[Channels 2:4, 1-8; July/August 2008]; ©2008 Landes Bioscience
Review
The STIM/Orai coupling machinery
Irene Frischauf, Rainer Schindl, Isabella Derler, Judith Bergsmann, Marc Fahrner and Christoph Romanin*
Institute for Biophysics; University of Linz; Linz, Austria
Key words: CRAC, STIM, Orai
This manuscript has been published online, prior to printing.Once the issue is complete and page numbers have been assigned, the citation will change accordingly.
Calcium (Ca2+) entry into non-excitable cells is mainly carried
by store-operated channels (SOCs), which serve essential func-
tions ranging from regulation of transcription to cell growth. The
best-characterized store-operated current, ICRAC, is the calcium
release-activated calcium (CRAC) current initially discovered
in T-lymphocytes and mast cells. The search for the molecular
components of the CRAC channel lasted over 20 years. Recently
STIM1 has been identified as the Ca2+ sensor in the endoplasmic
reticulum (ER) that accumulates into punctae close to the plasma-
membrane following store-depletion. The identification of STIM1
has been closely followed by the discovery of Orai1 as the CRAC
channel pore in human T-cells. Upon punctae formation STIM1
activates Ca2+ influx via Orai1 channels. This review covers func-
tional details concerning the activation cascade of the STIM1/
ON
Orai1 complex from ER Ca2+ sensing to Ca2+ influx through
.D
Orai1. Furthermore, functional domains within STIM1 and Orai1
in comparison to their structural homologs STIM2 as well as Orai2
CE
and Orai3, respectively, are displayed together with recent findings
IEN
on the pore architecture and selectivity filter of Orai channels. A
broad tissue expression of STIM and Orai proteins together with
substantial effects in STIM1/Orai1 knock-out mice suggests an
SC
essential physiological role in store-operated Ca2+ signalling in
human health and disease.
BIO
Discovery of STIM and Orai Proteins
Cytosolic calcium signals are adjusted by intracellular sources
ES
such as the endo-/sarcoplasmic reticulum, Ca2+ binding proteins and
via plasma-membrane Ca2+ permeable channels. The intracellular
ND
free calcium, Ca2+i, is essential for various cellular processes ranging
from contraction and secretion to transcription, cell growth and cell
LA
division.1,2 The endoplasmic reticulum (ER) functions as a store for
the intracellular pool of Ca2+ which is released when ER-localized
08
inositol-1,4,5-triphosphate (IP3) receptors are activated, a process
triggerd by the phospholipase C product IP3, allowing for Ca2+
20
efflux into the cytosol.3 Due to the limited capacity of the ER to
store Ca2+, it needs to be refilled following depletion. More than
©
two decades ago, in 1986, James Putney has proposed a ‘­capacitative
*Correspondence to: Christoph Romanin; Institute for Biophysics; University of
Linz; Altenbergerstr. 69; Linz 4040 Austria; Tel.: 43.732.2468.9272; Fax:
43.732.2468.9280; Email: christoph.romanin@jku.at
Submitted: 07/20/08; Accepted: 07/21/08
Previously published online as a Channels E-publication:
http://www.landesbioscience.com/journals/channels/article/6705
1 Channels
E .
Ca2+ entry’ hypothesis in which the depletion of ER Ca2+ stores
UT
itself activates Ca2+ channels in the plasma-membrane to refill
internal stores (later renamed store-operated calcium entry or
RIB
SOCE).4 Putney’s hypothesis has received strong support from Ca2+-
imaging experiments in various cell types where inhibitors of the
ER-Ca2+ uptake like thapsigargin have been shown to activate Ca2+
IST
entry from the extracellular milieu without the involvement of cell
surface receptors or IP3.5 Subsequently the use of the patch-clamp
D
technique has enabled to identify and characterize a store-operated
Ca2+ current, the so called Ca2+ release—activated Ca2+ (CRAC)
OT
current, or ICRAC, in mast cells6 as well as in Jurkat T-cells.7,8 The
distinctive biophysical characteristics of the CRAC channel comprise
a high selectivity for Ca2+, a pronounced inwardly rectifying current/
voltage relationship and a very low single channel conductance.9
Many studies have clearly demonstrated that the CRAC channel
is required for the response of T-cells to antigens,10,11 for prolif-
eration and cytokine secretion.12 However, despite intense studies
over 20 years, the decoding of the molecular choreography of the
CRAC channel has turned out to be very challenging. For example,
proteins of the canonical transient receptor potential (TRP) family
have been for a long time in the focus as components of CRAC
or SOC channels.9 A variety of CRAC blockers have so far been
identified,13 however, due to their non-specificity in the context
with other channels and signalling pathways, these compounds
have been less valuable for the identification of CRAC channels.14
Emphasized studies on the elucidation of the CRAC components
and their signalling machinery have remained inefficient until high-
throughput RNAi screens (searching for molecules involved in SOC
influx of Drosophila S2 and HeLa cells) have led to the identifica-
tion of a protein that is required for store-operated Ca2+ entry: the
stromal interaction molecule 1 (STIM1).15,16 STIM1 functions as an
ER calcium-sensor10,15 that translocates to defined punctae close to
the plasma-membrane following store-depletion thereby activating
CRAC currents.10 The elucidation of STIM1 as a Ca2+ sensor has
been closely followed by the identification of the CRAC channel’s
pore-forming subunit, named Orai1/CRACM1.17-19 Up to now,
three human homologs, termed Orai1-3 have been identified.
These proteins contain four transmembrane segments with both
N- and C-termini located intracellularly.18 Unveiling Orai1 as a
CRAC component has been additionally carried out by a modified
linkage analysis, where a naturally occurring single point mutation
at position 91 (R91W) results in impaired T-cell signalling, which
leads to severe combined immunodefiency (SCID) syndrome. This
single point mutation disrupts ICRAC in human T-cells of SCID
2008; Vol. 2 Issue 4
 The STIM/Orai coupling machinery
patients,11,17-19 however, the mechanism leading to complete loss of
function is still unresolved.
Molecular Structure of STIM1 and Orai Proteins
STIM1 domains. STIM1, initially characterised as a phospho-
protein,20 includes a single transmembrane domain located in the ER
in resting cells (Fig. 1A). The N-terminus of STIM1 stretches into
the ER lumen, while the C-terminus extends into the cytosol. Within
the former, a single EF-hand Ca2+-binding motif acts as luminal Ca2+
sensor.10,21 A sterile-alpha motif (SAM), including two N-linked
glycosylation sites, is followed by the transmembrane domain and a
cytosolic C-terminus with two coiled-coil regions overlapping with
an ezrin-radixin-moesin (ERM)-like domain. Subsequent gluta-
mate-, serine/proline-, serine/threonine- and lysine-rich regions are
additionally located at the C-terminus (Fig. 1A).10,22-24 STIM2,
the second protein within the STIM family, is structurally 61%
homologous to STIM1. Both proteins diverge significantly in their
C-terminal region after the ERM/coiled-coil domain.25
Orai domains. All three Orai proteins, containing four predicted
transmembrane segments (Fig. 1B), form Ca2+ selective plasma-
membrane channels. Within the cytosolic strands, the N-terminus
of only Orai1 includes a proline/arginine-rich region.26 In their
C-terminus all three Orai proteins contain a putative coiled-coil
domain, a common protein interaction motif (Fig. 1B), with varying
predicted probabilities which are 5–6 fold higher for Orai2 and Orai3
than for Orai1 [http://www.isrec.isb-sib.ch/webmarcoil/webmar-
coilC1.html; http://www.ch.embnet.org/software/COILS_form.
html]. The extracellular loop of Orai1 between the third and fourth
transmembrane segment comprises an N-glycosylation site.26,27
However, it has been shown not to be essential for Orai1 function.28
Based on coimmunoprecipitation experiments, Orai proteins are able
to assemble into homo- as well as heteromers.29,30 Co-expression of
STIM1 with Orai1 is required to fully reconstitute CRAC currents
while their expression alone results in no significant increase in or
even reduced store-operated Ca2+ entry.31,32 Additionally co-expres-
sion of STIM1 with Orai2 as well as Orai3 resulted in Ca2+ current
activation with biophysical characteristics similar to CRAC currents,
however, with slightly different selectivity profiles and differences
in feedback to intracellular Ca2+.33-35 The selectivity filter of Orai
pores are formed by acidic residues in transmembrane domain one
and three and the first loop domain.27,32,36,37 Also STIM2 is able to
activate all three Orai proteins.38
Store-Operated Activation of STIM/Orai
Store-depletion dependent STIM1 redistribution. STIM1
mediated robust Orai1 currents are initially stimulated by Ca2+
store-depletion sensed by the STIM1 luminal EF-hand. In resting
cells, STIM1 is uniformly distributed within the ER, displays
tubular structures and binds to the microtubule-plus-end-tracking
protein EB1 at those sites where microtubule ends come in contact
with the ER.39 Moreover, STIM1 co-localises with endogenous
α-tubulin.23,40 The STIM1 EF-hand is adequate to sense a decrease
in the ER Ca2+ level that is approximately 300–500 μM at rest.41-43
Subsequently, STIM1 forms oligomers before it redistributes with
an EC50 of 210 μM Ca2+,44 into punctuate clusters close to the
plasma-membrane.10,21,23,34,45-49 It has recently been shown that
oligomerisation of about four STIM1 proteins is the critical process
www.landesbioscience.com Channels
transmitting ER store depletion to STIM1/Orai1 clusters thereby
activating store-operated Ca2+ entry.50 TIRF microscopy in combi-
nation with electrophysiology has revealed that STIM1 translocation
proceeds CRAC activation by 6–10 seconds compatible with an
upstream step in CRAC channel activation.45 In addition, fluorescence
quenching and microscopic analysis visualise that STIM1 clusters
colocalise with areas of elevated Ca2+ concentrations from endog-
enous CRAC or coexpressed Orai1 channels47 and are located within
a distance of 10–25 nm from the plasma-membrane.45 Moreover,
upon store-depletion Orai1 proteins require STIM1 co-expression to
form a clustered localisation in the plasma-membrane at sites in close
proximity to STIM1 punctae.47,51 The process of store-operated
STIM1 aggregation has been demonstrated by FRET microscopy to
be fully reversible following store repletion.49,51 A STIM1 EF-hand
mutant that is no longer able to sense ER Ca2+ concentration,
already exhibits a punctuate pattern. Furthermore, expression of the
STIM1 EF-hand mutant results in constitutive CRAC currents even
when stores are full.10,16,19,34 It is under debate if store-depletion
mediated translocation of STIM1 results in insertion into or accu-
mulation underneath the plasma-membrane. A partial integration of
STIM1 into the plasma-membrane has been reported by a number
of laboratories employing biotinylation experiments.16,20,21,24,52,53
Gill and coworkers have proposed an interaction of endoplasmic
and plasma-membrane localised STIM1, due to a constant STIM1
plasma-membrane expression independent of store-depletion.16 In
contrast, an increase in plasma-membrane expression of STIM1
upon store-depletion has been observed with immunofluorescence,
immuno-electron microscopy and biotinylation experiments.21,53
Moreover, a small hexahistidine-Zn2+-tag fused to the N-terminus
of STIM1 has allowed demonstrating its plasma membrane inser-
tion following store-depletion. It is of note that large N-terminal
fusion proteins in contrast to C-terminal ones interfere with STIM1
plasma-membrane insertion.54 Functional evidence for plasma-
membrane STIM1 is presented by antibodies against an N-terminal
sequence of STIM1 that results in partial suppression of SOCs,16,55
but could not be confirmed in another study.52 Reports that observe
STIM1 distribution underneath but not in the plasma-membrane
have been widely based on TIRF microscopy using YFP-tagged
STIM1 as well as other, even larger tags. In line, antibodies against
these fluorophores fail to detect any insertion of YFP-STIM1 into
the plasma-membrane.10,34 As CFP or YFP N-terminally tagged
STIM1 is apparently able to activate Orai1 currents to a similar
extent as C-terminally tagged,51 a major role of plasma-membrane
inserted STIM1 for the activation of Orai1 currents seems unlikely.
Functional STIM1 domains. STIM1 displays multiple roles
in the CRAC signalling cascade, first sensing the decrease of ER
Ca2+ concentration via its EF-hand, followed by its aggregation
and punctae formation that culminates in activation of CRAC
channels.49 These diverse actions are accomplished by distinct
structures within both N- and C-termini of the STIM1 protein.
An N-terminal fragment consisting of both EF-hand and SAM
domain exists in vitro as a monomer when Ca2+ is bound and as an
oligomer when Ca2+ is absent.56 Hence, this correlates with in vivo
observations, where STIM1 is evenly distributed when Ca2+ stores
are filled, and following ER depletion it forms oligomers.45,49,51
In line, co-immunoprecipitation experiments suggest an essential
role of the N-terminal part, including the SAM domain in STIM1-
2
 The STIM/Orai coupling machinery

Figure 1. (A and B) Predicted structures of the Orai and STIM protein families. (A) Structure of STIM1 and STIM2. STIM proteins are single-transmembrane
spanning proteins located in the ER, with their N-termini extending into the ER lumen. The N-termini contain a Ca2+-sensing EF-hand and an SAM domain. The
single transmembrane domain is followed by the C-terminus containing two coiled-coil motifs and an overlapping ERM domain with a subsequent glutamate-,
serine/proline- and a lysine-rich region. Only STIM1 additionally comprises a glutamine- and a serine/threonine-rich region. (B) Structure of Orai1, Orai2
and Orai3, with only Orai1 containing two proline- and one arginine- rich region in its N-terminal strand. All three Orai proteins consist of four transmem-
brane regions and a putative coiled-coil domain in their C-terminus which is predicted with an about 5-6-fold higher probability for Orai2 and Orai3 than
for Orai1. Further, Orai3 displays a much longer second extracellular loop than Orai1 and Orai2. (C) Model for STIM1/Orai1 interaction. Following ER
store depletion and punctae formation, STIM1 directly or indirectly interacts with Orai1 triggering CRAC channel activation. On the one hand it is proposed
that the Orai1/STIM1 coupling is mediated by intermediate steps probably involving CIF.61 Nevertheless, this might be not required for the STIM1 C-terminal
fragment that activates Orai1 currents independent of store-depletion. On the other hand, the C-terminus of STIM1 may directly interact with the C-terminus
of Orai1 as shown by in vitro pulldown experiments, probably via coiled-coil interactions.51 Further auxiliary proteins including calmodulin (CaM) may also
regulate/modulate STIM1-Orai1 coupling.

3 Channels 2008; Vol. 2 Issue 4

 The STIM/Orai coupling machinery
STIM1 homomeric interaction.57 Deletion of the SAM domain (aa
122–199)23 prevents punctae formation and CRAC activation. The
C-terminus of STIM1 seems particularly important for Orai/CRAC
activation, as the cytosolic C-terminal strand alone is sufficient to
stimulate both endogenous CRAC24 as well as Orai1 currents.30,51
This fragment shows predominantly targeting to the plasma-
membrane when co-expressed with Orai151 in HEK293 cells, but
it is incapable of punctuate clustering yet closely co-localises with
Orai1.16,51 A STIM1 mutant lacking the C-terminus fails to co-loca-
lise with co-expressed Orai1 upon store depletion and is diffusely
distributed throughout the ER membrane.29 Deletion of the coiled-
coil domain results in much larger tubularvesicular arrangement
and this mutant lacks homo-oligomerisation and punctae formation
upon store-depletion as well as CRAC activation.23,24 Additionally
disruption of the C-terminus after the ERM domain similarly
impairs STIM1 redistribution and fails to activate both CRAC and
Orai1.29 However, deletion of a glutamate-rich domain that overlaps
with the first coiled-coil in the ERM domain, as well as the serine/
proline-rich regions (aa 600–629) has been reported to be less essen-
tial for CRAC activation.24 Deletion of the lysine-rich domain at the
very end of the C-terminus has been proposed to eliminate punctae
formation despite preserved STIM1-STIM1 interaction. Thus the
lysine-rich domain allows the separation of STIM1 homomeric
interaction from punctae formation.49 Moreover, this mutant results
in delayed stimulation when co-expressed with Orai1.29 Therefore
both coiled-coil domain and lysine-rich region of STIM1 are essen-
tial for Orai1/CRAC activation.
The role of STIM2. Worms and flies only possess one STIM
protein, while mammals include both STIM1 and STIM2.10,15 Both
proteins are approximately 61% homologous and show ubiquitous
tissue expression. Besides homomeric STIM1-STIM1 interaction,
STIM1 can form heteromers with STIM2.46,55 Co-expression of
both STIM proteins favors translocation of STIM2 into punctae
upon store depletion.46 Functionally Soboloff et al.,46 have also
revealed that STIM2 is a powerful SOC inhibitor when expressed in
HEK293, PC12, A7r5 and Jurkat T cells.
In the absence of Ca2+, STIM2 does not similarly aggregate as
STIM1.25 This functional difference is due to their distinct EF-hand
Ca2+ affinity. While the EF-hand of STIM1 binds Ca2+ with low
affinity in a range ideally adjusted to sense substantial changes in
ER Ca2+ concentrations, the EF-hand of STIM2 is more sensitive to
mild reduction of ER Ca2+.44 STIM2 already forms punctae with an
EC50 at 406 μM ER Ca2+ that represents resting levels. Accordingly,
STIM2 knock-down in Jurkat T-cells selectively lowers basal cyto-
solic and ER Ca2+ concentrations. Therefore STIM2 is partially
active at resting ER Ca2+ levels,44 covering both store-dependent
or -independent modes of Orai1 activation.38 A plausible model
suggests that STIM2 is part of a feedback module that keeps basal
cytosolic and ER Ca2+ concentrations within tight limits. STIM2
deficiency, however, imposes a smaller effect on Ca2+ influx than
STIM1 knock-out.
STIM1 and Orai1 coupling. Following store-depletion labeled
STIM1 proteins form clusters in close proximity to Orai1 leading
to CRAC channel activation.29,47,48 However, it remains unre-
solved if the two proteins interact directly or if the signal cascade
involves a third, intermediate component. While some laboratories
report co-immunoprecipitation of STIM1 and Orai1,32,36 others
www.landesbioscience.com Channels
have failed to detect an interaction.28 The distance between ER
and PM estimated in a range between 10–25 nm45 is in principle
small enough to allow direct STIM1/Orai1 interaction. Balla and
coworkers have utilized chemically inducible bridges of different
length to define the distance between plasma- and ER-membrane
in an attempt to identify whether STIM1, Orai1 and STIM1/Orai1
assemblies fit into distinct ER plasma-membrane space.58 Before
store-depletion STIM1 localises in regions of a 4–6 nm ER-PM
distance, while Orai1 requires distances in the range of about 11–14
nm. Upon store-depletion STIM1 moves into the larger regions
and colocalizses with Orai1 suggesting that Orai1 channels are part
of a larger macromolecular complex. An extended conformation of
STIM1 probably might span the distance to one of the Orai1 strands.
We and others demonstrated by FRET microscopy a distance closer
than 10 nm between CFP/YFP-labels linked to STIM1 and Orai1
in store-depleted cells.51,59 Moreover a cytosolic STIM1 C-terminal
fragment couples to Orai1 in in vitro as well as in vivo studies and
accordingly activates constitutive Orai1 currents.51 Thus, at least
in the case of STIM1 C-terminus we propose a rather direct than
indirect interaction of these two proteins via their C-termini. STIM1
coupling to Orai1 as well as STIM1 homomerization is reversed
when Ca2+ stores are refilled.10,51
Alternative to store-dependent direct coupling that may involve
the putative coiled-coil domains of STIM1 and Orai1 as depicted
in the model (Fig. 1C), a yet unidentified Ca2+ influx factor (CIF)
might additionally be involved. STIM1 has been proposed to
trigger the production of CIF,60 which could contribute to signal
transmission from STIM1 to Orai1.61 CIF has been suggested
to displace calmodulin (CaM) from phospholipase A2 (iPLA2),
stimulating lysophospholipid generation that in turn activates
CRAC.62 However, bromenolactone (BEL), an inhibitor of iPLA2
fails to prevent activation of heterologously co-expressed STIM1/
Orai1 currents14 in contrast to its blocking effect onto endogenous
CRAC in RBL cells and SOC in smooth muscle cells.63 Additionally
Ca2+ dependent CaM binding has been detected within a peptide
including residues 667–685 in STIM1 C-terminus.64 A CaM
binding domain search [http://calcium.uhnres.utoronto.ca/ctdb/
ctdb/sequence.html] has revealed additional putative CaM binding
sites both in the C-terminus of STIM1 as well as in the N-terminus
of Orai1 (Fig. 1C). Since CIF has been proposed to displace CaM
from PLA2, in the context with the STIM1/Orai1 system, it might
be interesting to see whether CIF would also be able to displace CaM
either from STIM1 or Orai1 as an additional way of modulation
within the STIM1/Orai1 signaling cascade.
In recent studies, Orai1 domains that are involved in the coupling
process to STIM1 and Ca2+ current activation have been identified.
While the C-terminus of Orai1 is required for STIM1 coupling and
subsequent current activation, Orai1 N-terminus is suggested to be
essential for Orai1 gating.29,51 A highly conserved 26 amino acid
long N-terminal region immediately before the first transmembrane
domain is essential for channel activation.29 Moreover, the proline/
arginine-rich domain in Orai1 N-terminus has a regulatory role
as Orai2 chimeras containing the N-terminus of Orai1 have been
reported to show enhanced Ca2+ entry.65 Orai1 C-terminal dele-
tion mutants fail to colocalize with STIM1 clusters upon Ca2+
store-depletion29,51 and accordingly lack ­store-operated ­activation.51
Pull-down experiments provide evidence for an interaction of STIM1
4
 The STIM/Orai coupling machinery
C-terminus with the C-terminus rather than the N-terminus of Orai1
further underscoring the role of Orai1 C-terminus for coupling to
STIM1. Especially a predicted putative coiled-coil domain within
Orai1 C-terminus has been suggested as functional coupling domain,
as a destabilizing single point mutation (L273S) eliminates coupling
to STIM1 and Ca2+ current activation.51 Therefore, it is tempting
to speculate that potential direct interaction of STIM1 and Orai1
is mainly mediated by their C-terminal coiled-coil domains—
possibly together with additional auxiliary components (see Fig. 1C).
Accordingly, a putative coiled-coil domain is also predicted within
the C-terminus of Orai2 and Orai3, even with a 5–6 fold higher
probability than that of Orai1. This may allow for a tighter or at least
distinct coupling to STIM1 that has still to be evaluated.
Channel Architecture of Orai Channels
Oligomerisation of Orai1 proteins. Many ion channel fami-
lies have been shown to require a multimeric assembly of various
numbers of individual subunits.66 Coimmunoprecipitation28,36
studies have shown for Drosophila Orai as well as mammalian
Orai1 that they assemble to dimers or higher order multimers.
Moreover all combinations of heteromeric Orai channel assemblies
are possible.28,30,33 A recent study67 has demonstrated that the func-
tional CRAC channel pore is formed by a tetrameric assembly of
Orai1 subunits. This evidence is based on maximal CRAC currents
developed after expression of preassembled tandem Orai1 multimers
comprising different numbers of subunits into cells stably overex-
pressing STIM1. Assembly of Orai1 channels is mainly mediated
by their transmembrane domains29,51 as deletion of one of their
cytosolic strands has not affected interaction of Orai1 channels.
Additionally the N-terminus of Orai1 functions dominant negative
on co-expressed Orai1 channels.65 However, discrete domains that
are involved in the multimerisation remain so far elusive.
Orai pore architecture. Further evidence that Orai channels form
the pore of CRAC channels is provided by point mutations in the
putative selectivity filter of Orai1. The narrowest region of native
CRAC, Orai1 or Orai3 pore is only ~3.9 Å wide.37,68,69 Amino
acids that form part of the selectivity filter in Orai1 include E106
in the first transmembrane domain, E190 in the third transmem-
brane domain and D110, D112 as well as D114 in the first loop
domain.27,32,36 While the glutamates within the transmembrane
domains are fully conserved in Orai2 and Orai3, the three aspar-
tates (D110, D112, D114) in the loop domain of Orai1 are either
glutamic acids or glutamines in Orai2 (E84, Q86, Q88) and Orai3
(E85, D87, E89). The glutamates and aspartates play an essential
role for the Orai1 channel’s high Ca2+ selectivity, its Ca2+ block,
regulation by Ca2+ and determine its pore geometry.36 Mutation
of the respective glutamates in Orai1-E106 and -E190 in the first
and third transmembrane region as well as corresponding E81 and
E165 in Orai3, alters ion selectivity, which underlines that Orai1 is a
pore subunit of the CRAC channel. Orai1-E106D36 and an analog
Orai3-E81D37 show reduced Ca2+ selectivity in comparison to wild-
type. In contrast Orai1-E106Q acts in a dominant negative manner
on all three STIM1 mediated Orai Ca2+ currents33,36 as well as
endogenous CRAC currents in T-cells.27,28 Yet this E106Q mutant
is able to couple to STIM1.70 The mutation E190Q or E190A in
the third transmembrane region of Orai1, as well as Orai3-E165Q
leads to a leftward shift in the reversal potential resulting in less Ca2+
5 Channels
selective channels.28,37,71 The more conservative Orai1-E190D27 as
well as Orai3-E165D70 mutations show similar pore characteristics
like wild-type Orai channels in both a Ca2+ and a monovalent bath
solution. The aspartates D110/112/114 in the first extracellular
loop additionally contribute to the increased Ca2+ selectivity of
Orai1 as their mutation to alanine (D110/112/114A) enhanced
monovalent cation permeation.36,68 Structurally these mutations of
the pore-relevant amino acids lower Ca2+ selectivity and result in a
striking increase in Cs+ permeation, which suggests an enlargement
of the unusually narrow pore of the CRAC channel, relieving steric
hindrance for Cs+ permeation. In addition, the mutations diminish
Ca2+-mediated fast inactivation, a key mode of CRAC channel
regulation.68 Pore mutants that modify Orai1 channel voltage sensi-
tivity include point mutation V102I as well as V105I close to the
selectivity filter.72 In conclusion these studies reveal that structural
elements contributing to ion permeation are located close to those
involved in the voltage gating of Orai1 channels.
Comparison of Orai1, Orai2 and Orai3. The three highly homol-
ogous Orai1, Orai2 and Orai3 proteins are widely expressed at the
mRNA level and display similar plasma-membrane expression.28,73
All three channels activate in a store-dependent manner when
co-expressed with STIM1, while the extent of their current ampli-
tudes is 2–3 fold smaller for Orai2 and Orai3 compared to Orai1.
These reduced currents may reflect differences in their expression
levels, efficiency in coupling to STIM1 and distinct single channel
properties.33 Moreover the three Orai homologs exhibit distinct
properties in terms of selectivity for Ca2+ and Na+ and strikingly
different feedback regulation by intracellular Ca2+.33 Orai3 chan-
nels undergo a lower degree of depotentiation than Orai1 or Orai2.
Nonetheless the Na+ currents through Orai1, Orai2 and Orai3 as well
as the endogenous store-operated Na+ currents in HEK293 cells, are
all inhibited by extracellular Ca2+ with a half-maximal concentration
of approximately 20 μM.35 Further, the three Orai proteins exhibit
differential pharmacological effects in response to 2-aminoethoxydi-
phenyl borate (2-APB). While STIM1-mediated Orai1 currents are
stimulated by low concentrations of 2-APB (5–10 μM), high concen-
trations (50 μM) completely inhibit Orai1 and partially suppress
Orai2 currents.30,33,35,37,71 In contrast, Orai3 has been demonstrated
to be exclusively activated by 2-APB independent of store-depletion
and STIM1, leading to an increased conductance for monovalent
ions.30,33,37,71 Substitution of the 2nd and 3rd transmembrane domain
of Orai1 with those of Orai3 conferred 2-APB stimulation onto
Orai1.30 Moreover single point mutations within the selectivity filter
of Orai3 have been reported to affect 2-APB sensitivity.37 Hence, the
positively charged 2-APB74 might exert part of its stimulatory action
via interaction with the Orai3 selectivity filter together with further
structural requirements located within transmembrane 2 and 3 that
finally result in stimulated currents with altered permeation proper-
ties. For a more detailed view on other drugs that affect STIM as well
as Orai proteins, we refer to Derler et al.14 Besides these three Orai
channels, further Orai2 splice variants have been identified on two
chromosomal loci,73 one containing an intronless gene and a second
locus that gives rise to the splice variants Orai2 long (Orai2L) and
Orai2 short (Orai2S). When co-expressed with STIM1 both Orai2S
and Orai2L represent Ca2+ selective channels.73
Physiological role of STIM and Orai channels. CRAC channels
are key mediators of sustained Ca2+ signaling in T-cells mediating
2008; Vol. 2 Issue 4
 The STIM/Orai coupling machinery
stable formation of immunological synapses. When a T-cell gets in
contact with an antigen-presenting dendritic cell, both STIM1 and
Orai1 redistribute to the immunological synapse, leading to Ca2+
influx.70 These elevated calcium levels induced by CRAC channels
prolong dephosphorylation of NFAT (nuclear factor of activated
T-cells) and accordingly its accumulation into the nucleus where it acts
as a transcription factor in the early immune response of T-cells.75,76
Moreover long-term function of CRAC channel activation includes
lymphocyte proliferation, effector functions as well as differentiation
of naïve T-cells.77 The importance of Ca2+ influx through CRAC
channels in T-cells is further highlighted by the existence of at least
three families of patients with SCID resulting in severe defects in
function of store-operated Ca2+ entry, impaired cytokine expression
and lymphocyte function.17,77,78 In addition CRAC channels play
an important role in mast cell activation, degranulation as well as
secretion of proinflammatory lipid mediators.79,80
Knock-down of either STIM or Orai proteins has been shown to
impair SOCE/CRAC entry in a variety of cell types.10,15-18,21,31,46,77,79‑82
STIM1 as well as STIM2 are detectable in various primary
lymphocytes such as Th, TC and B-cells.83 Furthermore both are
expressed at a low, uniform level in heart, brain, kidney, thymus,
lung, spleen, skeletal muscle, small intestine as well as in primary
aortic endothelial cells.83,84 High expression levels of STIM1 are
found in certain cell types and cellular regions such as the central and
the peripheral nervous systems as well as platelets.55 Nothern blot
analysis of heart, brain, kidney, thymus, lung, spleen, skeletal muscle,
small intestine, placenta, primary aortic endothelial and mast cells
suggests an ubiquitous expression of Orai1 and Orai3 proteins, while
Orai2 is predominantly expressed in brain and to a lower extent in
the lung, spleen and small intestine.28,73,84 Moreover all three Orai
proteins are expressed in primary lymphocytes. With the exception of
a lower expression of Orai3 in skeletal muscle and placenta, it seems
to be expressed in the same tissues as Orai1.
In mice the regulatory role of STIM as well as Orai proteins
has been recently examined in particular by knock-out and knock-
down studies. Mouse T-cells and fibroblasts lacking STIM1 (either
by knock-out or siRNA) exhibit impaired SOCE, while it was only
moderately diminished in STIM2-deficient T-cells.77 STIM1, as well
as STIM2-knock-out T-cells, show less cytokine production, nuclear
translocation of NFAT and additionally the number of regulatory
T-cells is decreased. Thus, these STIM proteins are required as a
prerequisite for T-cell development.77,79
Moreover STIM1 has been identified as critical to mast cell func-
tion, since STIM1-deficient mast cells of mice display much less
degranulation and cytokine production after FcεRI stimulation.
Hence, STIM1 acts as a key for mast cell activation and anaphy-
laxis.79 Besides the requirement of STIM1 in non-excitable cells,
there is further evidence that STIM1 has a regulatory role in excit-
able cells, such as skeletal muscle and myotubes. STIM1 has been
demonstrated to be expressed in both tissues. Mice lacking STIM1
die perinatally from a skeletal myopathy and their myotubes fail
to show SOC and fatigue rapidly.85 Further STIM1 is abundantly
expressed in platelets. STIM1-deficient platelets display a marked
defect in agonist-induced Ca2+ response, impaired activation and
thrombus formation under flow suggesting STIM1 as an impor-
tant mediator of ischemic cardio- and cerebrovascular events.86 A
mouse cell line expressing a constitutively CRAC current activating
www.landesbioscience.com
EF-hand mutant of STIM1 revealed macrothrombocytopenia and a
bleeding disorder. A preactivation state of platelets has been observed
as a result of an increase in basal intracellular calcium levels leading
to platelet consumption.87 Hence, STIM1 displays an essential role
in the regulation and function of platelets.
In hepatocytes bile acids lead to punctae formation of STIM1 and
activation of Ca2+-selective SOC currents. Further knock-down of
STIM1 has caused inhibited calcium entry activated by bile acids.88
Human T-cells that contain a point mutation in Orai1 R91W,
which has recently been identified in SCID patients result in
defective T-cell signalling.11,17,89 It causes a complete loss of store-
operated Ca2+ entry into T-cells, however, the detailed mechanism
for this impairment of Ca2+ flux across Orai1 R91W is so far elusive.
In contrast to human T-cells that require Orai1 for CRAC channel
formation, mice may utilize Orai2, as their thymocytes and T-cells
express much more Orai2 than Orai1 or Orai3. Orai1 plays a major
role in mice mast cells, as mice deficient in Orai1 exhibit defective
mast cell degranulation and cytokine secretion, lack allergic reactions
and are smaller in size.78,80
In contrast, store-operated calcium influx of naïve T cells, their
development and proliferation are unaffected in Orai1-deficient
mice.78,80 However, differentiated T-cells have been identified to
express higher amounts of Orai1 than of Orai2 and Orai3 consistent
with a severe but incomplete loss of store-operated Ca2+ entry. B-cells
express besides Orai1 and Orai2, Orai3 transcripts at highest amounts
among all lymphocytes. Orai1 deficient splenic B-cells exhibit also a
decrease in store-operated Ca2+ entry. Moreover, Orai1-/- mice suffer
from eyelid irritation as well as sporadic hair loss and display much
thinner skin with fewer, more elongated keratinocytes.78
Conclusions and perspectives. It has taken 20 years of exhaus-
tive studies on store-operated calcium entry before Orai1 and
STIM1 could be identified as the major components of ICRAC in
human T-cells. SOCs have been intensively examined over these past
years, revealing an involvement in a wide range of cellular processes
together with their pharmacological, physiological and pathophysi-
ological roles.9 In the focus of autoimmune and inflammatory
immune disorders an involvement of dysregulated Ca2+ signaling is
out of question. The recent identification of STIM1 and Orai1-3
and their role in calcium signaling through SOC/CRAC channels
opens the repertoire for targeting autoimmune diseases, e.g., rheuma-
toid arthritis, inflammatory disorders or allograft rejection.14 Orai1
seems to be of primary interest as in SCID patients the point muta-
tion Orai1 R91W abolishes CRAC currents only in T-cells, whereas
currents from B-cells and fibroblasts are only reduced.17 Yet there are
no specific inhibitors of the STIM1/Orai1 pathway available to test
the effect of blocking this pathway in vivo.14 With the discovery of
STIM1 and Orai1 as the key components of CRAC channels, a solid
basis is reached to study their molecular composition, stoichiometry
and activation mechanism. STIM1 as well as Orai1 have been shown
to interact with members of the canonical transient receptor potential
channels, a family of phospholipase C regulated channels.24,53,90-95
Further studies are required to reveal the nature of SOCE channels
in various tissues, possibly manifested by a combination of various
Orai and TRP channels. This will clarify the contribution of STIM1
and Orai1 to SOCE in different tissues and will provide a more
widespread view of store-operated channels and their physiological
roles in health and disease.
Channels 6
 The STIM/Orai coupling machinery
Acknowledgements
This work was supported by Austrian Science Fund project
P18169 to Christoph Romanin.
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