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Cell Calcium 42 (2007) 145156

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, Bos ton, 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 antigenantibody complexes, Ca 2+ 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 identifycritical 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

[14]. 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 ofcellular function.Increasesin intracellular free Ca2+ ([Ca2+]i)

levels modulate many intracellular processes by activat-

ing ubiquitous Ca2+ sensors such as calmodulin (CaM); in

Corresponding author at: Department of Pathology, Harvard Medical

School, The CBR Institutefor 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: [email protected] (A. Rao).

turn, calmodulin activates a large number of calmodulin-

dependent proteins including the kinases CaMKII and

CaMKIV and the phosphatase calcineurin which together

shape both the early and late phases of the subsequent cellularresponse [5].

The importance of Ca2+ as an intracellular second mes-

senger is emphasised by the many different mechanisms,

which work together to maintain Ca2+ homeostasis within

intracellular compartments (reviewed in [1,2]). The endo-

plasmic 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 100700M range, and about an order of magnitude

0143-4160/$ see front matter 2007 Elsevier Ltd. All rights reserved.

doi:10.1016/j.ceca.2007.03.007
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146 Y. Gwack et al. / Cell Calcium 42 (2007) 145156

more Ca2+ is bound to low-affinity sites in the ER lumen and

is therefore available for eventual mobilisation. Cytoplasmic

Ca2+ ([Ca2+]i) levels aretypically 70100 nM,whereas extra-

cellular Ca2+ levels ([Ca2+]o)are104-fold higher,12 mM.

Ca2+ entersthe cytoplasm through channels located in ER and

plasma membranes, and is extruded across the plasma mem-

brane and into ER Ca2+

stores by Ca2+

ATPases localisedin the ER and the plasma membrane (SERCA and PMCA

pumps, respectively). In some cells, Na+/Ca2+ exchangers in

the plasma membrane (NCX) contribute to maintaining the

low resting levels of [Ca2+]i.

Cells have several mechanisms for regulated Ca2+ entry;

the predominant mechanism utilised depends on the cell

type and manner of stimulation involved [14]. Four general

classes of Ca2+ entry channels have been described: voltage-

gated Ca2+ channels (Cav) such as the L-type Ca2+ channel

(LTCC); channels gated by physical parameters (tempera-

ture, mechanical forces, etc.), which are often members of

the TRP (transient receptor potential) family; channels gated

by ligand/receptor interaction, some of which are also TRP

family members; and store-operated Ca2+ channels gated by

depletion of intracellular Ca2+ stores (reviewed in [14,69].

Of these, voltage-gated Ca2+ channels, and the subtype of

store-operated Ca2+ channels found in immune cells and

termed Ca2+-release-activated Ca2+ (CRAC) channels, are

markedly Ca2+-selective, showing a preference for Ca2+ over

Na+ in physiological solutions that is estimated at 1000:1

[3,4,9].

Store-operated Ca2+ channels open upon stimulation of

receptors coupled to phospholipase C (reviewed in [14]).

Receptor tyrosine kinases (RTKs) and immunoreceptors

(antigen and Fc receptors on immune cells) activate phos-pholipase C gamma (PLC), while certain G protein-coupled

receptors (GPCR) activate PLC. Activated PLC hydrolyses

phosphatidylinositol-4,5-bisphosphate (PIP2), thereby gen-

erating the second messengers inositol-1,4,5-trisphosphate

(InsP3) and diacylglycerol. InsP3 releases Ca2+ from the ER

by binding to InsP3 receptors (InsP3R). The resulting deple-

tion of ER Ca2+ stores promotes a transient elevation of

[Ca2+]i, and opens store-operated Ca2+ channels through a

process whose molecular mechanism is not yet fully under-

stood (see below).

1. CRAC channels and the regulation of Ca2+ entry

in lymphocytes and mast cells

In this review, we focus on cells of the immune system, in

which the functional consequences of Ca2+ signalling have

been exhaustively studied [3,10]. Binding of antigen to T

and B cell antigen receptors (TCR, BCR), and binding of

antigenantibody complexes to Fc receptors on mast cells,

monocytes, macrophages and natural killer cells, 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. However, 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]). Since cytoplasmic Ca2+ is extruded by

PMCA pumps, release of Ca2+ from ER stores cannot by

itself support a sustained elevation of [Ca2+]i. Rather, as

mentioned above, store depletion triggers the opening of

a specific class of store-operated Ca2+

channels known asCRAC channels [3,4,9]. Under normal physiological con-

ditions, CRAC channels remain open as long as antigen is

present andstores remaindepleted, thus leading to a sustained

increase in [Ca2+]i that lasts until the antigenic stimulus dies

away.

Unlike TRP channels [7], the CRAC channels of mast

cells and lymphocytes have been unambiguously established

as store-operated Ca2+ channels [3,4,9]. The gating mecha-

nism for these channels involves depletion of Ca2+ stores per

se, rather than a response to Ca2+ released into the cytoplasm

or to second messengers such as diacylglycerol (reviewed

in [3,4,9,11]). The receptor-proximal events PLC acti-

vation, InsP3 generation that lead to store depletion canbe bypassed and store depletion can be achieved directly by

treating cells with a variety of agents: thapsigargin, which

blocks the SERCA pump responsible for maintaining the

stores; intracellular application of Ca2+ chelators such as

BAPTA, which promote passive store depletion by acting

as a cytoplasmic Ca2+ sink; or calcium ionophores such as

ionomycin, which, at the concentrations commonly used to

activate immune cells, operate primarily by providing an

additional pathway for Ca2+ efflux from stores (reviewed in

[3,4,9,11]).

The basic biophysical and electrophysiological features of

CRAC channels have beenwell established through studies inmany laboratories (reviewed in [3,4,9,11]). Fig. 1 illustrates

one of our own experiments, performed in collaboration with

Drs. Murali Prakriya and Richard Lewis at Stanford [12].

CRAC channels show a characteristic IV relationship with

pronounced inward rectification and a very high selectivity

for Ca2+ over monovalent cations. The ratio of the perme-

ability coefficients for Ca2+ and Na+ has been estimated at

1000:1, a degree of selectivity otherwise documented only

in voltage-gated (Cav) Ca2+ channels. Another hallmark of

the CRAC channel is its susceptibilty to potentiation by low

concentrations (35M) of 2-aminoethoxydiphenyl borate

(2-APB) [4,9] (Fig. 1).

T cells also express voltage-gated and Ca2+-activated

K+ channels in a pattern that depends on the activa-

tion/differentiationstatus of the cells[13]. 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. Kv1.3 channels maintain membrane potential and there-

fore the driving force on Ca2+: in turn Ca2+ entry and

the resulting increased [Ca2+]i levels are thought to acti-

vate the opening of Ca2+-activated IKCa1 channels. Toxins

and compounds that block Kv1.3 channels cause membrane

depolarisation, thus reducing thedriving force on Ca2+, atten-


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Y. Gwack et al. / Cell Calcium 42 (2007) 145156 147

Fig. 1. Electrophysiological characteristics of CRAC channels in human T cells. (A) Ca2+ and Na+ currents through CRAC channels in a control T cell (whole-

cell configuration). Thecell waspretreated with thapsigarginto activate CRACchannels, then exposedsequentially to 20mM Ca2+ or to a divalent-free solution

(DVF) extracellularly to record Ca2+ and Na+ currents, respectively. Peak currents during steps to 100 mV are shown. (B) Currentvoltage relationship of the

Ca2+ current recorded in A (average of 10 traces recorded at the time points indicated by the blue bar). Note the pronounced inward rectification characteristic

of ICRAC . (C) 2-APB (5M) strongly potentiates ICRAC. Reproduced with permission from ref. [12] (For interpretation of the references to colour in this figure

legend, the reader is referred to the web version of this article).

uating Ca2+ influx and [Ca2+]i increase, and inhibiting T cell

activation [13].

2. NFAT is a major target of Ca2+ signalling in many

cell types

Ca2+ signalling activates nuclear factor of activated T

cells (NFAT), a family of four transcription factors (NFAT1-

4, also known as NFATc1-c4) (reviewed in [10,1417])

(Fig. 2). NFAT regulates gene transcription during T cell

activation and differentiation, osteoclast differentiation, car-

diac valve development and differentiation of slow-twitch

skeletal muscle fibers, among others; it is also implicated

in many pathological processes, among them transplant

rejection, osteoporosis, myocardial hypertrophy, allergy and

autoimmune disease [10,1417]. NFAT proteins are heav-ily phosphorylated and reside in the cytoplasm; when cells

are stimulated, they are dephosphorylated by calcineurin,

a calmodulin-dependent serine/threonine phosphatase, and

translocate to the nucleus [10,1417]. The phosphorylated

serine residues of NFAT are located primarily within four

conserved sequence motifs in a conserved regulatory domain

[10,18]. Phosphorylated NFAT exposes a nuclear exportsequence (NES), which binds the exportin Crm1; dephos-

phorylation results in a conformational change that masks

the NES and exposes a nuclear localization sequence (NLS)

which binds importins [18]. The most N-terminal phosphory-

lated motif, SRR1, controls NLS exposure and accessibility

of the remaining phosphorylated residues to calcineurin [18],

while the two conserved motifs flanking the NLS (SP-2

and SP-3) control DNA-binding affinity, NES exposure and

nuclear export (reviewed in [10]).

The different serine-rich motifs of NFAT proteins are tar-

geted by different kinases, allowing for fine-tuned regulation

of NFAT activation [1820]. The kinases can be classified as

maintenance kinases which act in the cytoplasm of restingcells to keep NFAT in its phosphorylated state, and export

kinases which re-phosphorylate NFAT in the nucleus. Three

families of constitutively-active kinases DYRK, CK1 and

Fig. 2. Schematic view of the NFAT activation cycle.


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GSK3 act concertedly to phosphorylate NFAT [20]. CK1-

family kinases are both maintenance and export kinases,

which phosphorylate the SRR-1 region of NFAT1 and other

NFAT proteins [19]. DYRK-familykinases phosphorylate the

SP-3 motif of NFAT1, thereby priming for GSK3-mediated

phosphorylation at the SP-2 motif; DYRK2, which is cyto-

plasmic, serves as the maintenance kinase while DYRK1A,which resides in the nucleus, is the export kinase [20]. In

addition, other intracellular signalling pathways influence the

phosphorylation state of NFAT. For instance, MAP kinases

facilitate the nuclear export of NFAT proteins by potentiating

the ability of CK1 to phosphorylate the SRR1 motif; con-

versely, the activity of GSK3 is suppressed by Akt, a kinase

activated in response to diverse signalling pathways in dif-

ferent cell types (reviewed in [10]). Thus the level of active

nuclear NFAT depends both on the parameters of Ca2+ influx

as described below, and on which inducible kinases are active

under the particular stimulation conditions imposed.

3. Ca2+ signalling and transcriptional activation in

cells of the immune system

Uponstimulationthrough immunoreceptors, cells of the

immune system B cells which bind antigen through the

BCR, T cells which bind MHC/peptide complexes through

the TCR, mast cells which bind antigen-IgE complexes

through the Fc receptor, and natural killer (NK) cells which

bind antigen-IgG complexes through Fc receptors activate

similar downstream signalling pathways and transcription

factors (reviewed in [2124]). Each of these immunorecep-

tors is coupled to tyrosine kinases of the Src and ZAP70/Sykfamilies, whose activation results in tyrosine phosphoryla-

tion andactivation of PLC, andconsequent generation of the

second messengers InsP3 and diacylglycerol. InsP3-mediated

depletion of ER Ca2+ storesresults in CRAC channel opening

and Ca2+ influx across the plasma membrane, thus driving

activation of the transcription factor NFAT. Diacylglycerol

binds to two distinct classes of signalling enzymes, Ras-

GRP and protein kinase C, thus activating MAP kinase and

IKK (IB kinase) pathways, which lead to activation of the

AP-1 (Fos-Jun) and NFB transcription factors respectively

[2527]. MAP kinases are responsible for activation of the

AP-1 transcription factor, which consists of homo- and het-

erodimers of Jun family proteins, as well as heterodimers

of Fos and Jun [25,26]. In response to stimulation through

immunoreceptors, Fos and Jun proteins are synthesised and

also activated posttranslationally by site-specific phosphory-

lation; for instance, Jun is modified by members of the family

of Jun N-terminal kinases (JNK) [26]. Together, NFAT, AP-1

and NFB act in concert with secondary transcription factors

to drive the transcription of a large number of genes that reg-

ulate lymphocyte proliferation and differentiation (reviewed

in [10,2527]).

NFAT, AP-1 and NFB were shown to be optimally acti-

vated in response to different patterns of Ca2+ signalling in

JurkatT cells [2830]. Transient highCa2+ spikes evokedsus-

tained activation of JNK and NFB, but not NFAT, whereas

prolongedlow increasesin [Ca2+]i, which were insufficientto

activate JNK or NFB, sufficed to activate NFAT [28]. Acti-

vation of NFAT and NFB was also sensitive to the frequency

of [Ca2+]i oscillations: low frequency oscillations activated

NFB, whereas high frequencies activated both NFAT andNFB [29,30]. Moreover, oscillations enhanced signalling

efficiency specifically at low levels of stimulation [29,30].

4. 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, both

rapid and long-term. (i) The rapid responses are indepen-

dent of new gene transcription: they include degranulation

of allergen-exposed mast cells, which occurs in minutes,

and target cell killing by cytolytic T cells, which is com-plete within a few hours. Mast cells that are coated with

immunoglobulin E (IgE) degranulate and release proteases,

prostaglandins,leukotrienes,histamineand manyother medi-

ators when exposed to appropriate allergens [23]; similarly,

cytolytic T cells attack and lyse infected, malignant or trans-

planted cells by secreting the pore-forming protein perforin

and specialised proteases known as granzymes [31]. (ii) In

contrast, the long-term responses involve transcriptional pro-

grammes initiated by sustained Ca2+ signalling (reviewed

in [10,15,25]): they include proliferation, differentiation and

acquisition of effector function by nave T and B lympho-

cytes following their first encounter with antigen, as well astranscription of cytokine, chemokine and other activation-

associated genes by differentiated effector T cells upon

secondary exposure to antigen. The productive interaction of

T cells with antigen-presenting cells is accompanied by sus-

tained activation of Ca2+, phosphatidylinositol (PI) 3-kinase,

NFAT and other signalling pathways, as shown by monitor-

ing [Ca2+]i elevation and production of PI-3,4,5-phosphate

(PIP3) in individual T cells for many hours [32]. Moreover,

sustainedactivationis required to maintain ongoing transcrip-

tional responses, as shown by adding inhibitors (e.g. the PI

3-kinase inhibitor wortmannin or the calcineurin inhibitors

cyclosporin A (CsA) or FK506) to T cells after initiation

of T cell activation [3234]. (iii) A third and very inter-

esting 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(150200 nM), which nevertheless causes low-level activa-

tion of multiple signalling pathways, and sends a substantial

fraction of the total NFAT in the cell (30%) to the nucleus

[3537]. Continuous occupancy of the BCR activates potent

negative feedback mechanisms that cause the B cells to enter

an anergic or unresponsive state, such that they become

unable to respond to strong antigen stimulation with an ele-

vation of [Ca2+]i. In vivo, anergic B cells are very short-lived


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and thus are unable to provoke autoimmune destruction by

producing antibodies that react to self [3638]. The anergic

state decays rapidly upon dissociation of bound antigen from

the B cell surface [36]. Anergy is a transcriptional state that

is strongly dependent on NFAT, as shown by analysis of B

cell anergy in mice lacking NFAT1 [39].

The existence of a similar negative regulatory programmeis well established in T cells [40], and many of the relevant

target genes (anergy-associated genes) have been identified

[41,42]. A substantial number of these are known signalling

proteins or transcription factors thatfunction in negative feed-

back loops to attenuate T cell responses. Established negative

regulators that are transcriptional targets of the anergy pro-

gramme mediated by Ca2+, calcineurin and NFAT include:

(i) the diacylglycerol kinases DGK and DGK [43]; (ii) the

transcriptional regulators Egr2 and Ikaros [44,45]; (iii) the E3

ligases Itch, Cbl-b and Grail [46,47]; and (iv) an unidentified

palmitoyltransferase that anchors the transmembrane adap-

tor LAT in cholesterol-rich lipid microdomains in the plasma

membrane, thereby facilitating T cell signalling [48].

5. Severe combined immunodeficiencies resulting

from defects in CRAC channel function

Theimportance of Ca2+ influxthrough 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 [4951]. The children with SCID

were originally identified by their susceptibility to recur-

rent infections, and later shown to be completely deficientin store-operated Ca2+ entry as measured by Ca2+ imaging

in single cells. We have shown that T cells from the two

affected patients in one of these families show marked atten-

uation of NFAT dephosphorylation and nuclear translocation

in response to TCR stimulation or treatment with pharma-

cological agents such as ionomycin or thapsigargin, with no

obvious defect in activation of the transcription factors AP-1

and NFB [49,52,53]. As a result, the T cells are defective in

transcription of multiple cytokine and chemokine genes, fully

explaining the patients severe immune deficiency [52,53].

The cells are completely deficient in CRAC channel func-

tion as judged by electrophysiological measurements [12].

The molecular defect in these patients has been identified as

a point mutation in the CRAC channel pore subunit, Orai1

([54,55]; see below). One patient, whose older sibling suc-

cumbed to a severe infection, was rescued by administration

of recombinant IL-2 and bone marrow transplantation, and

he is now essentially normal except for a slight degree of

non-progressive muscle hypotonia and a mild case of a syn-

drome termed ectodermal dysplasia with anhydrosis [52].

This finding emphasises that although CRAC currents are

broadly apparent in non-excitable cells(includingDrosophila

S2 cells, [56]), there is clearly some lymphocyte selectivity

that could be exploited therapeutically.

Even brief nuclear entry of NFAT can have significant

biological consequences, as shown by using T cells from the

SCID patients mentioned above. Despite the fact that they

have almost imperceptible store-operated Ca2+ influx and

CRAC channel activation, release of Ca2+ from ER stores

is normal in these cells, and leads to transient NFAT dephos-

phorylation and nuclear import [12,52,53]. Surprisingly, thistransient activation permits almost normal induction of a

small number of NFAT-dependent genes [52]. Tomakeamore

quantitative assessment of the number of genes controlled by

Ca2+ signalling, cDNA microarrays were used to compare the

transcriptional profiles of normal T cells and SCID T cells

underboth resting and activated conditions [53]. As expected,

loss of CRAC channel function was linked to pronounced

changes in the expression of activation-associatedgenes. Sur-

prisingly, almost half of the genes whose expression was

altered upon activation showed increased expression in the

SCID patients cells, implying that calcium signalling acti-

vates a complex transcriptional programme in which nearly

as many genes are repressed (40%) as activated (60%).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) sig-

nalling pathway [53].

6. 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 [20,54]. The

first was a positional cloning approach aimed at identi-fying the gene defective in the SCID patients mentioned

above (Fig. 3). The second was a genome-wide Drosophila

RNAi screen (Fig. 4), made possible (i) by the fact

that Drosophila cells have a store-operated Ca2+ channel

with electrophysiological characteristics very reminiscent

of CRAC channels [56], and (ii) by the establishment of

the Drosophila RNAi Screening Centre by Norbert Perri-

mon and Bernard Mathey-Prevot at Harvard Medical School

[5760].

At the time that we initiated our efforts, the molecular

identity of the CRAC channel was not known, but there was

much discussion of thepossibilitythat theCRAC channel was

formed by one or more TRP family members (reviewed in

[4,7]). We therefore examined the SCID patients T cells and

fibroblasts for TRP family members known to be expressed in

these cell types, and sequencedthe relevant TRP genes. How-

ever, we did not observe altered expression of TRP mRNA

and/or protein, nor did we find any mutations in coding and

junctional sequences of the TRP genes that we analysed (S.F.,

A.R., unpublished).

In addition to identifying CRAC channel components and

regulators, we were interested in identifying the kinase that

phosphorylated the SP3 motif of NFAT1, since it had eluded

biochemical identification for many years [18,19]. Also, it


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Fig. 3. Identification by genome-wide SNP analysis of the region linked to the mutation in the SCID patients cells. (a) Pedigree of the SCID family. Black

squares, affected patients. Yellow and double-coloured symbols, unaffected and presumed heterozygous carriers of the disease gene based on experimentally

measured Ca2+ influx. (b) Map of the genomic region linked to the SCID mutation. The ORAI1 gene is indicated. Adapted with permission from ref. [54] (For

interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).

was clear that the positional cloning approach would yield

one component of the pathway leading from store depletionto CRAC channel opening, but identifying other components

obviously called for a more global approach. Because our

objective was to find all potential regulators of NFAT, we

decided to use NFAT nuclear translocation as the readout of

the global RNAi screen (Fig. 4), rather than thapsigargin-

evoked changes in [Ca2+]i as preferred by other groups that

performed similar screens [61,62]. This choice turned out to

be ideal in our screen of 21,800 gene products, we obtained

only 16 robust candidates whose depletion prevented NFAT

nuclear translocation, and as expected the candidates

included nuclear transport proteins and calcineurin sub-

units [20,63]. In contrast, two fluorescence-based screens

for candidates whose depletion abolished store-operated

Ca2+ influx, monitored by use of a Ca2+ indicator dye,

yielded1500 candidates and 75 filtered hits, respectively

[61,62].

7. Positional cloning of the mutation in the SCID

patients cells

The SCID patient family that we were investigating was

initially very small, and not amenable to standard positional

cloning approaches. We had access to T cells and DNA

from two parents who were first cousins and so were pre-

sumed to be heterozygous for the mutant gene (assumingan autosomal recessive mode of inheritance) and their two

affected children. The parents were clinically normal, and

initial testing of their T cells showed no obvious defect in

store-operated Ca2+ influx when 2 mM extracellular Ca2+

was used. However, when Ca2+ influx was monitored at

lower concentrations of [Ca2+]o (0.2 to 0.5 mM), a signif-

icant deficit became apparent, with lower peak [Ca2+]i as

well as a lower apparent rate of [Ca2+]i increase observed

in the parents cells [54]. This modification of the assay

allowed us to test a panel of additional family members,

including the grandparents and an unaffected sibling, bring-

ing the total number of family members analysed to 23, of

whom 13 appeared to be heterozygous carriers of the mutant

gene (Fig. 3a). DNA samples from all family members were

then used for genome-wide SNP (single nucleotide polymor-

phism) mapping using microarrays, permitting simultaneous

genotyping of more than 10,000 SNPs in each individuals

genome [54].

SNP data were evaluated using two independent link-

age analyses. The first (standard homozygosity mapping)

assumed an autosomal recessive mode of inheritance based

on the clinical phenotype, and evaluated SNP data just from

the two patients, their parents, their unaffected brother and

their grandparents (grey shaded area in Fig. 3a); the sec-


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Fig. 4. Genome-wide Drosophila RNAi screen to identify upstream regulators of NFAT. (Left) Candidates whose RNAi-mediated depletion ( ) would be

expected to prevent nuclear translocation of a GFP-NFAT fusion protein in thapsigargin-treated cells. (Right) Candidates whose RNAi-mediated depletion ( )

would be expected to cause aberrant nuclear localisation of NFAT in unstimulated cells.

ond utilized the remainder of the pedigree (green box in

Fig. 3a), and assumed an autosomal dominantmode of inher-

itance based on our identification of heterozygous carriers

of the mutant gene by phenotypic analysis of store-operated

Ca2+

influx. The dominant analysis identified a unique regionon chromosome 12q24 with a LOD score of3.8, clearly

overlapping with one of six regions (on six different chromo-

somes, LOD scores 1.51.9) identified in the homozygosity

mapping analysis. Because the two analyses were run on

different sets of individuals and were fully independent, it

was possible to add the parametric LOD scores to obtain

a statistically robust combined LOD score of 5.7 (odds of

500,000: one in favour of linkage), defining an 9.8 Mb

candidate region that was overwhelmingly likely to contain

the true gene (Fig. 3b). Genomic sequencing of six known

genes in this region with a potential role in Ca2+ signalling

or Ca2+ binding (shown in blue in Fig. 3b) did not reveal any

mutations in exons or immediately adjacent genomic regions,

but did allow us to narrow down the candidate homozygous

region from 9.8 to an 6.5 Mb interval which contained

74 genes [54].

8. Genome-wide Drosophila RNAi screen to identify

upstream regulators of NFAT

In parallel with genome-wide SNP mapping, we con-

ducted a genome-wide RNAi screen for NFAT regulators

in Drosophila. We chose to perform the RNAi screen in

Drosophila rather than in mammalian cells, 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, and more importantly, because

RNAi in Drosophila cell cultures is much more efficientthan in mammalian cells [64]. This is primarily because

long double-stranded RNAs (300700 basepairs) are used,

which yield multiple 21-nt siRNAs after processing by Dicer.

Long double-stranded nucleic acids cannot be used in mam-

malian cells since they elicit an interferon response. Another

advantage ofDrosophila RNAi screens is that there are typ-

ically fewer members of each protein family in Drosophila

than in mammals, thereby reducing redundancy; for instance,

Drosophila has one member each of the Stim and Orai pro-

tein families,whereas mammals have two and threemembers,

respectively.

Our decision to use NFAT in the Drosophila RNAi screen

was unusual because the four calcium-regulated NFAT pro-

teins emerged only in vertebrates and are not represented

in Drosophila. Drosophila does possess one protein termed

NFAT (Drosophila NFAT, dNFAT); however, this protein

is more closely related to mammalian NFAT5 (also termed

TonEBP or OREBP) than to the calcium-regulated vertebrate

NFATs [6567].

Only 2 of the 16 candidates, dOrai and dStim ( Fig. 5a,

b), were unambiguously identified as regulators of store-

operated Ca2+ influx in a secondary flow cytometry-based

screen [54,63]. Stromal interaction molecule (STIM) had

been previously identified as an essential regulator of


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Fig. 5. STIM1, Orai1, and CRAC channel activation. (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), followed by a SAM domain. The cytoplasmic segment contains coiled-coil regions and other regions whose

structure is not known (shown as blobs). The cytoplasmic region of STIM1 is long enough to span the distance between the ER and a closely apposed plasma

membrane. (b) Human Orai1 is an intrinsic plasma membrane protein with four transmembrane segments. The black rectangles in TM1 and TM3 show the

approximate locations of E106 and E190, glutamate residues with a key role in ion selectivity. The red rectangle shows the location of the R91W mutation in

SCID patients [54]. The approximate positions of the N-linked carbohydrate moiety and the HA epitope tag inserted into the TM3-TM4 loop are shown. (c)

The current model of STIM1-Orai1 signalling. The ER Ca2+ sensor STIM1 is distributed throughout the ER in resting cells, with its EF-hand occupied by Ca2+

(grey). On depletion of luminal Ca2+, bound Ca2+ dissociates from the EF-hand (red), eliciting a conformational change in STIM1 and causing it to localise

into puncta. At sites of ER-plasma membrane apposition, signals from STIM1 (red arrows) activate Ca2+ influx through the CRAC channel, a subunit of which

is Orai 1. Adapted with permission from ref. [11] (For interpretation of the references to colour in this figure legend, the reader is referred to the web version

of this article).

store-operated Ca2+ influx in RNAi screens performed inde-

pendently in Drosophila and in mammalian cells [68,69].

The protein product of the Drosophila gene olf186-F was

named Drosophila Orai by one of us (Y.G.); the cor-

responding names for the human proteins are Orai1, 2

and 3 [54] with an alternate proposed nomenclature being

CRACM1, M2 and M3 [61,70]. The genes encoding the

human proteins have been assigned thesymbols TMEM142A,

TMEM142B and TMEM142C (HUGO Gene Nomenclature

Committee).

A successful byproduct of the RNAi screen was the iden-

tification of the elusive SP-3 kinase for NFAT1 [20]. NFATkinases, and a multiude of other kinases, were identified in

a screen for proteins that sent NFAT-GFP to the nucleus

even under resting conditionsthe converse of the screen

used to identify Stim and Orai (Fig. 4). 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 same screen yielded diverse regulators of

calcium homeostasis, including SERCA pumps, Na+/Ca2+

exchangers, K+ channels, and various cation/Ca2+ channels

[20].

9. The ER Ca2+ sensor STIM

RNAi-mediated depletion of Drosophila Stim in S2R

cells, or depletion of either of its two human homologues,

STIM1 and STIM2, in HeLa cells, led to a markeddecreasein

store-operated Ca2+ influx [68,69]. In contrast, in Jurkat cells

STIM1 depletion suppressed thapsigargin-mediated Ca2+

influx whereas STIM2 depletion had little or no effect [68].

The question of the relative roles of STIM1 and STIM2 is

discussed in many of the other reviews in this volume, and

will not be addressed here. Analysis of gene-disrupted mice,

especially those bearing conditional alleles of the Stim1 andStim2 genes, will likely resolve the controversies.

STIM1 is a single-pass transmembrane protein (Fig. 5a)

localized both in the ER and in the plasma membrane.

There is considerable controversy about the role of plasma

membrane STIM1; this topic is also exhaustively reviewed

elsewhere in this volume and will not be discussed here. It

is clear, however, that ER STIM1 is sufficient for activation

of store-operated Ca2+ influx, and that there is no absolute

requirement for STIM1 in the plasma membrane, at least

in the initial activation of Ca2+ influx [71,72]. STIM pro-

teins possess conserved N-terminal Ca2+-binding EF hands


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localisedeither extracellularly, wherethey would be expected

to bind Ca2+ constitutively, or within the ER lumen, where

they would be expected to sense the luminal Ca2+ concentra-

tion. The EF hand of STIM1 has a Kd for Ca2+ binding that is

estimated at 200600M [73], closely matched to the Ca2+

concentration in the ER lumen (100700M) [74].

The mechanism by which STIM1 couples ER store deple-tion to CRAC channel opening is not clear at present. Store

depletion causes STIM1, which is normally diffusely dis-

tributed in the ER, to relocalise into regions of ER-plasma

membrane apposition, which are visible in the light micro-

scope as small clusters or aggregates that have been termed

puncta (Fig. 5c). EF hand mutants of STIM1 that do not

bind Ca2+ effectively are constitutively localised in puncta

[69,75]. The propensity of STIM1 to aggregate in conditions

of low Ca2+ concentration is recapitulated by a small frag-

ment containing only the functional EF hand, an adjacent

vestigial EF hand (P.G.H., unpublished), and the adjacent

SAM domain (sterile- motif, a domain that in many cases

mediates protein-protein interactions) [73]. The minimummean distance between the ER and the plasma membrane

at sites of punctum formation has been estimated by elec-

tron microscopy as 17 10 nm, close enough for a potential

direct interaction between STIM1 in the ER membrane and

Orai1 in the plasma membrane [71]. Elegant experiments

from Rich Lewis laboratory have shown that STIM1 aggre-

gation precedes the onset of store-operated Ca2+ entry and

CRAC channel opening, suggesting a causal role [76], and

there is evidence that overexpressed Orai1 coaggregates with

overexpressed STIM1 after store depletion [76,77]. In Jurkat

T cells treated with cytochalasin D, large STIM1 aggregates

were shown to correspond to sites of localised Ca2+

influx,and roughly, to sites of Orai1 aggregation as well [76].

Stim and Orai are clearly key components of the path-

way leading from from ER store depletion to CRAC channel

opening. Overexpression of dStim and dOrai in Drosophila

S2 cells, or Orai1 and STIM1 in HEK293 cells or RBL cells,

results in a striking increase in store-operated Ca2+ entry,

and more dramatically, in CRAC currents [62,72,78,79],

suggesting that these two proteins are the only limiting

components of the pathway. Other components may yet be

discovered: potential players include proteins involved in

Stim trafficking, proteins that recruit STIM1 to sites of ER-

plasmamembraneapposition,proteins that arepart of a larger

CRAC channel complex at the plasma membrane, and pro-

teins involved in organising Orai1 within this putative larger

complex. These presumed additional players may be abun-

dant and stable proteins that are difficult to deplete by RNAi.

Stim and Orai were the only ones found in common in the

three Drosophila genome-wide RNAi screens performed by

three independent groups [54,6163]; indeed Stim served as a

positive control based on previous reports [68,69]. 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]), our secondary screens suggested that it

was not a relevant player in the Ca2+ entry pathway [63].

Syntaxin 5, a SNARE protein involved in vesicle fusion, was

identified in only one of the screens, but was suggested to

have a role in the pathway based on the fact that its depletion

inhibited store-operated Ca2+ influx by 23-fold [62].

As an ER Ca2+ sensor, it is likely that STIM1 has roles in

a variety of pathways unrelated to CRAC. Indeed there have

been several reports that STIM1 couples to TRP channelsand is involved in their gating. Again, this point is thoroughly

covered elsewhere in this volume and we refer the reader to

these other reviews.

10. Biochemical properties of Orai1, Orai2 and

Orai3

All three Orai proteins can localise to the plasma mem-

brane, as unambiguously demonstrated by showing that

epitope-tagged Orai, in which theHA epitope tagwas inserted

into the predicted TM3-TM4 loop, was detected by surface

staining of unpermeabilised cells [54,63]. Orai1 has a con-sensus N-glycosylation sequence (NVS) and is glycosylated

when expressed in HEK293 cells, but mutation of this residue

does not impair either surface localisation or function [63]

(Fig.5b). In contrast Orai2 andOrai3do notpossess a consen-

sus sequence for N-glycosylation and are not glycosylated.

Orai1 is a stable dimer in non-ionic detergent solutions,

as shown by glycerol gradient centrifugation [63]. Moreover,

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, both before and

after treatment with thapsigargin. Co-immunoprecipitation,

a technique that provides no information about stoichiom-etry, also showed that Orai1 molecules bearing different

epitope tags could associate with one another in detergent

solution [63,70]. Further, Orai1 could co-immunoprecipitate

with Orai2 and Orai3 under the same conditions [63]. Further

experiments are needed to confirm these latter associations

and determine whether mixed dimers or multimers of Orai

proteins exist in cells, and whether they are functional ion

channels.

11. 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 [55,70,80]. All

three groups focused on conserved acidic residues, which

are essential for Ca2+ permeation through all Ca2+ channels

that have been analysed so far [6]. In our own experiments

[55], 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. We

then replaced the corresponding glutamates (E106 and E190)


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in human Orai1 with alanine (A), which truncates the side-

chain to a methyl group; aspartate (D), which decreases the

length of the side-chain by a single methylene but preserves

the negative charge; or glutamine (Q), in which the nega-

tively charged carboxylate (COO) has been replaced with

the uncharged but polar amide (CONH2). The mutant pro-

teins were introduced into SCID T cells, and store-operatedCa2+ influx was analysed. The E190Q substitution and all

three substitutions at residue 106 decreased store-operated

Ca2+ influxconsiderably. We usedthe whole-cellpatchclamp

technique to analyse SCID T cells reconstituted with E106D

and E190Q, two mutants whose function was substantially

impaired but which carried sufficient current for reproducible

electrophysiological measurements. Both mutants showed a

decrease in selectivity for Ca2+, measured as an increase in

the relative permeability for Na+ and Cs+ over Ca2+ [55].

In divalent-free solutions, Na+ can permeate through wild-

type CRAC channels but influx is half-maximally blocked by

20M Ca2+ [4,9]; howeverthe E106D mutantshows an10-

fold shift in the dose-response curve, requiring 200MCa2+ to block Na+ influx to the same extent [55]. Similar

results were obtained by Yeromin et al. using Drosophila

Orai, in a context in which it was overexpressed together with

Drosophila Stim in S2 cells [80]. Yeromin et al. also found

that when either of two specific aspartate and asparagine

residues in theextracellular loop ofDrosophila Orai,between

transmembrane segments 1 and 2, was replaced with alanine,

the ability of Gd3+ to block the CRAC current was decreased,

suggesting that these residues were located at the extracellu-

lar mouth of the pore [80]. Finally, Vig et al. overexpressed

human Orai1 and STIM1 in HEK293 cells, and showed again

that E106 and E190 were critical for CRAC channel func-tion [70]. They also confirmed that simultaneous mutation of

D110 and D112 in the extracellular TM1-TM2 loop to ala-

nine decreased the ability of micromolar Ca2+ concentrations

to block Na+ currents in divalent-free medium [70]. These

results are consistent, and show that Orai1 is a pore subunit

of the CRAC channel.

12. Conclusion and perspectives

The stage is now set for detailed analysis of the mecha-

nism of store-operated Ca2+ entry through CRAC channels.

Previously proposed models were: (i) direct conformational

coupling of proteins which sense store depletion in the

ER (previously thought to be InsP3 receptors, now most

likely STIM1) to components of the CRAC channel com-

plex (now, Orai1 and associated proteins); (ii) an insertional

model in which CRAC channels located in intracellular vesi-

cles 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], but not consistent with the fact that Orai1 is primarily

located in the plasma membrane [63]); and (iii) a diffusible

messenger model in which CRAC channels are gated by

a small, diffusible Ca2+ influx factor that is released from

depleted stores (possibly as a result of STIM1 aggrega-

tion into puncta). Each of these models was supported by

some circumstantial evidence, but conflicting opinions and

reports abounded (reviewed in [81]). With the molecular

players in hand, however, the controversies should soon be

resolved.Several questions remain. (i) The first is the structure of

the CRAC channel itself: is it composed of homomultimers

or heteromultimers of the Orai, 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, its high selectivity for Ca2+

over Na+, its sensitivity to removal of all divalent cations, its

calcium-dependent inactivation, its dualsensitivity to 2-APB,

etc.? (ii) A second outstanding question is how CRAC chan-

nels are gated; the mechanism clearly involves STIM1 but

it remains to be determined how STIM1 and Orai1 interact,

how their interaction is regulated by store depletion, and how

STIM1 reaches sites of ER-plasma membrane apposition.

In T cells, both store-operated Ca2+ entry and NFAT-driven

reporter activity were substantially diminished by RNAi-

mediated depletion of WAVE2, an effector protein which

regulates actin polymerisation downstream of Rac1 [82]. Itis

now feasible to ask whether WAVE2 depletion interferes with

the function of known components in the Orai-Stim pathway,

for instance by impairing STIM1 aggregation in response to

store depletion. (iii) A third major question involves the bio-

logical roles of STIM1, STIM2, Orai1, Orai2 and Orai3 in

different cell types and tissues: are the interactions among

these proteins promiscuous or selective, do they interact withother types of proteins (e.g. TRPs), and do they regulate func-

tions other than store-operated Ca2+ entry? (iv) Finally, the

surviving SCID patient has only mild extra-immunological

phenotypes [52], suggesting that blocking the Orai1 channel

will be valuable therapeutically. This possibility remains to

be tested. Given the existence of Orai2 and Orai3, it will be

necessary to identify inhibitors selective for channels con-

taining Orai1, especially if functional channels are formed

that contain heteromultimers of Orai1 with Orai2, Orai3 or

other unrelated proteins.

Acknowledgements

This work was supported by NIH grants GM075256 and

AI40127 to A.R., and NIH grant AI066128 and grants from

the March of Dimes and Charles H. Hood Foundations to S.F.

References

[1] M.J. Berridge, P. Lipp, M.D. Bootman, The versatility and universality

of calcium signalling, Nat. Rev. Mol. Biol. 1 (2000) 1121.

[2] E. Carafoli, The calcium-signalling saga: tap water and protein crys-

talis, Nat. Rev. Mol. Cell. Biol. 4 (2003) 326332.


11/12


[3] R.S. Lewis, Calcium signalling mechanisms n T lymphocytes, Annu.

Rev. Immnol. 19 (2001) 497521.

[4] A.B.Parekh,J.W.PutneyJr., Store-operatedcalciumchannels, Physiol.

Rev. 85 (2005) 757810.

[5] P. James, T. Vorherr, E. Carafoli, Calmodulin-binding domains: just

two faced or multi-faceted? Trends Biochem. Sci. 20 (1995) 3842.

[6] W.A. Sather, E.W. McCleskey, Permeation and selectivity in calcium

channels, Annu. Rev. Physiol. 65 (2003) 133159.

[7] D.E. Clapham, TRP channels as cellular sensors, Nature 426 (2003)

517524.

[8] C. Montell, L. Bimbaumer, V. Flockerzi, The TRP channels, a remark-

ably functional family, Cell 108 (2002) 595598.

[9] M. Prakriya, R.S. Lewis, CRAC channels: activation, permeation, and

the search for a molecular identity, Cell Calc. 33 (2003) 311321.

[10] P.G. Hogan, L. Chen, J. Nardone, A. Rao, Transcriptional regulation

by calcium, calcineurin, and NFAT, Genes Dev. 17 (2003) 22052232.

[11] P.G.Hogan,A. Rao,DissectingICRAC, a store-operatedcalcium current,

Trends Biochem. Sci. 32 (2007) 235245.

[12] S. Feske, M. Prakriya, A. Rao, R.S. Lewis, A severe defect in CRAC

Ca2+ channel activation and altered K+ channel gating in T cells from

immunodeficient patients, J. Exp. Med. 202 (2005) 651662.

[13] K.G. Chand, H. Wulff, C. Beeton, M. Pennington, G.A. Gutman, M.D.

Cahalan,K+ channelsas targetsfor specific immunomodulation, Trends

Pharmacol. Sci. 25 (2004) 280289.

[14] G.R. Crabtree, E.N. Olson, NFAT signalling: choreographingthe social

lives of cells, Cell 109 (Suppl.) (2002) S67.

[15] F. Macian, NFAT proteins: key regulators of T-cell development and

function, Nat. Rev. Immunol. 5 (2005) 472484.

[16] J. Heineke, J.D. Molkentin, Regulation of cardiac hypertrophy by

intracellular signalling pathways, Nat. Rev. Mol. Cell Biol. 8 (2006)

589600.

[17] V. Horsley, G.K. Pavlath, NFAT: ubiquitous regulator of cell differen-

tiation and adaption, J. Cell Biol. 156 (2002) 771774.

[18] H. Okamura, J. Aramburu, C. Garcia-Rodriguez, J.P. Viola, A. Ragha-

van, M. Tahiliani, X. Zhang, J. Qin, P.G. Hogan, A. Rao, Concerted

dephosphorylation of the transcription factor NFAT1 induces a con-

formational switch that regulates transcriptional activity, Mol. Cell 6

(2006) 539550.[19] H. Okamura, C. Garcia-Rodriguez, H. Martinson, J. Qin, D.M. Vir-

shup, A. Rao, A conserved docking motif for CK1 binding controls the

nuclear localization of NFAT1, Mol. Cell Biol. 24 (2004) 41844195.

[20] Y. Gwack, S. Sharma, J. Nardone, B. Tanasa, A. Iuga, S. Srkanth, H.

Okamura, D. Bolton, S. Feske, P.G. Hogan, A. Rao, A genome-wide

Drosophila RNAi screen identifies DYRK-family kinases as regulators

of NFAT, Nature 441 (2006) 646650.

[21] R.T. Abraham, A. Weiss, Jurkat T cells and development of the T-cell

receptor signalling paradigm, Nat. Rev. Immunol. 4 (2004) 301308.

[22] S.B. Gauld, J.M. Dal Porto, J.C. Cambier, B cell antigen receptor sig-

nalling: roles in cell development and disease, Science 296 (2002)

16411642.

[23] H. Turner, J.P. Kinet, Signalling through the high-affinity IgE receptor

Fc epsilonRI, Nature 402 (1999) 6760 Suppl. B24-B30.

[24] L.L. Lanier, NK cell recognition, Annu. Rev. Immunol. 23 (2005)225274.

[25] F. Macian, C. Lopez-Rodriguez, A. Rao, Partners in transcription:

NFAT and AP-1, Oncogene 20 (2001) 24762489.

[26] M. Karin, E. Gallagher, From JNK to pay dirt: jun kinases, their bio-

chemistry, physiology and clinical importance, IUBMB Life 57 (2005)

283295.

[27] J. Schulze-Luehrmann, S. Ghosh, Antigen-receptor signalling to

nuclear factor kappa B, Immunity 5 (2006) 701715.

[28] R.E. Dolmetsch, R.S. Lewis, C.C. Goodnow, J.I. Healy, Differential

activation of transcription factors induced by Ca2+ response amplitude

and duration, Nature 386 (1997) 855858.

[29] R.E. Dolmetsch, K. Xu, R.S. Lewis, Calcium oscillations increase

the efficiency and specificity of gene expression, Nature 392 (1998)

933936.

[30] W. Li, J. Llopis, M. Whitney, G. Zlokamik, R.Y. Tsien, Cell-permeant

caged InsP3 ester shows that Ca2+ spike frequency can optimize gene

expression, Nature 392 (1998) 936941.

[31] C. Nagler-Anderson, N.L. Allbriton, C.R. Verret, H.N. Eisen, A com-

parison of the cytolytic properties of murine primary CD8 + cytotoxic

T lymphocytes and cloned cytotoxic T cell lines, Immunol. Rev. 103

(1988) 111125.

[32] J.B. Huppa, M. Gleimer, C. Sumen, M.M. Davis, Continuous T cell

receptor signalling required for synapse maintenance and full effector

potential, Nat. Immunol. 4 (2003) 749755.

[33] C. Loh, K.T.Y. Shaw, J.A. Carew, J.P.B. Viola, B.A. Perrino, A. Rao,

Calcineurin binds the transcription factor NFAT1 and reversibly regu-

lates its activity, J. Biol. Chem. 271 (1996) 1088410891.

[34] L.A. Timmerman, N.A. Clipstone, S.N. Ho, J.P. Northrop, G.R. Crab-

tree, Rapid shuttling of NF-AT in discrimination of Ca2+ signals and

immunosuppression, Nature 383 (1996) 837840.

[35] J.I. Healy, R.E. Dolmetsch, L.A. Timmerman, J.G. Cyster, M.L.

Thomas, G.R. Crabtree, R.S. Lewis, C.C. Goodnow, Different nuclear

signals are activated by the B cell receptor during positive versus neg-

ative signalling, Immunity 4 (1997) 419428.

[36] S.B. Gauld, R.J. Benschop, K.T. Merrell, J.C. Cambier, Maintenance

of B cell anergy requires constant antigen receptor occupancy and

signalling, Nat. Immunol. 6 (11) (2005 Nov.) 11601167.

[37] K.T. Merrell, R.J. Benschop, S.B. Gauld, K. Aviszus, D. Decote-

Ricardo, L.J. Wysocki, J.C. Cambier, Identification of anergic B cells

within a wild-type repertoire, Immunity 6 (2006) 953962.

[38] D.A. Fulcher, A. Basten, Reduced life span of anergic self-reactive B

cells in a double-transgenic model, J. Exp. Med. 179 (1994) 125134.

[39] R. Barrington, M. Borde, A. Rao, M. Carroll, NFAT1 involvement in

B cell self-tolerance, J. Immunol. 177 (2006) 15101515.

[40] R.H. Schwartz,T cellanergy, Annu. Rev. Immunol.21 (2003) 305334.

[41] F. Macian, F.J.G. Cozar, S.-H. Im, H.F. Horton, M.C. Bryne, A. Rao,

Transcriptional mechanisms underlying lymphocyte tolerance, Cell

109 (2002) 719731.

[42] V. Heissmeyer, A. Rao, E3 ligases in T cell anergy: turning immune

responses into tolerance, Science STKE 241 (2004) 15.

[43] B.A. Olenchock, R. Guo, J.H. Carpenter, M. Jordan, M.K. Topham,

G.A. Koretzky, X.P. Zhong, Disruption of diacylglycerol metabolismimpairs the induction of T cell anergy, Nat. Immunol. 7 (2006)

11741181.

[44] M. Safford, S. Collins, M.A. Lutz, A. Allen, C.T. Huang, J. Kowalski,

A. Blackford, M.R. Horton, C. Drake,R.H. Schwartz,J.D. Powell, Egr-

2 and Egr-3 are negative regulators of T cell activation, Nat. Immunol.

6 (2005) 472480.

[45] S. Bandyopadhyay, M. Dure, M. Paroder, N. Soto-Nieves, I. Puga, F.

Macian, Interleukin 2 gene transcription is regulatedby Ikaros-induced

changes in histone acetylation in anergic T cells, Blood (2006) [Epub

ahead of print].

[46] V. Heissmeyer,F. Macian,S.-H. Im, R. Varma,S. Feske, K. Venuprasad,

M.-S.Jeon, H. Gu, Y.-C.Liu, M.L. Dustin, A. Rao,Calcineurin imposes

T cell unresponsiveness through targeted proteolysis of signalling pro-

teins, Nat. Immunol. 5 (2004) 255265.

[47] D.L. Mueller, E3 ubiquitin ligases as T cell anergy factors, Nat.Immunol. 5 (2004) 883890.

[48] M. Hundt, H. Tabatha, M.S. Jeon, K. Hayashi, Y. Tanaka, R. Krishna,

L. De Giorgio,Y.C. Liu, M. Fukata, A. Altman, Impairedactivationand

localization of LAT in anergic T cells as a consequence of a selective

palmitoylation defect, Immunity 24 (2006) 513522.

[49] S. Feske, J.M. Muller, D. Graf, R.A. Kroczek, R. Drager, C. Niemeyer,

P.A. Baeuerle, H.H. Peter, M. Schlesier, Severe combined immunod-

eficiency due to defective binding of the nuclear factor of activated T

cells in T lymphocytes of twomalesiblings, Eur. J. Immunol.26 (1996)

21192126.

[50] M. Partiseti, F. Le Deist, C. Hivroz, A. Fischer, H. Korn, D. Choquet,

The calcium current activated by T cell receptor and store depletion in

human lymphocytes is absent in a primary immunodeficiency, J. Biol.

Chem. 269 (1994) 3232732335.


12/12


[51] F. Le Deist,C. Hivroz, M. Partiseti, C. Thomas, H.A. Buc, M. Oleastro,

B. Belohradsky, D. Choquet, A. Fischer, A primary T-cell immunodefi-

ciency associated with defective transmembrane calcium influx, Blood

85 (1995) 10531062.

[52] S. Feske, R. Draeger, H.H. Peter, K. Eichmann, A. Rao, The dura-

tion of nuclear residence of NFAT determines the pattern of cytokine

expression in human SCID T cells, J. Immunol. 165 (2000) 297305.

[53] S. Feske, G. Giltnane,R. Dolmetsch, L. Standt, A. Rao,Gene regulation

mediated by calcium signals in T lymphocytes, Nat. Immunol.2 (2001)

316324.

[54] S. Feske, Y. Gwack, M. Prakriya, S. Srikanth, S.-H. Puppel, B. Tanasa,

P.G. Hogan, R.S. Lewis, M. Daly, A. Rao, A mutation in Orai1 causes

immune deficiency by abrogating store-operated Ca2+entry and CRAC

channel function, Nature 441 (2006) 179185.

[55] M. Prakriya, S. Feske, Y. Gwack, S. Srikanth, A. Rao, P. Hogan, Orai1

is an essential pore subunit of the CRAC channel, Nature 443 (2006)

230233.

[56] A.V. Yeromin, J. Roos, K.A. Stauderman, M.D. Cahalan, A store-

operated calcium channel in Drosophila S2 cells, J. Gen. Physiol. 123

(2004) 167182.

[57] N. Perrimon, B. Mathey-Prevot, Applications of high-throughput RNA

interference screens to problems in cell and developmental biology,

Genetics 175 (2007) 716.

[58] C.J. Echeverri, N. Perrimon, High-throughput RNAi screening in cul-

tured cells: a users guide, Nat. Rev. Genet. 7 (2006) 373384.

[59] I. Flockhart, M. Booker, A. Kiger, M. Boutros, S. Armknecht, N.

Ramadan, K. Richardson, A. Xu, N. Perrimon, B. Mathey-Prevot, Fly-

RNAi: the Drosophila RNAi screening center database, Nucl. Acids

Res. 34 (2006) D489D494 (Database issue).

[60] S. Armknecht, M. Boutros, A. Kiger, K. Nybakken, B. Mathey-Prevot,

N. Perrimon, High-throughput RNA interference screens in Drosophila

tissue culture cells, Meth. Enzymol. 392 (2005) 5573.

[61] M. Vig, C. Peinelt, A. Beck, D.L. Koomoa, D. Rabah, M. Koblan-

Huberson, S. Kraft, H. Turner, A. Fleig, R. Penner, J.P. Kineet,

CRACM1 is a plasma membrane protein essential for store-operated

Ca2+ entry, Science 312 (2006) 12201223.

[62] S.L. Zhang, A.V. Yeromin, X.H. Zhang, Y. Yu, O. Safrina, A. Penna, J.

Roos, K.A. Stauderman, M.D. Cahalan, Genome-wide RNAi screenof Ca2+ influx identifies genes that regulate Ca2+ release-activated

Ca2+ channel activity, Proc. Natl. Acad. Sci. U S A 103 (2006) 9357

9362.

[63] Y. Gwack, S. Srikanth, S. Feske, F. Cruz-Guilloty, M. Oh-hora, D.

Neems, P.G. Hogan, A. Rao, Biochemical and functional characteriza-

tion of Orai-family proteins, J. Biol. Chem. 282 (2007) 1623216243.

[64] J.C. Clemens, C.A. Worby, N. Simonson-Leff, M. Muda, T. Maehama,

B.A. Hemmings, J.E. Dixon, Use of double-stranded RNAinterference

in Drosophila cell lines to dissect signal transduction pathways, Proc.

Natl. Acad. Sci. U S A 97 (2000) 64996503.

[65] C. Lopez-Rodriguez, C.L. Antos, J.M. Shelton, J.A. Richardson, F. Lin,

T.I. Novobrantseva,R.T. Bronson,P.Igarashi,A. Rao,E.N. Olson, Loss

of NFAT5 results in renal atrophy and lack of tonicity-responsive gene

expression, Proc. Natl. Acad. Sci. U S A 101 (2004) 23922397.

[66] J. Aramburu, K. Drews Elger, A. Estrada-Gelonch, J. Minguillon,B. Morancho, V. Santiago, C. Lopez-Rodriguez, Regulation of the

hypertonic stress response and other cellular functions by the Rel-

like transcription factor NFAT5, Biochem. Pharmacol. 72 (2006)

15971607.

[67] I.A. Graef, J.M. Gastier, U. Francke, G.R. Crabtree, Evolutionary

relationships among Rel domains indicate functional diversification

by recombination, Proc. Natl. Acad. Sci. U S A 98 (2001) 5740

5745.

[68] J. Roos, P.J. DiGregorio, A.V. Yeromin, K. Ohlsen, M. Lioudyno,

S. Zhang, O. Safrina, J.A. Kozak, S.L. Wagner, M.D. Cahalan, G.

Velicelebi, K.A. Stauderman, STIM1, an essential and conserved com-

ponent of store-operatedCa2+ channelfunction, J. CellBiol. 169(2005)

435445.

[69] J. Liou, M.L. Kim, W.D. Heo, J.T. Jones, J.W. Myers, J.E. Ferrell Jr.,

T. Meyer, STIM is a Ca2+ sensor essential for Ca2+-store-depletion-

triggered Ca2+ influx, Curr. Biol. 15 (2005) 12351241.

[70] M. Vig, A. Beck, J.M. Billingsley, A. Lis, S. Parvez, C. Peinelt,

D.L. Koomoa, J. Soboloff, D.L. Gill, A. Fleig, J.P. Kinet, R. Penner,

CRACM1 multimersform the ion-selective pore of the CRAC channel,

Curr. Biol. 16 (2006) 17.

[71] M.M. Wu, J. Buchanan, R.M. Luik, R.S. Lewis, Ca2+ store depletion

causes STIM1 to accumulate in ER regions closely associated with the

plasma membrane, J. Cell Biol. 174 (2006) 803813.

[72] J.C. Mercer, W.I. Dehaven, J.T. Smyth, B. Wedel, R.R. Boyles, G.S.

Bird, J.W. Putney Jr., Large store-operated calcium selective currents

due to co-expression of Orai1 and Orai2 with the intracellular calcium

sensor, Stim1, J. Biol. Chem. 281 (2006) 2497924990.

[73] P.B.Stathopulos,G.Y. Li, M.J.Plevin, J.B.Ames, M. Ikura, Stored Ca2+

depletion-induced oligomerization of stromal interaction molecule 1

(STIM1) via the EF-SAM region. An initiation mechanism for capaci-

tive Ca2+ entry, J. Biol. Chem. 281 (2006) 3585535862.

[74] J. Meldolesi, T. Pozzan, The endoplasmic reticulum Ca2+ store: a view

from the lumen, Trends Biochem. Sci. 23 (1998) 1014.

[75] S.L. Zhang, Y. Yu, J. Roos, J.A. Kozak, T.J. Deerinck, M.H. Ellisman,

K.A. Stauderman, M.D. Cahalan, STIM1 is a Ca2+ sensor that acti-

vates CRAC channels and migrates from the Ca2+ store to the plasma

membrane, Nature 437 (2005) 902905.

[76] R.M. Luik, M.M. Wu, J. Buchanan, R.S. Lewis, The elementary

unit of store-operated Ca2+ entry: local activation of CRAC chan-

nels by STIM1 and CRACM1 (Orai1), Nat. Cell Biol. 8 (2006) 815

825.

[77] P. Xu, J. Lu, Z. Li, X. Yu, L. Chen, T. Xu, Aggregation of STIM1underneath the plasma membrane induces of Orai1, Biochem.Biophys.

Res. Commun. 350 (2006) 969976.

[78] C. Peinelt, M. Vig, D.L. Koomoa, A. Beck, M.J. Nadler, M. Koblan-

Huberson, A. Lis, A. Fleig, R. Penner, J.P. Kinet, Amplification of

CRAC current by STIM1 and CRACM1 (Orai1), Nat. Cell Biol. 8

(2006) 771773.

[79] J. Soboloff, M.A. Spassova, X.D. Tang, T. Hewavitharana, W. Xu,

D.L. Gill, Orai1 and STIM reconstitute store-operatedcalcium channel

function, J. Biol. Chem. 281 (2006) 2066120665.

[80] A.V. Yeromin, S.L. Zhang, W. Jiang, Y. Yu, O. Safrina, M.D. Cahalan,

Molecular identification of the CRAC channel by altered selectivity in

a mutant of Orai, Nature 443 (2006) 226229.

[81] J.T. Smyth, W.I. Dehaven, B.F. Jones, J.C. Mercer, M. Trebak, G.

Vazquz, J.W. Putney Jr., Emerging perspectives in store-operated Ca 2+

entry: roles of Orai, Stim, and TRP.[82] J.C. Nolz, T.S. Gomez, P. Zhu, S. Li, R.B. Medeiros, Y. Shumizu, J.K.

Burkhardt, B.D. Freedman, D.D. Billadeau, The WAVE2 complex reg-

ulates actin cytoskeletal reorganization and CRAC-mediated calcium

entry during T cell activation, Curr. Biol. 16 (2006) 2434.

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