding glutamates (E106 and E190) . Orai1 has a consensus N-glycosylation sequence (NVS) and is glycosylated when expressed in HEK293 cells. Elegant experiments from Rich Lewis’ laboratory have shown that STIM1 aggregation precedes the onset of store-operated Ca2+ entry and CRAC channel opening. as unambiguously demonstrated by showing that epitope-tagged Orai. where they would be expected to sense the luminal Ca2+ concentration. where they would be expected to bind Ca2+ constitutively.. a domain that in many cases mediates protein-protein interactions) [73]. results in a striking increase in store-operated Ca2+ entry. Further. Gwack et al. All three groups focused on conserved acidic residues.80]. our secondary screens suggested that it was not a relevant player in the Ca2+ entry pathway [63]. this point is thoroughly covered elsewhere in this volume and we refer the reader to these other reviews. Store depletion causes STIM1. proteins that are part of a larger CRAC channel complex at the plasma membrane.70. and whether they are functional ion channels. and more dramatically. Again.78. The propensity of STIM1 to aggregate in conditions of low Ca2+ concentration is recapitulated by a small fragment containing only the functional EF hand.69].G. Orai1 is a pore subunit of the CRAC channel Mutational analyses by several groups have confirmed that Orai1 is a pore subunit of the CRAC channel [55. Overexpression of dStim and dOrai in Drosophila S2 cells. In contrast Orai2 and Orai3 do not possess a consensus sequence for N-glycosylation and are not glycosylated.motif.

its dual sensitivity to 2-APB. both store-operated Ca2+ entry and NFAT-driven reporter activity were substantially diminished by RNAimediated depletion of WAVE2. suggesting that blocking the Orai1 channel will be valuable therapeutically. In divalent-free solutions. Gwack et al. Vig et al.. With the molecular players in hand. do they interact with other types of proteins (e. Rev. suggesting that these residues were located at the extracellular mouth of the pore [80]. Lipp. Conclusion and perspectives The stage is now set for detailed analysis of the mechanism of store-operated Ca2+ entry through CRAC channels. the surviving SCID patient has only mild extra-immunological phenotypes [52]. TRPs). how their interaction is regulated by store depletion. etc. Nat. measured as an increase in the relative permeability for Na+ and Cs+ over Ca2+ [55]. M. or glutamine (Q). aspartate (D). in which the negatively charged carboxylate (–COO− ) has been replaced with the uncharged but polar amide (–CONH2 ). We used the whole-cell patch clamp technique to analyse SCID T cells reconstituted with E106D and E190Q. using Drosophila Orai.9]. / Cell Calcium 42 (2007) 145–156 in human Orai1 with alanine (A). and does it contain other channel subunits or intracellular components that serve to organise the complex? How do residues in the pore determine the characteristic properties of the CRAC channel: its very low single-channel conductance. (ii) an insertional model in which CRAC channels located in intracellular vesicles traffic to the plasma membrane in response to store depletion (potentially consistent with the identification of Syntaxin 5 as a verified candidate in a Drosophila screen [62]. Mol. its sensitivity to removal of all divalent cations. and (iii) a diffusible messenger model in which CRAC channels are gated by a small. also found that when either of two specific aspartate and asparagine residues in the extracellular loop of Drosophila Orai. the controversies should soon be resolved. Each of these models was supported by some circumstantial evidence.? (ii) A second outstanding question is how CRAC channels are gated. and store-operated Ca2+ influx was analysed. 1 (2000) 11–21. It is now feasible to ask whether WAVE2 depletion interferes with the function of known components in the Orai-Stim pathway. 12. and NIH grant AI066128 and grants from the March of Dimes and Charles H. the ability of Gd3+ to block the CRAC current was decreased.F. overexpressed human Orai1 and STIM1 in HEK293 cells. Nat. Given the existence of Orai2 and Orai3. and show that Orai1 is a pore subunit of the CRAC channel. The mutant proteins were introduced into SCID T cells. P. between transmembrane segments 1 and 2.D. now most likely STIM1) to components of the CRAC channel complex (now. Several questions remain. Both mutants showed a decrease in selectivity for Ca2+ . Yeromin et al. Berridge. and do they regulate functions other than store-operated Ca2+ entry? (iv) Finally. and showed again that E106 and E190 were critical for CRAC channel function [70]. Na+ can permeate through wildtype CRAC channels but influx is half-maximally blocked by 20 M Ca2+ [4. (i) The first is the structure of the CRAC channel itself: is it composed of homomultimers or heteromultimers of the Orai. 4 (2003) 326–332. Rev. however. . Previously proposed models were: (i) direct “conformational coupling” of proteins which sense store depletion in the ER (previously thought to be InsP3 receptors. was replaced with alanine. References [1] M. Bootman. an effector protein which regulates actin polymerisation downstream of Rac1 [82]. two mutants whose function was substantially impaired but which carried sufficient current for reproducible electrophysiological measurements. Orai3 or other unrelated proteins. Biol. diffusible Ca2+ influx factor that is released from depleted stores (possibly as a result of STIM1 aggregation into puncta). Cell. Similar results were obtained by Yeromin et al. but not consistent with the fact that Orai1 is primarily located in the plasma membrane [63]). Biol. requiring ∼200 M Ca2+ to block Na+ influx to the same extent [55]. Orai1. especially if functional channels are formed that contain heteromultimers of Orai1 with Orai2. Finally. Orai2 and Orai3 in different cell types and tissues: are the interactions among these proteins promiscuous or selective. Carafoli. In T cells. its calcium-dependent inactivation. Mol. Hood Foundations to S. Orai1 and associated proteins). The E190Q substitution and all three substitutions at residue 106 decreased store-operated Ca2+ influx considerably. for instance by impairing STIM1 aggregation in response to store depletion. it will be necessary to identify inhibitors selective for channels containing Orai1. STIM2. Acknowledgements This work was supported by NIH grants GM075256 and AI40127 to A. These results are consistent.J. but conflicting opinions and reports abounded (reviewed in [81]). its high selectivity for Ca2+ over Na+ . however the E106D mutant shows an ∼10fold shift in the dose-response curve. (iii) A third major question involves the biological roles of STIM1. in a context in which it was overexpressed together with Drosophila Stim in S2 cells [80].g. The versatility and universality of calcium signalling. They also confirmed that simultaneous mutation of D110 and D112 in the extracellular TM1-TM2 loop to alanine decreased the ability of micromolar Ca2+ concentrations to block Na+ currents in divalent-free medium [70].R. This possibility remains to be tested. and how STIM1 reaches sites of ER-plasma membrane apposition. which decreases the length of the side-chain by a single methylene but preserves the negative charge. [2] E.154 Y. which truncates the sidechain to a methyl group. The calcium-signalling saga: tap water and protein crystalis. the mechanism clearly involves STIM1 but it remains to be determined how STIM1 and Orai1 interact.

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