Download store-operated calcium channels

Physiol Rev 95: 1383–1436, 2015
Published September 23, 2015; doi:10.1152/physrev.00020.2014
STORE-OPERATED CALCIUM CHANNELS
Murali Prakriya and Richard S. Lewis
Department of Pharmacology, Feinberg School of Medicine, Northwestern University, Chicago, Illinois; and
Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, California
Prakriya M, Lewis RS. Store-Operated Calcium Channels. Physiol Rev 95: 1383–1436,
2015. Published September 23, 2015; doi:10.1152/physrev.00020.2014.—Storeoperated calcium channels (SOCs) are a major pathway for calcium signaling in virtually
all metozoan cells and serve a wide variety of functions ranging from gene expression,
motility, and secretion to tissue and organ development and the immune response.
SOCs are activated by the depletion of Ca2⫹ from the endoplasmic reticulum (ER), triggered
physiologically through stimulation of a diverse set of surface receptors. Over 15 years after the
first characterization of SOCs through electrophysiology, the identification of the STIM proteins as
ER Ca2⫹ sensors and the Orai proteins as store-operated channels has enabled rapid progress in
understanding the unique mechanism of store-operated calcium entry (SOCE). Depletion of Ca2⫹
from the ER causes STIM to accumulate at ER-plasma membrane (PM) junctions where it traps and
activates Orai channels diffusing in the closely apposed PM. Mutagenesis studies combined with
recent structural insights about STIM and Orai proteins are now beginning to reveal the molecular
underpinnings of these choreographic events. This review describes the major experimental advances underlying our current understanding of how ER Ca2⫹ depletion is coupled to the activation
of SOCs. Particular emphasis is placed on the molecular mechanisms of STIM and Orai activation,
Orai channel properties, modulation of STIM and Orai function, pharmacological inhibitors of SOCE,
and the functions of STIM and Orai in physiology and disease.
L
INTRODUCTION
STORE-OPERATED CALCIUM ENTRY:...
MOLECULAR CHOREOGRAPHY OF...
CRAC CHANNEL GATING
CRAC CHANNEL PERMEATION AND...
CRAC CHANNEL MODULATION
PHARMACOLOGY
PHYSIOLOGICAL FUNCTIONS...
FUTURE DIRECTIONS
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I. INTRODUCTION
Calcium as an intracellular second messenger serves a remarkable diversity of roles that span a range of biological
processes from birth through the development, function,
and death of cells, tissues, and organisms (23, 42, 59). In
metazoans, one of the primary sources of Ca2⫹ signals in
both excitable and particularly in nonexcitable cells is the
family of store-operated calcium channels (SOCs). These
channels are typically activated by the engagement of cell
surface receptors that through G proteins or a tyrosine
kinase cascade activate phospholipase C to cleave phosphatidylinositol 4,5-bisphosphate (PIP2) and produce
inositol 1,4,5-trisphosphate (IP3). SOCs are so named
because they respond to the reduction of ER intraluminal
Ca2⫹ ([Ca2⫹]ER), a consequence of IP3-induced Ca2⫹ release through IP3 receptors in the ER membrane (283)
(FIGURE 1A).
Store-operated channels are unique among ion channels,
from their molecular basis to their biophysical properties
and mode of regulation. Because of their intimate physical
and functional connections to the ER, they play a homeostatic role in providing Ca2⫹ to refill the ER after Ca2⫹ has
been released and pumped out across the plasma membrane
(PM) (95, 163, 261, 283). Importantly, however, Ca2⫹ entry through SOCs also serves a much wider set of signaling
functions by elevating the cytosolic Ca2⫹ concentration
([Ca2⫹]i). Because of the finite Ca2⫹ capacity of the ER,
Ca2⫹ release can only generate transient signals; however,
prolonged store depletion can evoke Ca2⫹ entry through
SOCs that is sustained for minutes to hours, driving a wide
assortment of basic biological processes such as secretion,
gene transcription, and modulation of enzymatic activity
and motility. Because they are not voltage dependent, SOCs
can conduct Ca2⫹ at negative membrane potentials at
which depolarization-sensitive channels [e.g., voltage-gated
Ca2⫹ (CaV) or NMDA-R channels] are inactive, thus enabling complementary roles. In addition, their small but
selective conductance for Ca2⫹, coupled with their tight
localization at ER-PM junctions gives SOCs preferential
access to Ca2⫹ response pathways within microdomains
(23, 279). Together, these features enable SOCs to fulfill
unique functional roles within the panoply of cellular Ca2⫹dependent pathways.
For two decades following the first proposal of the storeoperated Ca2⫹ entry (SOCE) pathway (307), its molecular
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MURALI PRAKRIYA AND RICHARD S. LEWIS
B
A
3
Ag
Ca2+
Extracellular
SOC
G/TK
PLC
IP3
PIP2
Cytosol
ICRAC (pA/pF)
R
Ca2+
TPEN
Ca2+
“leak”
2
1
Ca2+-TPEN
ER
Ionomycin
SERCA
2+
Ca -EGTA
2+
Ca
EGTA
Ca
Thapsigargin
0
0
200
400
600
[Ca2+]ER (µM)
FIGURE 1. Store-operated calcium entry. A: a diagram describing the SOCE mechanism as of 1990, prior
to the discovery of STIM and Orai. Under physiological conditions, extracellular agonists (Ag) bind to receptors
(R) and activate PLC through a G protein or tyrosine kinase-coupled pathway (G/TK). PLC cleaves PIP2 to
produce IP3, which releases Ca2⫹ from the ER. Store-operated channels (SOCs) are activated by the consequent reduction of ER luminal [Ca2⫹]. SOCs can be activated experimentally without receptor engagement by
chelating intracellular Ca2⫹ with EGTA, inhibiting SERCA pumps with thapsigargin, releasing Ca2⫹ from the ER
with ionomycin, or chelating intraluminal Ca2⫹ with TPEN. B: the steady-state input-output relation of the CRAC
channel, a prototypic SOC. CRAC current is negligible at the resting [Ca2⫹]ER of ⬃400 ␮M but increases as
[Ca2⫹]ER is reduced, with a K1/2 of ⬃170 ␮M and a Hill coefficient of ⬃4. [From Luik et al. (209).]
basis remained a complete mystery (282, 283, 298). The
identification of STIM proteins as ER Ca2⫹ sensors in
2005 (199, 321) and Orai proteins as SOC subunits a
year later (103, 398, 446) were major breakthroughs that
spurred a wave of advances in elucidating the molecular
mechanisms and functions of SOCs in many cells and
tissues (192). Since then, the combined efforts of many
groups have revealed the key cellular events involved in
SOC activation by store depletion as well as the molecular determinants of SOC biophysical properties. Most
recently, structural models for STIM1 (368, 370, 424)
and Orai (153) are beginning to illuminate the molecular
mechanisms that control activation and ion permeation
of store-operated Ca2⫹ channels.
This review begins with a brief historical overview of some
of the milestones in the study of store-operated Ca2⫹ entry
that provide context for more recent mechanistic studies.
Thereafter, the focus is on our current understanding of the
molecular and cellular mechanisms underlying SOCE; how
specific molecular determinants of STIM and Orai proteins
control SOC channel gating, permeation, modulation, and
pharmacology; and how STIM and Orai as molecular
probes have been used to reveal many previously unknown
functions of SOCE in health and disease. We have included
tables compiling the many mutations that have been found
to affect STIM and Orai function, to provide both an overall view of the functionally important domains and to help
guide future studies. While SOCE operates in many species,
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we focus here on mammalian systems where the most work
has been done, with appropriate references to differences in
other species. Our overall goal is to describe key findings
that have helped establish our current level of understanding, while highlighting significant controversies or uncertainties that remain to be resolved.
II. STORE-OPERATED CALCIUM ENTRY:
AN HISTORICAL OVERVIEW
A. The Capacitative Ca2ⴙ Entry Hypothesis
Soon after the discovery of IP3 and its role in triggering
Ca2⫹ release from the ER (24, 374), it was well recognized
that an almost universal consequence of IP3 production was
a transient increase in [Ca2⫹]i resulting from the release of
Ca2⫹ from intracellular stores, followed by a sustained
[Ca2⫹]i plateau generated by influx across the plasma membrane (21, 22, 57, 307). While the molecular mechanism of
Ca2⫹ release was understood to result from IP3 acting on
the IP3 receptor/Ca2⫹ channel in the ER (21, 374), the
mechanism underlying Ca2⫹ entry was considerably more
mysterious. Several studies suggested that the Ca2⫹ permeability of the plasma membrane was more closely related to
the degree of filling of internal stores than to either agonist
receptor occupancy or IP3 levels. Thus, after transient activation of phospholipase C (PLC)-coupled receptors, a PM
pathway that allowed extracellular Ca2⫹ to reload the
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2+
STORE-OPERATED CALCIUM CHANNELS
The first direct identification of the SOCE permeation pathway came from electrophysiological studies, conducted
shortly after the original CCE proposal, aimed at identifying Ca2⫹ channels underlying secretion in mast cells and
mitogenic activation of T cells. Using a combination of
patch-clamp and Ca2⫹ imaging techniques, Penner, Matthews, and Neher (230, 293) described a small (1–2 pA),
low-noise current that was activated by agonists such as
substance P and intracellular dialysis with IP3 in rat mast
cells. This small current developed in parallel with a rise in
[Ca2⫹]i, leading to the suggestion that it was responsible for
the [Ca2⫹]i rise caused by agonists and IP3. Lewis and Cahalan (193) described a similarly small, highly selective
Ca2⫹ current that developed slowly during whole cell recordings in Jurkat human leukemic T cells with buffered
low-Ca2⫹ internal pipette solutions. Interestingly, in perforated-patch recordings, the same current was activated by
phytohemagglutinin (PHA), a T cell mitogen that crosslinks
the T cell receptor (TCR) and generates IP3. A number of
unique features of the Ca2⫹ current were described, including a lack of voltage-dependent gating, high Ca2⫹ selectivity
with an inwardly rectifying current-voltage relationship,
low conductance based on the absence of obvious current
noise, feedback inhibition by Ca2⫹, and blockade by Ni2⫹
and Cd2⫹. At the time, the mechanism for its activation was
not understood, although the possibility that the current
flowed across the PM and directly through the ER on its
way to the cytosol as depicted by the original CCE model
was considered (193).
The link between these Ca2⫹ currents and SOCE became
clearer following the introduction of SERCA inhibitors as
experimental tools. The most widely used of these was thapsigargin (TG), a plant-derived lactone that inhibits SERCA
family ATPases in the ER and SR with high selectivity and
potency (384). The main advantage of TG and other
SERCA inhibitors (cyclopiazonic acid, BHQ) is that they
directly deplete Ca2⫹ from the ER while bypassing receptors and associated biochemical signals. By inhibiting ongoing uptake, SERCA inhibitors unmask an ongoing Ca2⫹
“leak” from the ER, leading to luminal Ca2⫹ depletion
without concomitantly generating IP3 (FIGURE 1A). The
sources of the leak are not yet well defined but may
include the protein translocon that spans the ER membrane (271, 392). The introduction of fura 2 and related
Ca2⫹-sensitive fluorescent dyes by Tsien and colleagues
(129) made it possible to detect Ca2⫹ release in response
to TG added in the absence of extracellular Ca2⫹ followed by Ca2⫹ entry through SOCs after subsequent readdition of Ca2⫹. These kinds of experiments led to an
avalanche of reports of SOCE in diverse cell types, primarily nonexcitable cells (oocytes, hepatocytes, lymphocytes, epithelial acinar cells, etc.), but in some excitable
cells as well (282, 310). With these tools, Putney and
colleagues (381) made the critical observation that Ca2⫹
influx across the plasma membrane was triggered by the
depletion of an intracellular Ca2⫹ pool and importantly
that IP3 and TG had nonadditive effects, suggesting that
they activate the same Ca2⫹ influx pathway.
It soon became evident that the Ca2⫹ entry induced by TCR
agonists and SERCA inhibitors shared key properties, suggesting that the currents in T cells and most likely in mast
cells were store operated (122, 228, 333). Hoth and Penner
(151) demonstrated this first in mast cells by identifying a
Ca2⫹-selective current activated in whole cell recordings by
intracellular EGTA, IP3, or ionomycin, and named it the
Ca2⫹ release-activated Ca2⫹ (CRAC) channel (151). In Jurkat T cells, Zweifach and Lewis (458) used perforatedpatch recording and TG to identify a similar Ca2⫹-selective
current that appeared identical to the PHA-activated current in terms of ion selectivity, sensitivity to Ni2⫹, and an
extremely small unitary conductance (10 –25 fS as estimated from noise analysis). The similarity between store
depletion-mediated and TCR-stimulated currents (302,
458), as well as the parallel loss of CRAC current and
TCR-mediated Ca2⫹ influx in T cells from human SCID
patients (286) and in mutant Jurkat T cells (95, 342),
argued strongly that the TCR is coupled to the CRAC
channel. Similarly, in mast cells, ICRAC was shown to be
activated by stimulation through Fc receptors (442). Together, these early studies established physiological links
between ICRAC and receptor stimulation in mast cells and
T cells and laid the basis for more than a decade of
detailed biophysical studies of CRAC channel properties
and behavior. By enabling the direct measurement of
channel activity, patch-clamp current recordings made it
possible to unravel intrinsic channel properties and understand their modulation, which was simply not possible based on [Ca2⫹]i measurements alone because of the
many additional parameters that regulate [Ca2⫹]i, such
as membrane potential, buffering, and ATP-driven
pumps.
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stores remained active long after IP3 or its metabolites returned to baseline levels (8, 308). Based on these and related
results from Casteels and Droogmans (44), Putney (307)
proposed that Ca2⫹ store depletion rather than IP3 was
responsible for triggering influx. This process was initially
called “capacitative Ca2⫹ entry” (CCE) to reflect the idea
that Ca2⫹ entered the cytoplasm from outside by traversing
the ER (the “capacitor”), which was assumed at that time to
be directly connected to the external milieu. Subsequent
experiments showed that Ca2⫹ actually enters the cytoplasm before it is taken up into the stores, leading to a
revision of the original hypothesis (308). However, the nature of the coupling between ER depletion and PM Ca2⫹
entry, as well as the permeation pathway itself, was unknown (FIGURE 1A). In 1995, CCE was renamed “storeoperated Ca2⫹ entry” (SOCE) to refer more explicitly to its
mode of activation and to distinguish it from receptor-,
ligand-, and voltage-operated Ca2⫹ channels.
MURALI PRAKRIYA AND RICHARD S. LEWIS
B. The Ca2ⴙ Release-Activated Ca2ⴙ (CRAC)
Channel Is a Prototypic SOC
A variety of ionic currents have been described that satisfy a
limited set of criteria for being store operated in various
cells (e.g., activation by TG or intracellular Ca2⫹ chelators),
but have not been extensively characterized and their molecular basis is still not understood (reviewed in Ref. 283).
One complication in testing whether a channel is storeoperated is that agents that release Ca2⫹ from the ER (e.g.,
TG or IP3) will elevate [Ca2⫹]i, which in some cases can by
itself activate channels directly or through Ca2⫹-induced
changes in membrane potential. Likewise, intracellular dialysis with Ca2⫹ chelators can in principle activate channels
by removing tonic inhibition by resting cytosolic [Ca2⫹]i.
C. The Fingerprint of the CRAC Channel
Electrophysiological studies from 1989 –2006 revealed essentially all that is known about the biophysical and pharmacological properties of the CRAC channel (283, 298).
There are several notable characteristics in addition to its
activation by Ca2⫹ store depletion that distinguish the
CRAC channel among the large number of known Ca2⫹permeable channels. It is extremely Ca2⫹ selective with a
Ca2⫹:Na⫹ permeability ratio of ⬎1,000, comparable to the
most selective Ca2⫹ channels known (e.g., the voltage-gated
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D. The Search for the CRAC Channel and Its
Activation Mechanism
From 1992–2005, numerous candidate genes were proposed to encode the CRAC channel, and many mechanisms
were hypothesized to explain its coupling to ER Ca2⫹ content (283). Based on early speculation that Drosophila TRP
channels were store operated (419), the most prominent
CRAC channel candidates were mammalian TRP homologs
(including TRPC1, TRPC3, and TRPV6) (67, 251, 295,
435). However, when expressed heterologously their Ca2⫹
selectivity and conductance failed to match those of ICRAC
(399), and it was not clear in many cases whether the channels were truly store operated (74). In the meantime, ironically the founding member of the family, dTrp, was definitively shown to be store independent (313). Additional confusion arose when the use of Mg2⫹-free intracellular
solutions inadvertently resulted in the slow induction of
Mg2⫹-inhibited cation (MIC/TRPM7 channel) currents
(140, 177, 301) that were initially mistaken for CRAC
channel currents (169, 170).
The mechanism coupling ER Ca2⫹ depletion to CRAC
channel activation was also obscure. Among over a dozen
proposed mechanisms (282, 283), the three most extensively studied were a diffusible activating factor released
from the ER, called calcium influx factor (CIF) (316), insertion of active CRAC channels in the PM by membrane
fusion (426), and conformational coupling between the
CRAC channel and a Ca2⫹ sensor protein in the ER (22,
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CRAC channels are considered to be SOCs because they are
activated by agents that deplete Ca2⫹ from the ER lumen
without a significant change in cytosolic [Ca2⫹]i. By this
criterion the most direct evidence for the store dependence
of CRAC channels is their activation by TPEN, a membrane-permeant Ca2⫹ chelator that reduces luminal Ca2⫹
(144), as well as by TG or ionomycin (a membrane-permeant ionophore that transports Ca2⫹ out of the ER) when
applied in the presence of high concentrations of intracellular Ca2⫹ buffers to clamp cytosolic Ca2⫹ at its resting
level (96, 458) (FIGURE 1A). The essential operating characteristic of the CRAC channel is its dependence on [Ca2⫹]ER.
This input-output relation has been examined directly by
measuring [Ca2⫹]ER with Ca2⫹-sensitive dyes loaded into
the ER (144) or ER-targeted cameleons (209) while recording ICRAC in whole cell or perforated-patch mode. A quantitative analysis by Luik et al. (209) showed that ICRAC
increases steeply (Hill coefficient of ⬃4) as [Ca2⫹]ER is reduced from its resting value of ⬃400 ␮M, with half-maximal activation at ⬃200 ␮M luminal Ca2⫹ (FIGURE 1B, and
discussed below). Together, these results demonstrate that
CRAC channels are activated by reduction of free [Ca2⫹]ER
rather than by changes in total ER Ca2⫹ or [Ca2⫹]i; this,
together with extensive characterization of their biophysical and pharmacological properties, led to the widespread
acceptance of the CRAC channel as the prototypic SOC and
the primary target in the search for a molecular mechanism
for SOCE.
L-type Ca2⫹ channel) (148). However, its unitary conductance is ⬎100-fold smaller than that of CaV channels, making single-channel currents too small to measure directly;
based on noise analysis, the unitary Ca2⫹ conductance is
only ⬃10 –35 fS (299, 458). In the absence of extracellular
divalent ions, CRAC channels conduct Na⫹ with a unitary
conductance of ⬃1 pS and low permeability to Cs⫹ (299).
Ca2⫹ modulates CRAC channel activity in several distinctive ways. On a milliseconds time scale, the channels undergo fast Ca2⫹-dependent inactivation (CDI) due to binding of Ca2⫹ close to the intracellular face of the pore (105,
152, 459). Over seconds to tens of seconds, intracellular
Ca2⫹ accumulation causes slow inactivation (276, 460),
while extracellular Ca2⫹ potentiates channel activity by
severalfold (54, 461). CRAC channels are insensitive to
most of the common CaV channel inhibitors, but are inhibited by a number of weakly selective compounds (reviewed
below) as well as by submicromolar concentrations of lanthanides (9, 324, 430). Overall, the combination of storedependent activation, lack of V-dependent activation, high
Ca2⫹ selectivity and low conductance, low Cs⫹ permeability, Ca2⫹-dependent inactivation and potentiation, and
high sensitivity to lanthanide block emerged as a stringent
set of criteria by which to screen candidates for the CRAC
channel gene.
STORE-OPERATED CALCIUM CHANNELS
158). Most proposals were supported at best by indirect
evidence, and were beset by conflicting data from followup
studies so that none achieved wide acceptance (283). In
retrospect, the conformational coupling model (158) came
closest to the actual mechanism as we now know it, although the molecular components as originally proposed
were incorrect (IP3 receptors in the ER serving as Ca2⫹
sensors that interact with and open IP4 receptor channels in
the PM; Ref. 158).
E. Identification of STIM Proteins as Ca2ⴙ
Sensors for SOCE
STIM (stromal interaction molecule) proteins were initially
discovered in a screen for stromal cell transmembrane and
secreted proteins that bind to pre-B lymphocytes (273).
Mammals express two homologs, STIM1 and STIM2. Both
are type I single-pass ER membrane proteins with a luminal
NH2 terminus and a cytoplasmic COOH terminus with
⬎74% sequence similarity (54% sequence identity) and
molecular weights of 77 kDa (STIM1) or 84 kDa (STIM2)
(for reviews, see Refs. 43, 61, 357, 371). While early studies
linked STIM proteins to suppression of tumor growth (285,
327), a role in Ca2⫹ signaling was unsuspected until two
groups rediscovered them in focused RNAi screens for inhibitors of TG-evoked SOCE. Following the recognition
that Drosophila S2 cells express a CRAC-like current (430),
Stauderman, Cahalan, and colleagues screened these cells
for suppression of SOCE using 170 dsRNA probes that
included channel-like domains, transmembrane regions,
Ca2⫹-sensing domains and TRP and other putative SOC
genes. They identified STIM dsRNA as the sole potent inhibitor of SOCE and the CRAC current in their library
(321). While Drosophila expresses only one STIM isoform
(dSTIM), vertebrates express two homologs, STIM1 and
STIM2. Importantly, they showed that knockdown of
The first clue that STIM proteins function as the longsought ER Ca2⫹ sensors came from their intracellular location and the organization of their functional domains.
STIM1 is primarily localized throughout the ER in resting
(i.e., Ca2⫹-replete) cells (223, 244), but fluorescently labeled STIM1 was seen to redistribute into clusters
(“puncta”) near the PM upon store depletion (199, 443).
STIM1 also was known to be a single-pass transmembrane
(TM) protein with a Ca2⫹-binding EF hand domain predicted to lie within the ER lumen (FIGURE 2). The notion
that STIM proteins act as ER Ca2⫹ sensors for SOCE was
quickly accepted, largely because SOCE was not merely
reduced by STIM knockdown but was also activated by
mutagenesis. Overexpression of STIM1 proteins with EF
hand mutations designed [and later confirmed (155)] to
reduce Ca2⫹ affinity (D76A, D76A/D78A, E87A) triggered constitutive formation of puncta as well as activation of SOCE and ICRAC in unstimulated cells (199, 239,
362, 443), essentially mimicking the effects of ER Ca2⫹
depletion (TABLE 1).
In the years since these pioneering studies, quantitative in
vivo and in vitro measurements have added further support
for the role of STIM proteins as ER Ca2⫹ sensors for SOCE.
The Ca2⫹ affinity of the isolated EF-SAM domains of
STIM1 (⬃200 ␮M) (367) and STIM2 (⬃500 ␮M) (449) in
vitro are comparable to the K0.5 values for the redistribution of STIM1 (187–210 ␮M) and STIM2 (406 ␮M) into
puncta in intact cells (32, 209). Furthermore, the [Ca2⫹]ER
dependence of STIM1 redistribution into puncta and ICRAC
activation are indistinguishable (209). These complementary studies demonstrated that STIM1 is the Ca2⫹ sensor
for SOCE and that the store-dependent activation of the
CRAC channel can be entirely accounted for by the release
of Ca2⫹ from STIM1.
STIM1 and STIM2 are glycosylated and are constructed
from a number of structural modules, including several protein interaction motifs (FIGURE 2). Among the most critical
for SOCE are the EF hand and sterile alpha motif (SAM)
domains on the luminal side, and on the cytoplasmic side
several coiled-coil domains, including the CRAC activation
domain (CAD; also known as STIM-Orai activation region,
or SOAR) which binds to Orai, and a polybasic domain at
the extreme COOH terminus which interacts with the PM.
The functions of these and other protein interaction domains are described in greater detail below. For simplicity
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A number of factors compounded the difficulty of identifying molecular constituents in the SOCE pathway. No biological tissue was known to express a sufficient abundance
of SOCE components to support an approach based on
biochemical purification. The absence of specific, high-affinity inhibitors for SOCE precluded approaches based on
ligand binding. Cells commonly used as heterologous gene
expression systems display endogenous SOCE so that expression cloning was undermined by high background activity, and attempts to use CRAC-deficient mutants (95,
342) for expression cloning were unsuccessful. Finally, the
properties of the CRAC channel are unique compared with
those of all other known channels, making a homologybased search risky at best (which was later borne out by the
unique sequences of the Orai gene family members). Fortunately, the introduction of RNAi screening and advances in
human linkage analysis methods ultimately made possible
the identification of the STIM and Orai protein families in
2005–2006.
STIM1 suppressed SOCE and ICRAC in Jurkat T cells and
SOCE in HEK 293 cells, firmly linking the protein to CRAC
channel function. During the same period, Liou, Meyer,
and colleagues (199) performed a similar siRNA screen for
suppression of SOCE in HeLa cells based on 2,304 proteins
with known signaling domains and independently identified STIM1 and STIM2 as the strongest hits.
MURALI PRAKRIYA AND RICHARD S. LEWIS
STIM1
Luminal
Cytoplasmic
CAD/SOAR
SP
cEF nEF
SAM
132
1
22
63
96
TM
128
CC1
α2 α3
α1
200 213 233
238
271 278
304 308
CC2
345
CC3
437
408
337 343
E136X
P/S EB PBD
470
491
601
STIM1L insert
(106 aa)
R429C
L251
STIM2
ID
391
629
671
685
TRIP
L416
L423
Luminal
Cytoplasmic
SP
cEF nEF
1 14
SAM
136
67
100
131
TM
204 217 237
240
α1
275
CC1
α2 α3
285 307 312
342
CC2
349
CC3
ID
P/S
PBD
395
347
412
441
474
495
521
566
730 746
Long SP
insert
(87 aa)
STIM2β
insert
(8 aa)
FIGURE 2. Domain organization of STIM1 and STIM2. Colored regions indicate structural and functional
domains (SP, signal peptide; cEF, canonical EF hand; nEF, noncanonical EF hand; SAM, sterile alpha motif; TM,
transmembrane domain; CC1-3, coiled-coil domains 1–3; CAD, CRAC activation domain; SOAR, STIM-Orai
activating region; ID, inactivation domain; P/S, proline-serine rich domain; EB, EB1 binding domain; PBD,
polybasic domain). Colored lines mark the sites of mutations that activate STIM (yellow), inhibit STIM (red), alter
CDI (orange), prevent binding to EB1 (purple), and inhibit SOCE during mitosis (blue). Note that clusters of
gain-of-function mutations define regions (EF hands and CC1) that help maintain STIM in the inactive state, while
clusters of loss-of-function mutations identify a region (CC2) that is proposed to interact with Orai. The sites of
alternatively spliced insertions for STIM1 and STIM2 and human mutations E136X (premature stop) and
R429C are shown. Closed circles show glycosylation sites at N131, N171 for STIM1 (409) and N135 for
STIM2. The effects of the STIM1 mutations and references are listed in TABLES 1–3. Sequences are indicated
for selected regions with known functions; see text for details. Domains are drawn to scale, and residue
numbering for STIM2 is based on the protein without the long signal peptide insertion.
the CAD/SOAR domain will be referred to as the CAD
domain throughout this review.
F. Identification of Orai Proteins as
Store-Operated Ca2ⴙ Channels
Soon after the emergence of STIM proteins as SOCE Ca2⫹
sensors, several groups independently converged on the
CRAC channel gene using RNAi approaches and human
genetic linkage analysis. Beginning in the 1990s, several
human patients were identified with severe combined immunodeficiency (SCID) that was attributable to the absence
of CRAC channel function (102, 104, 286). Rao, Feske and
colleagues (103) used modified linkage analysis and positional cloning to localize the mutated gene to a region of
chromosome 12 covering ⬃74 genes (103). In the same lab,
a genome-wide RNAi screen in S2 cells for inhibition of
nuclear translocation of NFAT (a marker for sustained
1388
SOCE) yielded a number of hits, one of which (olf186-F)
had a homolog in the region of human chromosome 12
identified by the linkage analysis. This molecule, a widely
expressed and previously uncharacterized 33-kDa cell surface protein with four predicted transmembrane domains
and intracellular NH2 and COOH termini, was named
Orai1. At the same time, the Kinet and Cahalan groups
(398, 446), conducting their own screens of the S2 RNAi
library for suppression of SOCE, identified the same
olf186-F gene in S2 cells, and Vig et al. (398) named the
closest mammalian homolog CRACM1 (CRAC modulator 1). In all, three Orai homologs (Orai1-3, or
CRACM1-3) were identified in the human genome. Although all three proteins are highly homologous to each
other (⬃62% overall identity, increasing to ⬃92% in the
transmembrane domains), they exhibit negligible sequence similarity to other ion channels, which likely explains the failure of earlier homology-based searches to
identify them. An overview of the transmembrane topol-
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CAD/SOAR
STORE-OPERATED CALCIUM CHANNELS
ogy and functional domains of the Orai family members
is depicted in FIGURE 3.
Initial studies quickly linked Orai1 and Drosophila Orai
(dOrai) to CRAC channel function. First, the SCID patients
were found to be homozygous for a single missense mutation in Orai1 (R91W) that abrogated CRAC channel activity, and transformation of the patient’s T cells with the
wild-type Orai1 gene rescued SOCE and ICRAC (103).
Knockdown of dOrai suppressed the endogenous CRAC
current in S2 cells (446). Overexpression of Orai1 with
STIM1 in HEK293 cells generated store-operated currents
up to ⬎100 times larger than endogenous ICRAC that displayed the ion selectivity, pore dimensions, and pharmacological sensitivity of native CRAC channels (239, 291, 358,
420, 446). These results suggested that STIM and Orai
alone may be sufficient to reconstitute the basic SOCE
mechanism, an idea that was later supported by in vitro
reconstitution studies (453). However, one should note that
these studies were conducted with massively overexpressed STIM and Orai; at the much lower expression
levels typical of native cells, STIM and Orai function may
be regulated in important ways by other proteins (242,
363, 364) (discussed below). An interesting footnote is
that overexpression of Orai1 alone actually suppresses
endogenous SOCE in many cells (147, 206, 239, 291,
358) by reducing the STIM1:Orai1 binding stoichiometry (147). Thus, in retrospect, it was fortuitous that
STIM1 was discovered before Orai1; otherwise, Orai1
might have been identified as a CRAC channel suppressor
based on effects of overexpression alone.
While the overexpression and knockdown studies as well as
the inhibitory effect of the R91W mutation suggested that
Orai1 could be the CRAC channel structural gene, they did
not exclude the possibility that it merely encoded an accessory protein required for channel opening. The known role
of glutamate side chains in tuning the selectivity of Ca2⫹selective channels (334) suggested a way to distinguish these
possibilities. Several groups soon reported that mutagenesis
of highly conserved acidic residues in the transmembrance
domains of hOrai1 and dOrai significantly reduced the
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FIGURE 3. Functional organization of Orai1, 2, and 3. The topology of Orai1 is illustrated, with selected
functional domains noted by colored bars or circles. Orai1-3 protein sequences are highly conserved in the 4
TM regions. Sequences for Orai1-3 are compared in the NH2-terminal region, the I–II and II–III loops, and the
COOH terminus. The thick orange bars in the NH2 and COOH termini depict putative STIM1 binding sites on
Orai1. Yellow and red lines show the locations of mutations causing gain-of-function (i.e., STIM1-independent
Orai1 activation) or loss-of-function phenotypes, respectively. Black lines indicate mutations affecting ion
selectivity. Intracellular residues important for CDI are marked in purple (NH2 terminus and II–III loop); selected
residues important for STIM binding and gating are marked in red (NH2 and COOH termini). On the extracellular
side, charged residues in the I–II loop are black, and the target of redox inhibition (C195) and the glycosylation
site of Orai1 (N223) are shown in blue (both in III–IV loop). The effects of the Orai1 mutations and references
are listed in TABLES 1–3.
MURALI PRAKRIYA AND RICHARD S. LEWIS
Ca2⫹ selectivity and lanthanide sensitivity of CRAC channels or blocked conduction (297, 396, 429) (FIGURE 3; discussed in sec. V). Just as mutagenesis of the EF hand helped
establish STIM1 as a Ca2⫹ sensor for SOCE, the effects of
altering acidic residues on selectivity and block quickly led
to the acceptance of Orai1 as a key component of the CRAC
channel pore.
It is important to note that identification of the CRAC channels and Orai proteins as store-operated does not preclude
other channels from functioning in a similar way. For example, several TRPC channels have been proposed to act as
SOCs (3, 203, 413), although this assignment continues to
be debated (53, 58, 74). The most compelling argument in
favor of TRPCs as SOCs comes from charge-swapping experiments by Muallem and colleagues in which charge reversal of either the last two lysines in STIM1 (K684/K685)
or two key aspartate residues in TRPC1 (D639/D640) abrogated TRPC1 activity, while the two mutants expressed
together restored activity (189, 441). On this basis, Zeng et
al. (441) proposed an electrostatic model in which positive
and negative charges on either protein must interact for
STIM1 to activate TRPC1. Despite this evidence, it has been
difficult to build a widely accepted case for TRPCs as SOCs,
largely due to a number of confounding factors (53, 125).
TRPC channels respond to diverse stimuli, including DAG,
G proteins, Ca2⫹, and redox compounds, and several of the
TRPC proteins form heteromultimers with other TRPC
members which can alter their preferred mode of activation
(434a). The complexities introduced by heteromultimer
formation have been put forward to explain the inconsistent conclusions among laboratories regarding TRPC
1390
G. Evolution, Tissue Distribution, and
Diversity of STIM Isoforms
STIM proteins are highly conserved in metazoans from C.
elegans and Drosophila to Homo sapiens. STIM homologs
are not found in lower organisms including yeast and Dictyostelium, and first appeared during evolution with the
choanoflagellates (Monosiga brevicollis) (61). While the
Ca2⫹-sensing NH2-terminal regions of STIM proteins
are fairly well conserved from worms and flies to humans,
there is wide divergence in the cytosolic domains. Invertebrate STIM lacks several domains COOH-terminal to the
coiled-coil domains, including the proline-rich and polybasic domains. A phylogenetic analysis revealed that STIM
may have evolved in two stages from invertebrate to vertebrate forms (37). The missing COOH-terminal domains
first appeared in the primordial vertebrate form of STIM
expressed in urochordates (sea squirts). Subsequently, a
gene duplication event that is thought to have occurred at
the start of the vertebrate Euteleostomi lineage created the
STIM1 and STIM2 isoforms. Interestingly, a second duplication in bony fishes generated four divergent STIM homologs that may have evolved to carry out additional functions (37). The presence of specific regulatory domains in
STIM proteins varies according to species and protein isoform; although in most cases the consequences of this variation are unknown, the differences between STIM1 and
STIM2 are believed to create distinct but overlapping functions as described below.
In mammals, STIM proteins are broadly expressed in many
organs and tissues. Northern blot analysis in human tissue
samples reveals robust expression of STIM1 and STIM2
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While Orai1 is most closely associated with the endogenous
CRAC channel of mast cells and T cells, all three Orai
homologs produce Ca2⫹-selective store-operated channels
when coexpressed with STIM1 in HEK293 cells (72, 126,
133, 200, 239), although differences in ion selectivity, inactivation, depotentiation, pharmacology, affinity for STIM1,
redox sensitivity, and activation by STIM2 have been reported (29, 72, 109, 190, 200) (discussed below). Overall,
Orai1 is the best characterized of these proteins and has
proven the most popular for structure-function studies of
SOCs due to its close similarity to the single Orai proteins
found in Drosophila and C. elegans, well-established functional links with endogenous CRAC channels and effector
functions in many immune cells including human T cells, as
well as its relatively high expression level in heterologous
systems. Despite the ability of homomeric Orai proteins to
form SOCs, it is noteworthy that Orai1 and Orai3 proteins
may also assemble as heteromultimers to form STIM1-dependent but store-independent Ca2⫹ channels that are regulated by arachidonic acid (ARC channels) (245, 247, 386)
or leukotriene C4 (LRC channels) (120). In this way, heteromultimeric assembly may serve to increase the functional diversity of the Orai channel family.
store dependence (53). Added to this is the complication
that many studies use agonists of PLC-linked receptors to
activate TRPC channels, making it difficult to separate
the stimulatory effects of biochemical pathways from
those of [Ca2⫹]ER. As a result, there is as yet no definitive
measurement of the dependence of TRPC activity on
[Ca2⫹]ER, or on store depletion alone without parallel
activation of receptors and associated biochemical pathways. A final complication is that TRPC channel activity
in some contexts also depends on Orai1 activity, which
has been attributed by Ambudkar and colleagues to the
insertion of TRPC channels into the PM in response to
Ca2⫹ influx through Orai1 (48) but may also involve
activation by Ca2⫹ entering through Orai1 (125). A complex picture is emerging in which the behavior of TRPC
channels in a particular cell may depend on the relative
amounts and accessibility of STIM1, Orai1, and the various TRPC homologs. The difficulty of measuring and
controlling these parameters poses persistent challenges
to achieving wider acceptance of the function of TRPCs
as SOCs in a physiological as opposed to an experimental
setting (53, 58, 74).
STORE-OPERATED CALCIUM CHANNELS
The diversity of STIM protein function is further increased by
alternative splicing. An alternatively spliced long variant of
STIM1 (STIM1L) has been described in which an extra 106
residues are inserted in the STIM1 cytosolic domain (FIGURE
2) (69). STIM1L is expressed in several tissues but most prominently in muscle, and its abundance increases during myotube
differentiation. The insert in STIM1L is an actin-binding domain and leads to altered localization and activation kinetics
(69). Two recent reports describe a STIM2 splice variant
(STIM2␤, or STIM2.1) in which a highly conserved set of
eight residues is inserted in the CAD domain (FIGURE 2), converting it into the first known inhibitory STIM protein (243,
315). STIM2␤ cannot bind appreciably to Orai1 by itself, but
is recruited to Orai1 by forming heterodimers with STIM1 or
STIM2, and inhibits SOCE in part through sequence-specific,
direct inhibition of Orai channel function (315). Considering
⬎95% of all multiexon loci in vertebrates are alternatively
spliced to greatly expand the functional diversity of a limited
number of genes, more STIM splice variants are to be expected.
H. Evolution, Tissue Distribution, and
Diversity of Orai Isoforms
Orai proteins are expressed in metazoans from nematodes
to primates (38). Orai homologs first appeared in evolution
with nematodes and insects, but proteins with distant homology to Orai are found even in organisms such as green
algae and moss that do not express recognizable STIM proteins, suggesting activation through other mechanisms (61).
Invertebrates express a single Orai isoform (38, 61). The
first appearance of two Orai homologs is associated with
vertebrates, and Orai3 appears even later, only in mammals, and appears to have evolved from Orai1 from a gene
duplication event. Thus, in evolutionary terms, Orai1 is the
oldest family member, and Orai3 the newest (347). Orai3
has a significantly longer III–IV loop than Orai1 and little
sequence similarity in this region, as well as a shorter
COOH terminus that exhibits a greater coiled-coil probability than the COOH terminus of Orai1 (109).
Evidence from a variety of techniques (Northern blot, Western blot, rtPCR, immunohistochemistry) indicates broad
tissue expression of Orai transcripts or proteins in mammals (126, 132, 133, 411). A monoclonal antibody that
specifically targets the second extracellular loop of Orai1
has provided evidence that rodents and primates have similar expression patterns of Orai1 except in male gonad tissue (132). In addition to immune cells such as macrophages
and lymphocytes, significant expression of Orai1 was also
reported in the brain, pancreas, kidney, and skin. Genetic
studies in humans and knockout studies in mice complement these expression profiles and confirm an important
role for Orai1 in a variety of immune cells (T cells, mast
cells, B cells, NK cells) as well as nonimmune cells (fibroblasts, skeletal muscle, platelets, microglia, endothelial
cells, smooth muscle, hepatocytes, and neural stem cells)
(12, 34, 103, 133, 233, 269, 360, 373, 393). The expression
of Orai1 in the nervous system, especially in the cortex and
hippocampus is noteworthy and raises the prospect of important functions in the brain (see sect. VIII). Comparable
expression studies of Orai2 and Orai3 are lacking, due to
the scarcity of high quality antibodies that can distinguish
among the Orai isoforms. However, from analysis of RNA
levels and ability of the small molecule 2-aminodiphenylborate (2-APB) to stimulate Orai3 channel activity (see sect.
VII), some reports have indicated that Orai3 expression is
enhanced in cancerous cells and promotes tumorigenesis
(252, 253). These differences in expression may have important implications for cell function as well as development of isoform-specific drugs for therapeutic applications.
Expression studies have revealed that the three mammalian
Orai homologs are often present in overlapping patterns in
different tissues, including but not limited to the brain, the
lung, and immune cells (126, 133, 411). This begets the question of whether there exist heteromultimeric channels, and if
so, what are their unique functional features? Recombinant
overexpressed Orai homologs clearly interact with each other,
as shown by coimmunoprecipitation experiments and the ability of a nonconducting mutant of Orai1 to act as a dominant
negative for all three Orais (200). The functional diversity of
multimeric Orai channels, however, remains largely unknown. In one study, coexpression of Orai1 and Orai3 channels yielded store-operated currents with reduced Ca2⫹ selectivity compared with homomeric Orai1 or Orai3 channels
(336; but see Refs. 245, 247). The reduction in Ca2⫹ selectivity
was attributed to differences in acidic residues in the first extracellular loop of Orai1 and Orai3 (E85 in Orai3 vs. D110 in
Orai1). However, Orai1 and Orai3 channels also differ in
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RNA in pancreas, skeletal muscle, brain, and heart as well
as modest levels in liver and lung (407). Expression analysis
with affinity-purified antibodies confirms the presence of
both STIM1 and STIM2 in a wide variety of human cells
and cell lines (223, 407). Additionally, a STIM1-LacZ fusion protein is strongly expressed in all types of muscle as
well as cerebellum, spleen, and thymus (373). There is also
evidence that the expression may be developmentally regulated, as STIM1-LacZ expression increases significantly
during myotube differentiation and correlates with increased SOC activity (373). Coexpression of STIM1 and
STIM2 in the same tissues and cells raises the possibility
of heteromeric association. Indeed, immunoprecipitation
analyses of endogenous proteins in cell lines expressing
both isoforms as well as FRET between labeled STIM1 and
STIM2 indicates that they can form heterooligomers in situ
(70, 315, 407). Given differences in the response of the two
isoforms to Ca2⫹ store depletion and their ability to activate
CRAC channels (32, 402), heteromerization of these proteins may have important implications for the amplitude
and dynamics of SOCE-mediated Ca2⫹ signals (270).
MURALI PRAKRIYA AND RICHARD S. LEWIS
tions (272, 414) where the ER is held close enough to the
PM that STIM1 can bind to Orai1, trapping and opening
the channel to trigger Ca2⫹ entry (FIGURE 4).
While alternatively spliced isoforms of Orai proteins have
not yet been described, a short form of Orai1 is generated
by translation initiation at a second site (M64/71) (113).
Both forms are expressed in most human cell lines, and with
heterologous expression the relative amount of each is determined by the strength of the Kozak sequence in the expression vector. The shorter form lacks a polybasic sequence that may interact with negatively charged phospholipids and appears to diffuse faster than its “full-length”
counterpart, although the functional consequences of this
are not known (113).
SOCE is terminated following the reuptake of Ca2⫹ by
sarco/endoplasmic reticulum Ca2⫹-ATPases (SERCA
pumps) which may achieve high efficiency by accumulating at or near ER-PM junctions (2, 163, 221, 222, 332).
Upon store refilling, STIM1 and Orai1 revert to their
diffuse distributions (199, 219, 256, 332, 354). Interestingly, one study suggests that ER refilling by itself may
not be sufficient for reversing the SOCE process and may
require Ca2⫹ entry through Orai1 into the cytosol in
addition to the rebinding of luminal Ca2⫹ to the STIM1
EF hand (344) (see also Ref. 219). The Ca2⫹ sensor for
this process is not known, but SARAF (274), CRACR2A
(364), and CaM bound to the PBD (17) or other parts of
STIM1 (111) are possible candidates.
III. MOLECULAR CHOREOGRAPHY OF
STORE-OPERATED CALCIUM CHANNELS
A. Overview
The activation process of CRAC channels is highly unusual
in that it involves contact between membrane proteins located in two different cell compartments, the ER and the
PM. In most cells, 75– 85% of endogenously expressed
STIM1 resides in the ER membrane, with the remainder in
the PM (223, 244). Early studies were divided on the issue
of whether STIM1 must translocate to the PM to evoke
SOCE (142, 208, 362, 443) or whether it functions within
the ER membrane (199, 239, 414, 418). This debate was
eventually settled by the demonstration that labels attached
to the NH2 terminus of STIM1 block transport to the PM
(137) yet do not prevent robust generation of SOCE in
normal cells and reconstitution of SOCE in STIM1-deficient cells (11; see also Ref. 244). Thus ER-localized STIM1
appears to be sufficient for the activation of SOCE. In contrast, PM-localized STIM1 is thought to be required for
activation of store-independent, arachidonate-regulated
Ca2⫹ (ARC) channels through a constitutive association
with the Orai3 subunit of the channel (244, 386). However,
a recent report shows that this is true during whole cell but
not perforated-patch recording, suggesting that ER-localized STIM1 may be sufficient for ARC channel activation
under physiological conditions (447).
Activation of SOCE by store depletion is a highly dynamic
process requiring the redistribution of STIM and Orai in the
cell. In resting cells with replete Ca2⫹ stores, fluorescently
labeled STIM1 and Orai1 are mobile and diffusely distributed throughout the ER and plasma membrane, respectively (FIGURE 4) (11, 64, 113, 198, 199, 284, 416, 443).
Within seconds to minutes after depletion of ER Ca2⫹,
STIM1 and Orai1 redistribute within their respective membranes and coaccumulate in clusters visualized as “puncta”
of fluorescently labeled proteins (11, 199, 210, 239, 414,
418, 443). The sites of coaccumulation are ER-PM junc-
1392
It is interesting to note that while the choreographic process
underlying SOCE is unique, functional coupling between
Ca2⫹ signaling proteins in the PM and internal organelles is
not without precedent. In the triadic junctions of skeletal
muscle, depolarization-activated CaV channels (DHPR) in
the PM bind directly to ryanodine receptors (RyR) in the
sarcoplasmic reticulum (SR) to trigger Ca2⫹ release, a process called excitation-contraction (e-c) coupling. While
SOCE and e-c coupling are similar in terms of signal transmission by protein contacts across a narrow 10- to 20-nm
gap, they differ in that signal propagation during e-c coupling is outside-in rather than inside-out, and RyR-DHPR
complexes in muscle are preassembled while the SOCE
complexes form only “on demand,” i.e., after ER Ca2⫹
depletion. Accordingly, SOCE in most cells develops over
seconds to tens of seconds, being limited by the time it takes
STIM1 and Orai1 to accumulate by passive diffusion, as
well as possible delays in binding and activation (43, 414).
In contrast, SR Ca2⫹ release occurs within milliseconds of
PM depolarization, being limited only by the kinetics of
protein conformational changes and thereby ensuring the
precision, reliability, and speed of e-c coupling (83, 323).
Interestingly, STIM1 and Orai1 in skeletal muscle appear to
be prelocalized at the triad (373, 406), and SOCE is reported to activate within milliseconds of SR Ca2⫹ depletion
(91, 187), most likely because of the removal of protein
diffusional delays. The underlying mechanisms that prelocalize STIM1 and Orai1 to these sites are not well understood.
B. The ER-PM Junction: the Nexus of StoreOperated Calcium Entry
Initial light microscopy studies showed that upon store depletion YFP-STIM1 puncta formed near the PM (within
⬃200 nm, the length constant of the TIRF evanescent field)
(199). Subsequent electron microscopic localization of a
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their COOH termini, which can affect the strength of STIM
binding (109), potentially generating secondary effects on ion
selectivity (237).
STORE-OPERATED CALCIUM CHANNELS
A
mCherry-STIM1
D
GFP-Orai1
Extracellular
Orai1
Stores full
PM
Cytosol
ER
10 μm
STIM1
Ca2+
Stores depleted
Store depletion
B
C
Ca2+ entry
ER
2 μm
ER
mCh-STIM1
ER
200 nm
FIGURE 4. STIM1 and Orai1 accumulate at ER-PM junctions to form the elementary unit of SOCE. A:
mCherry-STIM1 and GFP-Orai1 are distributed throughout the ER and PM of a resting HEK293 cell (top).
Several minutes after store depletion with TG, the two proteins have redistributed and are colocalized in puncta
(bottom) seen in confocal images of the cell footprint. B: at the ultrastructural level, puncta correspond to
ER-PM junctions, visualized in this transmission EM image by the accumulation of HRP-STIM1 in a Jurkat T cell
after store depletion (left). C: fluo-5F imaging reveals hotspots of Ca2⫹ influx that colocalize precisely with
STIM1 puncta. [Images adapted from Luik et al. (210) and Wu et al. (414).] D: diagram depicting the events
in A–C, leading from store depletion to coclustering of STIM1 and Orai1 at junctions, to opening of Orai1
channels and localized Ca2⫹ entry. Formation of new junctions is indicated in the middle panel.
STIM1-horseradish peroxidase chimera (HRP-STIM1) indicated that in store-depleted cells STIM1 accumulates at
ER-PM junctions that correspond to the puncta visible at
the light microscopic level (FIGURE 4) (414). Together with
observations that STIM1 and Orai1 are colocalized in
puncta and that Ca2⫹ entry is precisely restricted to these
sites, these studies defined the elementary unit of SOCE to
be clusters of closely apposed STIM1 and Orai1 at ER-PM
junctions (210). EM studies in different cells are in general
agreement that STIM1 accumulates at sites where the average ER-PM gap ranges from 10 to 17 nm (212, 272, 414). A
detailed immunoelectron microscopic analysis by Orci et al.
(272) delineated different types of junctional ER, including
“cortical” (cER) and “thin cortical” structures where the
tubules narrow from a diameter of ⬃70 to ⬃25 nm and
exclude KDEL-containing ER proteins. In this study, store
depletion caused STIM1 accumulation only in thin cER,
whereas in the initial work by Wu et al. (414), cER containing HRP-STIM1 appeared similar to cER visualized with an
ER-targeted, KDEL-containing HRP. The basis for this dif-
ference and possible functional distinctions betweeen cER
and thin cER are not yet understood.
The ability of STIM1 to return to the same sites after multiple rounds of depletion and refilling suggests that ER-PM
junctions are stable over minutes, at least at room temperature (199, 354). In general agreement, EM studies have
reported that the majority of cortical ER-PM junctions preexist in resting cells, but that their number increases to
varying extents after store depletion (272, 414). This increase is not due to a bulk translocation of ER towards the
PM (414) but is instead likely to involve movements of
individual tubules. Junction stability and the store dependence of junction abundance may vary among cells, as polymerization of cortical actin, which is thought to interfere
with junction formation, inhibits SOCE in some cells but
not others (210, 289).
The proteins involved in creating and maintaining ER-PM
junctions are a subject of intense interest (306). STIM1 itself
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ER
MURALI PRAKRIYA AND RICHARD S. LEWIS
Another promising candidate for enabling ER-PM junction
formation is the extended synaptotagmin family (45, 119).
These ER membrane proteins localize to ER-PM junctions
and interact via C2 domains with PIP2 in the PM (119).
Interestingly, the E-Syts may link Ca2⫹ signaling and PIP2
homeostasis, as a local rise in [Ca2⫹]i appears to recruit
E-Syt1 to junctions (119) where it pulls the ER closer to the
PM and recruits Nir2, a phosphatidylinositol transfer protein which helps replenish PIP2 in the PM (45). Surprisingly,
while knockdown of all three E-Syts greatly reduced the
number of junctions in HeLa cells, it had little effect on
SOCE, raising the possibility that E-Syts may be more directly involved in controlling lipid transfer than Ca2⫹ entry
(119). The ER membrane protein junctate is a third candidate for promoting ER-PM contacts, as it localizes to junctions, and junctate overexpression increases the number
and sizes of junctions (388, 389) while knockdown reduces
SOCE (363, 388). Junctate is also reported to have a luminal EF hand which senses [Ca2⫹]ER and may enhance the
accumulation of STIM1 at junctions after store depletion by
binding to the luminal STIM1 domain (363).
Further studies and approaches will be needed to positively
identify the mechanisms involved in ER-PM junction formation and turnover. Unfortunately, existing tools for
studying junctional dynamics in living cells are quite limited; all of the current marking methods involve probes that
interact in some way with both the ER and PM and therefore perturb junctional stability. This caveat applies to
STIM1 as well as a rapamycin-inducible heterodimerizer
system that irreversibly links the ER and PM (394). A recent
study described an ER membrane protein engineered to
constitutively localize to junctions through binding of a
1394
COOH-terminal polybasic domain to the PM, much like
STIM1/2, but without interacting with STIMs (45). The
general challenge in applying such tools to study junction
dynamics will be to reduce expression to a low enough level
that they do not affect junctional stability or dynamics yet
generate enough signal to illuminate junctional contacts
with high sensitivity.
C. STIM Activation and Accumulation With
Orai at ER-PM Junctions
The maintenance of ER Ca2⫹ homeostasis and generation
of receptor-activated SOCE signals depends critically on the
precise control of STIM protein activity. In cells with replete
Ca2⫹ stores, STIM1 must be kept in an inactive state and
diffusely localized throughout the ER, yet after ER Ca2⫹
depletion it must effectively adopt an active conformation
and become highly concentrated at ER-PM junctions where
it can interact with Orai1 to generate SOCE. Many of the
critical events in this process are becoming understood and
support a diffusion trap mechanism that commences with
Ca2⫹ release from STIM1, followed by oligomerization and
conformational changes that trap STIM1 at junctions and
enable it to bind and trap Orai1. This section reviews the
current understanding of how these events are controlled at
a molecular level.
1. The resting (inactive) state of STIM: structural
basis and mobility
Multiple lines of evidence suggest that STIM1 is a dimer in
resting cells with full Ca2⫹ stores. STIM1 coimmunoprecipitates with itself through interactions involving the cytoplasmic region (11, 408), and isolated fragments of the
cytosolic domain generally form dimers in solution (154,
254, 453, 455). Although the CC1 domain by itself can
support some dimerization, these oligomers are unstable
(64) as CC1-CC1 interactions are inherently weak (see also
Ref. 455). The CAD domain appears to play the dominant
role in determining the dimeric stoichiometry of STIM1, as
STIM1-CAD (residues 1– 448) is a stable dimer in situ (64),
and cytosolic fragments containing CC1⫹CAD are dimeric
in solution (254, 455).
The physical basis for the dimerizing ability of CAD is not
yet clear. A crystal structure of human CAD (residues 345–
444) reveals several reciprocal hydrophobic and hydrogen
bond interactions between the two CAD monomers which
have been proposed to hold the dimer together (FIGURE 5, D
AND E) (424). Alanine substitutions at these locations prevented STIM1 accumulation in puncta and interactions
with Orai1 (424), but the effects on STIM1 dimerization
need to be tested directly. The structure also revealed extensive interactions between the CC2 and CC3 domains within
each monomer that are expected to stabilize the closed
structure of the protein (FIGURE 5D).
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could be involved through multiple mechanisms. STIM1 is
known to bind to the microtubule (MT) tip attachment
protein EB1 through its TRIP sequence (FIGURE 2), and
STIM1 knockdown reduces the rate of ER tubule formation, suggesting that endogenous STIM1 regulates the formation and extension of new ER tubules (123). In this way,
STIM1 could promote junction formation by enabling MTs
to drag nascent ER tubules towards the PM. Such a role
would be consistent with observations of microtubules
closely associated with precortical and cortical ER (272,
345). STIM proteins could also promote junction formation through interactions of their COOH-terminal polybasic domains with the PM. Overexpression of STIM1 is well
known to increase the spatial extent of junctions; with high
enough expression, this extent can increase from a baseline
level of a few percent of the cell surface to cover the entire
footprint of the cell (272, 394, 414). STIM2 may also play
a role (94) as it has a higher affinity for PIP2 (26) and is
prelocalized at junctions in resting cells (32). Thus STIM
proteins could potentially function both in the extension of
ER towards the PM and in promoting adhesion forces to
help stabilize ER-PM junctions.
STORE-OPERATED CALCIUM CHANNELS
A
B
Ca2+
Extracellular
PIP2
PIP2
Cytosol
Orai1
STIM1
Start
CAD
CC1
End
PBD
1 μm
SAM
EF
ER lumen
Ca2+
STIM1
trapping
Orai1
trapping
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Resting
Extended
state Store
state
depletion
C
D
CC2
F394
SAM
E
CC2’
CC3’
K386
L436
K385
K382
K384
W350
A380
90°
α10
L347
A369
nEF
L416
α10
L423
cEF
D76
I433
R429
T354
W430
L351
CC2
Y362
CC3
CC3’
N80
D78 D87
C
N
FIGURE 5. A diffusion trap model for SOCE. A: a possible model of the events leading from store depletion
to the trapping and activation of STIM and Orai. At far left, STIM1 is in its resting state bound to Ca2⫹ and freely
diffusing in the ER membrane. A single dimer is shown, with the conformation of CAD borrowed from the
crystal structure of Yang et al. (424) (shown in D). Colors of CC1, CC2, and CC3 match those in FIGURES 5D
AND 7A. Store depletion and Ca2⫹ unbinding from the EF hand initiates a conformational change of the
cytosolic domains that allows the PBD to bind to PIP2 in the PM, trapping STIM1 at the ER-PM junction. In
addition, CC2 helices in STIM1 move into an antiparallel configuration, and the CC3 helix disengages from CC2 to
create a binding interface for the Orai1 COOH termini (based on results of Ref. 368, detailed in FIGURE 7; the CC3
helix has been foreshortened to simplify the display). STIM1 binding traps and activates the Orai1 channel. Only a
single dimer is shown for clarity; higher-order STIM1 oligomers are thought to form after store depletion, and
binding of multiple STIM1 dimers to Orai1 hexamers is required for full activity (see text). B: the trajectory of a single
GFP-labeled Orai1 channel (yellow) is shown as it diffuses freely in the plasma membrane of a HEK cell with depleted
Ca2⫹ stores, becomes trapped at an ER-plasma membrane junction marked by mCherry-STIM1 (red), and after
⬃15 s escapes the junction. [From Wu et al. (416).] C: solution NMR structure of the human STIM1 EF-SAM
domain (2K60.pdb) (370). Colors distinguish the 3 functional domains (cEF, violet; nEF, orange; SAM, green).
Bound Ca2⫹ (yellow) is coordinated by interactions with the indicated cEF side chains. The complex is tightly packed
with the ␣10 helix of SAM nestled in a hydrophobic groove created by the paired EF hands. L195 and L199 of SAM
are seen descending from the helix in the structure on the right. D: crystal structure of the human CAD domain
(amino acids 345– 444 with mutations L374M, V419A, and C437T; 3TEQ.pdb) (424). Each of the two monomers
consists of CC2 (teal) and CC3 (green) domains linked by a pair of short helices (gray). NH2 and COOH termini are
indicated for the monomer in front. Labeled residues where mutations promote STIM and Orai activation are shown
in yellow, and those where mutations inhibit STIM and Orai function are in red. The magnified structure at right,
viewed from the base, shows the hydrophobic and hydrogen bond interactions between CC2 and CC3 of the
neighboring monomer that have been proposed to stabilize the CAD dimer (424).
The structure of dimeric full-length STIM1 in its resting
inactive state is not known. However, the conformation of
the luminal EF hand and SAM domains and how it regulates the stability of the resting state is relatively well under-
stood. In the presence of Ca2⫹, the isolated EF-SAM domain in solution is a relatively compact and stable monomer
(367). Based on NMR solution structures of Ca2⫹-bound
EF-SAM fragments from STIM1 and STIM2, the EF-SAM
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MURALI PRAKRIYA AND RICHARD S. LEWIS
domain is predominantly ␣-helical and consists of a canonical EF hand (cEF) with a typical helix-loop-helix structure
that binds one Ca2⫹ ion between loops 1 and 2 (amino acids
73– 88 in STIM1, amino acids 87–103 in STIM2); a noncanonical EF hand (nEF) that does not bind Ca2⫹ (but which
helps stabilize the canonical EF hand through hydrogen
bonding); and the SAM domain (FIGURE 5C) (370, 450).
With Ca2⫹ bound in the resting state, the two EF hands of
each monomer are in the “open” conformation, creating a
hydrophobic cleft which binds hydrophobic residues extending from the SAM domain to stabilize the compact
monomeric structure (FIGURE 5C).
Mobility of STIM1 in cells has been studied using fluorescence recovery after photobleaching (FRAP) as well as single-particle tracking. FRAP studies suggest that STIM1 in
cells with full Ca2⫹ stores diffuses passively within the ER
membrane at a slow rate (⬃0.1 ␮m2/s) compared with
many single-pass ER membrane proteins (64, 198). Its
speed is greatly affected by the cytosolic domain; while
STIM1-⌬C (STIM1 amino acids 1–237) diffuses at a similar
speed as many other ER membrane proteins, the addition of
1396
A second mode of STIM1 transport arises from association
with MTs. This mode is particularly evident when fluorescently tagged STIM1 is overexpressed, generating a striking
pattern of fluorescent “comets” that radiate in relatively
linear paths from a central site, presumably the microtubule
organizing center (11, 198, 415). This behavior has been
attributed to STIM1 binding via its TRIP sequence to endbinding (EB) proteins that associate with the plus ends of
microtubules (123) (FIGURE 2A). A combination of immunohistochemistry, coimmunoprecipitation, biochemistry,
and in vitro studies of purified proteins indicates that
STIM1 associates with EB1 and EB3 at MT tips (123, 146).
While STIM1 moves both along existing microtubules and
with the growing plus-end of microtubules, only the association with MT ends is enhanced by STIM overexpression.
Although this mode of MT-associated transport was initially thought to be involved in the delivery of STIM1 to
ER-PM junctions (11), later work deemed this unlikely, as
knockdown of EB1 or stabilization of microtubules with
taxol failed to affect puncta formation or SOCE (123). In
addition, single-particle tracking of GFP-STIM1 failed to
find evidence for active transport, suggesting that MTs
make a negligible contribution to STIM1 transport at endogenous levels of expression (416).
2. Sensing of ER Ca2⫹ depletion by STIM:
conformational changes and oligomerization
Activation of STIM proteins is initiated by the release of
Ca2⫹ from the luminal cEF hand, which triggers unfolding
of the EF-SAM domain and conformational changes in both
luminal and cytosolic domains. The KD for Ca2⫹ binding of
the isolated EF-SAM domains of STIM1 and STIM2 in
solution are ⬃200 and ⬃500 ␮M, respectively (450). Structural and biochemical studies of the isolated STIM1 EFSAM domain show that unfolding of the EF-SAM domain
exposes hydrophobic surfaces that promote the formation
of dimers and higher order aggregates in solution (367).
These findings led Ikura, Stathopulos, and colleagues (367)
to make the initial proposal that oligomerization of the
EF-SAM domains initiates the STIM activation process, an
idea supported later by observations that mutations designed to destabilize interactions of the EF hands with the
SAM were shown to create dimers or aggregates of the
peptide in solution and promote puncta formation and
SOCE when introduced into full-length STIM1 (370) (TA2⫹
BLE 1). The monomeric Ca -bound EF-SAM domain may
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STIM1 and STIM2 differ in their functional properties, and
these differences may be traced in large part to differential
stability of their resting states. Unlike STIM1, STIM2 is
partially active in resting cells, attributable to its lower overall affinity for Ca2⫹ (32). Its lower Ca2⫹ affinity allows
STIM2 to respond to changes of [Ca2⫹]ER around the resting level and engage in homeostatic functions, whereas
STIM1 responds only to the larger extent of store depletion
generally resulting from stimulation of PM receptors (32,
209). A series of cEF and SAM chimeras (450) showed that
for STIM1 a higher affinity cEF hand is paired with a relatively less stable SAM domain (producing weaker cEF-SAM
binding). In contrast, the cEF hand of STIM2 has a lower
affinity for Ca2⫹, but increased hydrophobic and electrostatic interactions make its EF-SAM domain more stable.
Interestingly, these properties create metastable states, such
that STIM1 responds only to larger degrees of store depletion but does so rapidly, while STIM2 responds to smaller
changes in [Ca2⫹]ER but in a slower, more damped fashion.
These characteristics appear to be well matched to the agonist response versus homeostatic roles of the two proteins
(369, 450). The poorly conserved random coil STIM sequences NH2-terminal to the EF-SAM domain also enhance
the stability of STIM1 and STIM2 resting conformations
(369). Chimeric swap experiments show that the NH2-terminal region of STIM2 reduces the speed and extent of
CRAC channel activation relative to that of STIM1 (452).
Thus, together with the EF-SAM domain, the NH2 terminus acts to dampen activation of SOCE by STIM2. These
results raise the possibility that large variations observed in
the sequence and length of the NH2-terminal regions of
STIM from different species may generate species-specific
differences in the behavior and functions of STIM proteins.
CC1 (amino acids 1–344) or the entire cytosolic domain
(amino acids 1– 685) slows it down by approximately twofold (64). These results suggest that STIM proteins are
slowed significantly by interactions of their cytosolic domains with elements of the cytosolic environment. These
ensemble measurements have been recently extended by
single-particle tracking methods, which confirm that
STIM1 moves by Brownian diffusion in the resting state
(416).
STORE-OPERATED CALCIUM CHANNELS
Table 1. Mutations that activate STIM1 or Orai1 independently of store depletion
Mutation
Reference Nos.
STIM1 cEF hand
STIM1 nEF hand
D76A/N, D78A/N, N80A, D84G, E87A/Q
F108D/G110D
I115F
L167R, T172R, L195R
I220W, C227W
L248S
L251S
L258S/G
L261G
R304W
Y316A/S/T/D/E
E318A-E319A-E320A-E322A (4EA)
A369K
A380R
L416S-L423S
L416G, V419G, L423G
G98D/P/S
V102A/C/S/T
L138F
W176C
P245L/X (any amino acid)
11, 124, 199, 239, 260, 359, 362, 370, 443
370
224
370
214
255
255, 455
214, 255
214
262
439
175, 255
64
368
255
214
445
237
93a
366
262, 275
STIM1 SAM
STIM1 TM
STIM1 CC1
STIM1 CC2
STIM1 CC3
Orai1 TM1
Orai1 TM2
Orai1 TM3
Orai1 TM4
Naturally occurring human mutations are indicated in bold.
act as a “brake” on STIM activation, as its removal and
replacement with a fluorescent protein promotes spontaneous puncta formation and SOCE (206).
The role of STIM1 oligomerization as an early event in
SOCE also received support from FRET studies of fulllength STIM1 in cells. FRET between YFP-STIM1 and
CFP-STIM1 increases upon store depletion and is reversed
when stores refill (64, 198, 256). The increased FRET occurs at locations within the cell that are distinct from
ER-PM junctions and prior to accumulation of STIM1 at
the cell periphery and FRET between STIM1 and Orai1,
indicating that STIM1 oligomerization precedes binding
and activation of Orai1. Likewise, refilling of stores caused
STIM1-STIM1 FRET to decrease prior to STIM1-Orai1
FRET, again supporting a link between oligomerization
and Orai1 binding (256).
The nature of active STIM oligomers is at this point an open
question. Are they formed from the association of multiple
STIM1 dimers (“interdimeric oligomers”) or do they represent dimers in which the EF-SAM domains of a single dimer
are bound to each other (“intradimeric oligomers”)? An
early interpretation of FRET studies was that the moderate
degree of resting FRET comes from dimers containing a
donor and an acceptor fluorophore, and the FRET increase
after store depletion reflects the formation of interdimeric,
higher order oligomers that bring more donors and accep-
tors together. This interpretation is supported to some degree by an approximately twofold reduction of the STIM1
diffusion rate after store depletion (64, 198), although this
effect may be more directly related to increased interactions
with the local environment as the cytosolic domain extends
than to increased stoichiometry per se (64). Formation of
higher order oligomers is also supported by the ability of
STIM1-⌬C (STIM1 1–237) proteins lacking the cytosolic
domain to be recruited to ER-PM junctions by wt-STIM1
after store depletion (64), presumably through interactions
with the transmembrane/luminal regions of full-length
STIM1.
On the other hand, the observed increases in STIM-STIM
FRET could simply result from a conformational change
that brings the fluorophores in a single dimer closer together
(intradimeric oligomerization; for an example see FIGURE
5A). Based on the crystal structure of the CAD domain,
Yang et al. (424) suggested that the CC1 helices in the
resting state may be splayed apart. In support of this idea,
FRET studies of the CT-STIM1 fragment in solution suggest that the ER-proximal ends of the dimer are well separated (455). Interestingly, bringing the two NH2 termini
together via crosslinking of introduced cysteines creates a
COOH-terminal conformational change that may allow
the CAD domain to bind to Orai (455). A recent study by
Zhou and colleagues (214) provides a different view as to
how this conformational change may occur. They found
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Domain
MURALI PRAKRIYA AND RICHARD S. LEWIS
In addition to the EF hand and SAM domains, the CAD
region of STIM1 is also critical for STIM1 oligomerization
in response to store depletion. Based on FRET measurements of STIM1 proteins truncated at various cytosolic locations, it appears that CC1 alone (STIM1 1–344) is not
sufficient to support oligomerization, but addition of CAD
(STIM1 1– 448) restores the normal response (64). Within
CAD, both CC2 and CC3 domains are essential for stabilizing the oligomerized state following store depletion. It is
not clear whether this requirement reflects the role of CAD
in stabilizing the resting dimeric state which is likely to be
necessary for further conformational changes during activation, or rather a specific role of CAD in stabilizing the
conformation of the activated cytosolic structure. With regard to the latter possibility, a recent FRET study has demonstrated homomultimerization to explain the role of CC3
in higher order oligomerization of STIM1 (94a).
A fundamental question is how the conformational changes
associated with luminal sensing of Ca2⫹ depletion by EFSAM drive changes in the cytosolic regions of STIM that
enable targeting to ER-PM junctions and binding and activation of Orai. A concept that is emerging involves the
operation of two “brakes” on STIM activation. The first is
the EF-SAM domain itself, which is a compact monomer in
the Ca2⫹-bound resting state (see above), and the second is
an interaction between CC1 and CAD. With store depletion, Ca2⫹ release causes EF-SAM to separate, and hydrophobic interactions could bring together the two EF-SAM
domains of the dimer. This propagates a conformational
change through the STIM TM domains to the cytoplasmic
domain to override a CC1 structural clamp that keeps CAD
1398
in an inactive conformation, allowing it to adopt the active
conformation.
The notion that CC1 interacts with CAD to keep it in an
inactive state was first elaborated by Balla and colleagues
(175) in the form of an electrostatic clamp between a highly
conserved acidic region in CC1 (318EEELE322) and basic
region in CC2 (382KIKKK386). This was based largely on the
inhibitory effects of neutralizing the quartet of glutamates
in CC1. Later studies showed that this CC1 acidic region is
unlikely to be in proximity to the basic region in CC2 (424,
455) and that the amphipathic properties of the helix determine its inhibitory capacity rather than charge (438). However, the concept of CC1 as an inhibitory clamp on CAD
activity has continued to garner support. Romanin, Hogan
and colleagues have convincingly shown that the isolated
STIM1 cytosolic domain assumes a compact form in the
resting state, based on high FRET or LRET values observed
with STIM1 fragments labeled at the ER membrane insertion site and the COOH terminus (255, 455). FRET values
decline upon binding the wild-type fragments to Orai1 or
introducing mutations in CC1 that activate STIM1 (4EA,
L248S, L251S, L258S, or the deletion of CC1 ␣1 helix)
leading to the idea that the cytosolic domain elongates as it
transitions to the active state (94a, 255, 455). In addition,
the L251S mutation increases the binding of the STIM1
fragment to PIP2, suggesting that the polybasic domain also
becomes exposed as a result of elongation (455).
The structural details of the interaction between CC1 and
CAD that controls activation are not yet fully understood.
Yang et al. (424) initially proposed that amino acids 308 –
337 (helix ␣3) constituted an “inhibitory helix” that suppresses CAD activity, based on the 4EA results described
above, a crystal structure of C. elegans CAD in which a
short segment of CC1 interacts with CAD, and the activating effects of deleting residues 310 –337. Mutation of Y316
in CC1 ␣3 also causes partial activation of STIM1 (439).
However, other regions are also clearly involved in stabilizing the inactive structure. Mutations of hydrophobic residues in helix ␣1 of CC1 (L248, L251, L258) activate (255,
455), as do mutations of residues predicted to contribute to
coiled-coil structures and likely to be involved in intramolecular interactions between CC2 and CC3 of CAD (e.g.,
A369, L416, L423; FIGURE 5D) (64, 255). Thus evidence
points to both the ␣1 and ␣3 helices of CC1 as part of the
inhibitory clamp, although their sites of interaction with
CC2 and/or CC3 of CAD remain to be identified.
Zhou, Hogan, and colleagues (455) have proposed a model
to explain how store depletion leads to the release of the
inhibitory clamp, based on studies of STIM1 cytosolic fragments in solution. They observed that bridging the NH2
termini (the ER insertion sites) of the CT-STIM1 peptide
through cysteine crosslinking caused peptide elongation
similar to that described by Muik et al. (255) upon Orai1
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that the TM domains associate with each other in the membrane and that a C227W mutation in the STIM1 TM domain reorients the TM helices and causes elongation of the
cytoplasmic domain and constitutive STIM1 activity (214).
Thus, with the assumption that the C227W mutant mimics
the structure of depletion-activated WT STIM1, Ca2⫹ depletion may activate STIM by driving the intradimeric binding of 2 EF-SAM domains, which then triggers conformational changes in the TM domain that propagate further
downstream to expose the PBD and allow CAD to bind and
activate Orai (FIGURE 5A). It should be noted that intradimeric binding does not preclude interdimeric associations
that could create larger clusters of STIM proteins, and it is
not yet clear which of these events is responsible for the
FRET changes that have been reported. Clearly more work
will be needed to resolve these issues, in particular to establish the conformation and stoichiometry of full-length
STIM1 proteins in intact membranes. For purposes of this
discussion, we will hereafter use the term oligomerization to
refer to the EF-SAM-mediated process that causes the FRET
increase and leads to STIM activation, without implying
any particular type of oligomer.
STORE-OPERATED CALCIUM CHANNELS
3. SOCE is a self-organizing process
The ability of STIM oligomerization to drive the process of
SOCE was tested directly by studies in which the EF-SAM
domains were replaced by FKBP and FRB protein modules
(209). Heterodimerization of these two domains with a
rapamycin analog triggered accumulation of FRB-STIM1
and FKBP-STIM1 in puncta as well as activation of Ca2⫹
entry and ICRAC, without affecting Ca2⫹ store content.
These results are compatible with both the inter- and intradimeric STIM activation models described above; while
the observed formation of high-molecular-weight forms of
STIM1 by rapamycin is consistent with higher-order oligomers, it is also possible that rapalog activated STIM1 and
SOCE by bringing together the NH2 termini within single
STIM1 dimers. This study demonstrated that STIM1 oligomerization is the key triggering event that couples store
depletion to the activation of SOCE, and that the underlying movements and interactions of STIM and Orai which
culminate in CRAC channel activation are self-organizing.
Several studies support the view that the primary if not sole
function of ER Ca2⫹ depletion in SOCE is to promote STIM
oligomerization, and that once oligomers have formed, all
further events leading ultimately to Ca2⫹ entry are autonomous. For example, mutations of the cEF hand that reduce
the Ca2⫹ affinity of STIM1 as well as mutations in the SAM
domain that promote STIM1 oligomerization (TABLE 1)
evoke constitutive formation of STIM1 puncta (32, 199,
370). Because these mutations function without a change in
[Ca2⫹]ER, their effects demonstrate the sufficiency of STIM
proteins to act as the Ca2⫹ sensors for SOCE. One question
from these studies is whether the EF-SAM domain is needed
to impose a particular conformational change on the
STIM1 luminal domains, or whether oligomerization is all
that is necessary to activate SOCE. However, the ability of
artificial dimerization of FRB/FKBP-STIM1 chimeras to
evoke SOCE shows that oligomerization itself, independent
of the EF-SAM domain, is the key triggering event. The
[Ca2⫹]ER dependence of STIM1 redistribution to peripheral
puncta (K0.5 ⫽ 187 ␮M, nH ⫽ 4) closely matched that of
ICRAC activation (209) (FIGURE 1B), adding further evidence that ER Ca2⫹ is critical only for controlling early
events (i.e., oligomerization) leading to STIM1 activation,
rather than directly modulating Orai channels at ER-PM
junctions. Puncta formation for both STIM1 and STIM2
are highly nonlinear functions of [Ca2⫹]ER (for STIM1,
K0.5 ⫽ 210 ␮M, nH ⫽ 8; for STIM2, K0.5 ⫽ 406 ␮M, nH ⫽
5; Ref. 32). The high cooperativity of puncta formation has
not been fully explained, although it may reflect the release
of two Ca2⫹ ions per dimer, the association of multiple
dimers to form higher order STIM oligomers, or cooperative allosteric changes in STIM.
Importantly, these studies reveal that SOCE is a self-organizing process held in check by the state of STIM oligomerization. Self-organization is a prevalent driving force that
contributes to many life processes, including the cell cycle
and the induction of cell polarity and control of cytoskeletal
structures and cell shape, among others (166). As described
below, current evidence argues for a diffusion-trap mechanism as the basis for the self-organization of the SOCE
machinery.
4. A diffusion-trap model for SOCE
After store depletion, STIM and Orai remain mobile in their
membrane compartments and accumulate passively at
ER-PM junctions by a two-part diffusion-trap mechanism.
These events have been examined in detail using singleparticle tracking techniques to follow single STIM1 oligomers and Orai1 channels as they enter ER-PM junctions (416)
(FIGURE 5B). In store-depleted cells, STIM moves by diffusion in the ER membrane at an average rate (D ⬃0.07
␮m2/s) that is roughly half that of STIM in resting (i.e.,
store-replete) cells (64, 198). Orai also moves by diffusion
in the PM with an average diffusion coefficient D of ⬃0.1
␮m2/s (284, 416). Single-particle tracking shows that Orai
channels are slightly subdiffusive, indicating interactions
with proteins or lipids in their environment (416).
STIM redistributes to ER-PM junctions independently of
Orai, as STIM puncta form in store-depleted cells expressing only a nominal amount of endogenous Orai1 (e.g., HEK
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binding. In addition, while CC1 by itself appeared monomeric in solution and bound to CAD, crosslinking CC1
induced formation of a coiled-coil dimer and loss of CAD
binding. These results support a model in which the CC1
monomer interacts with CAD to maintain it in a resting
(closed) conformation, and store depletion and consequent
dimerization of the EF-SAM domains releases the clamp by
triggering formation of the CC1 coiled-coil dimer. CC1 ␣1
and specifically the residue Leu-251 is critical for maintenance of the inactive state, as truncation of the CC1 ␣ helix
or the L251S mutation elongates the STIM1 cytosolic domain and activates STIM1 in the absence of store depletion
(94a, 255, 455). While L251S prevents coiled-coil formation it also weakens the interaction of CC1 with CAD,
potentially explaining its ability to activate STIM1. Thus, in
full-length STIM1, Ca2⫹ depletion has been proposed to
release the inhibitory clamp on CAD by sequestering L251
within the newly formed CC1 coiled-coil (455). In a very
recent extension of this work, Zhou and colleagues (214)
suggest that the inhibitory clamp includes coiled-coil interactions of L258 and L261 of the CC1 ␣1 helix with V419
and L416 of the CC3 helix of CAD. Although these contacts have yet to be shown exprimentally, and the insights
from this work are derived from studies of isolated and
mutagenized STIM1 fragments, they provide a strong basis
for new studies to determine the structural interactions that
maintain the resting state and how they change to trigger
STIM1 activation, particularly for full-length STIM1 embedded in the ER membrane.
MURALI PRAKRIYA AND RICHARD S. LEWIS
Like STIM, Orai moves by Brownian diffusion in the
plasma membrane and accumulates at ER-PM junctions
after store depletion (210, 418), but unlike STIM it is
trapped solely through interactions with its partner protein
(FIGURE 5, A AND B) (284, 418). FIGURE 5B shows an example of a single Orai1 channel being trapped by STIM1 in
a punctum before it eventually escapes. The requirement for
STIM in trapping Orai is shown most clearly by observations that Orai1 alone fails to form puncta but colocalizes in
puncta with STIM1-⌬K, which by itself is unable to form
puncta without its PBD domain (284). Trapping at junctions occurs primarily by binding to the Orai1 COOH terminus, as the COOH terminus but not NH2 terminus appears to be necessary (206, 284). Close interactions between STIM and Orai in situ after store depletion have been
shown by coimmunoprecipitation of Orai1 with STIM1
(287, 292, 396, 429) and STIM2 (287) and by FRET between STIM1-YFP and CFP-Orai1 (256, 260, 429). Orai1
is trapped through direct interactions with the CAD domain
of STIM1 (284). Experiments in which the ratio of accumu-
1400
lated STIM:Orai at junctions was measured under conditions of excess Orai1 suggest that only 0.3– 0.6 STIMs per
Orai are needed to trap a channel (147). Given a hexameric
channel stoichiometry, this result suggests that binding of
two to four STIM1 proteins (1–2 STIM1 dimers) is sufficient for trapping, although this is not enough to trigger
channel opening (147). Current views of how it opens the
Orai channel are described in more detail below.
Most of the work on the choreography of SOCE has been
done in cells overexpressing fluorescently labeled STIM and
Orai at levels probably orders of magnitude greater than
endogenous levels. To understand SOCE signaling in native
cells, a question of interest is the number of endogenous
CRAC channels that operate at the ER-PM junction. Electrophysiological and EM data collected from Jurkat T cells
(299, 414) allows a rough estimate to be made (see also Ref.
145). From the maximal whole cell Na⫹-ICRAC currents
measured under divalent-free conditions (⫺190 pA), the
unitary Na⫹ current amplitude (⫺110 fA), and an open
probability of ⬃0.8, one can estimate roughly 2,000 functional CRAC channels in a typical Jurkat T cell, or ⬃2/␮m2
given the average surface area of the cell of 1000 ␮m2 (299).
Considering that 2-APB can increase the current by up to
fivefold, the estimate increases to ⬃10,000 channels (10/
␮m2). EM measurements indicate the average size of the
ER-PM junction profile in these cells is ⬃200 nm and in
total they account for ⬃4% of the cell circumference (414).
If one assumes that each profile is the cross-section of a circular
disc (272), then the area of a junction is ⬃0.03 ␮m2, and this
will contain 0.03 ␮m2 ⫻ 2–10 channels/␮m2 ⫻ 1/.04 ⫽ 2–10
channels. While these are certainly rough estimates, they do
indicate that the number of channels is likely to be quite small,
far lower than the 1,300 channels/punctum estimated in
HEK cells overexpressing STIM1 and Orai1 (162).
IV. CRAC CHANNEL GATING
A. Subunit Stoichiometry of Orai Channels
Given the small size of the Orai proteins (⬃300 amino
acids), it was natural to assume that functional CRAC channels would have to be multimers of several Orai1 subunits.
This expectation is supported by dominant negative effects
of Orai1 pore mutants (E106Q and E190Q) on channel
function (133, 396), coimmuniprecipitation of orthogonally
tagged subunits, and FRET between CFP- and YFP-tagged
Orai1 molecules (256, 260). The precise stoichiometry, however, has drawn considerable disagreement. Attempts to evaluate the stoichiometry of the channel from purely biochemical
assays have not provided an easily interpretable answer, and
as described below, early conclusions regarding the tetrameric stoichiometry of CRAC channels have been called
into question by the hexameric stoichiometry reported for
the Drosophila Orai channel.
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293) (199, 284, 418). A key region involved in STIM1
redistribution is the polybasic domain (PBD), a lysine-rich
stretch of 15 residues at the extreme COOH terminus (FIGURE 2). Deletion of the PBD prevents redistribution but not
oligomerization, consistent with the idea that oligomerization triggers conformational changes that expose the PBD
to the PM (154, 198, 284). By promoting accumulation of
STIM1 at junctions, the PBD enchances the rate at which
STIM1 activates Orai1 (206). Several studies suggest that
the by promoting accumulation of STIM1 at junctions, the
PBD enhances the rate of which STIM1 activates Orai1
(206). PBD interacts electrostatically with negatively
charged phospholipids in the plasma membrane, in particular PIP2 and phosphatidylinositol (3, 4, 5)-trisphosphate
(PIP3). Depletion of PIP2 or PIP3 together in cells inhibits
puncta formation and SOCE, and PBD-containing fragments from STIM1 and STIM2 bind to PIP2-containing
liposomes in vitro (94, 400, but see Ref. 174). The trigger
for PBD binding to PIP2 in the PM may involve changes in
conformation (as extension of the STIM cytosolic domain
moves the PBD towards the PM) as well as avidity (as STIM
oligomerizes). The STIM1 PBD has only marginal affinity
for
PIP2containing liposomes unless it is presented as a dimer or
tetramer (26); in contrast, the STIM2 PBD has a higher
affinity and can bind as a monomer, perhaps because of its
ability to form an amphipathic helix. PIP2 has recently been
detected at locations where STIM1 puncta form using
PLC␦-PH domain-based indicators (343). Interestingly,
PIP2 levels appear to decline within puncta as STIM-Orai
complexes accumulate, possibly as a result of rearrangements of septins near junctions. These new findings raise the
possibility that PIP2 could play a dynamic role in the formation and maintenance of signaling complexes at ER-PM
junctions.
STORE-OPERATED CALCIUM CHANNELS
A
B
C
Outer vestibule
out
extracellular
TM1 helix
TM2 helix
TM3 helix
TM4 ext helix
E178 (E106)
Selectivity filter
F171 (F99)
G170 (G98)
Gating hinge
Hydrophobic
L167 (L95)
L319
(L276)
55 Å
Acidic
V174 (V102)
Hydrophobic gate
K163 (R91)
in
I316
(L273)
K159 (K87)
K157 (K85)
R155 (R83)
intracellular
Basic
Q152 (Y80)
CAD binding
domain (73-85)
W148 (W76)
10 Å
Among early studies to directly address the issue of Orai
channel stoichiometry, Shuttleworth and colleagues (246)
used concatenated Orai1 subunits and the effects of overexpressing dominant negative Orai1 subunits to conclude
that functional CRAC channel is a homotetramer of Orai1
subunits (246). Likewise, single-molecule photo-bleaching,
diffusional analysis of labeled Orai1 proteins, and FRET
measurements in tandem dimers and tetramers led to conclusions that STIM1-bound Orai1 channels are tetramers of
Orai1 subunits (162, 213, 292). Interestingly, two studies
reported that STIM1-free Orai1 channels are dimers (76,
292), leading to the suggestion that STIM1-gated tetramers
arise from STIM1-induced dimerization of resting Orai1
dimers. The stoichiometry reported by all of these reports
was ultimately challenged by the X-ray crystallographic
structure of Drosophila Orai (153) which showed that the
Orai channel is a hexamer (FIGURE 6B, discussed further
below).
These differing results underscore the need for further study
to establish the stoichiometry of the resting and active states
of the channel. Stoichiometry of channels is often difficult to
determine based on a single method. Concatemer studies
can be confounded by aberrant channel assembly from incomplete or degraded translation products, or subunit
swapping between concatemers (235, 328). Reliable interpretation of single-molecule photobleaching experiments
using GFP tags is limited to low numbers of subunits because of the flickery nature of GFP fluorescence which can
make up to 30 – 80% of the single-particle bleaching events
uninterpretable (76, 162, 292) and potentially bias the results towards lower numbers of bleaching steps (348). Stoichiometries greater than 4 cannot be ruled out from such
studies, and in fact, larger complexes have been detected
after oxidizing di-cysteine-substituted Orai1 monomers expressed in HEK cells (454) and crosslinking dOrai expressed in HEK cells (153).
B. Overview of Orai Structure
Determining the molecular structures of ion channels is
essential for understanding how they work, their roles in
disease, and to guide development of small molecular
therapeutics. Early efforts using cryoelectron microscopy
did not yield easily interpretable structural models (227),
but the recently solved structure of the Drosophila Orai
channel by Long and colleagues (153) has enabled detailed visualization of the CRAC channel architecture.
This model of a modified channel (with truncated NH2
and COOH termini and a mutated III–IV loop) showed a
complex composed of six dOrai subunits, whose transmembrane domains are arranged in concentric layers
around a central aqueous pore (FIGURE 6). The TM1
helix directly flanks the length of the pore, while TM2
and TM3 surround TM1, shielding it from the surrounding lipid bilayer, and TM4 forms the outermost and presumably the most lipid-exposed segment. The pore is
relatively long at 55 Å and is composed of both the TM1
segment as well as the membrane proximal Orai NH2
terminus (FIGURE 6C). The extracellular opening of the
pore has a highly negative electrostatic potential derived
from the presence of the six TM1 E178 (Drosophila
equivalent of human E106) residues comprising the selectivity filter. The side chains of the Glu ring appear to
all point towards the central symmetry axis (pore) in the
structure, with the oxygen atoms of the carboxylates separated by only ⬃6 Å. Crystal soaking experiments re-
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FIGURE 6. The crystal structure of Drosophila Orai. A: side view of the crystal structure of dOrai, showing the
arrangement of the transmembrane domains of the subunits (4HKR.pdb; Ref. 153). The Structure encompasses amino acids 132–341 and C224S/C283T/P276R/P227R mutations for facilitating crystallization.
B: a top view of dOrai, showing the arrangement of 6 subunits around a central pore with coiled-coil interactions
between the 3 pairs of TM4 extensions. Inset shows the antiparallel arrangement of the TM4 extension helices
and the hydrophobic interactions between I316 and L319 of each helix (equivalent hOrai1 residues in
parentheses). C: a side view of the TM1 helix and the NH2-terminal extension helix from two opposed subunits
in the crystal structure, showing pore-lining residues. Equivalent human Orai1 residues are shown in parentheses. The density corresponding to Ba2⫹ is illustrated in blue above E178. The anion density with the Fe
atoms modeled into the structure are shown in yellow and gray in the inner pore.
MURALI PRAKRIYA AND RICHARD S. LEWIS
vealed that in this closed structure, cations such as Ca2⫹
and Gd3⫹ bind at or slightly above the filter (FIGURE 6C).
Below this region is a three helical turn hydrophobic
section. Interestingly, the intracellular opening of the
pore has three adjacent rings of positively charged side
chains that extend into the lumen.
In addition to a hexameric stoichiometry, another unforeseen feature of the crystal structure is that the cytosolic
termini of the TM4 helices are arranged in pairs, with each
helix bending in opposite directions to form an antiparallel
coiled-coil with its neighbor (FIGURE 6). This association
appears to be maintained by hydrophobic interactions between the Drosophila equivalents of the residues L273
(I316) and L276 (L319) in human Orai1, creating a threefold symmetry at the channel periphery. Previous studies
have shown that mutations at these sites that lower the
coiled-coil probability of the COOH terminus, such as
L273S/D (205, 256) and L276D (260), inhibit STIM1 binding and disrupt channel activation. It is currently unclear
whether effects arise because L273 and L176 are needed to
maintain the coiled-coil TM4 pairs or that they directly
stabilize interaction with STIM. Hou et al. (153) have hypothesized that the individual Orai COOH termini may
straighten during STIM1 binding, breaking the anti-parallel
coiled-coil configuration and allowing the individual Orai
COOH termini to interact with STIM1. In contrast, a recent
NMR complex structure between STIM1 (amino acids
312–387) and Orai1 (amino acids 272–292) fragments suggests an alternative model where the Orai1 COOH termini
remain in a similar orientation to the coiled-coil form of the
Drosophila crystal structure, but with some minor alterations in the angles of the Orai1 helices (FIGURE 7) (368).
1402
Resolving the series of molecular steps from STIM binding
to the activation of the Orai channel is a major challenge
that has been approached using a range of technigues, especially mutational analysis. The interpretation of mutagenesis studies aimed at identifying and distinguishing
domains involved in binding and gating is often not
straightforward. Because gating of CRAC channels is allosterically coupled to STIM binding, mutations that affect the
gating steps will also affect STIM binding (and vice versa),
complicating attempts to distinguish between binding, gating, and coupling domains (62). However, recent studies
that have provided structural snapshots of putative binding
interfaces between STIM and Orai, and mutations causing
constitutive channel activation in the absence of STIM offer
promising leads to understand the molecular-structural basis of CRAC channel gating.
1. STIM binding to Orai
The initial observations that Ca2⫹ influx occurs locally at
sites where STIM and Orai are coclustered and separated
only by the narrow gap of the ER-PM junction supported
the idea that SOCE is driven by physical contact between
STIM and Orai (210). Based on the finding that the isolated
cytosolic domain of STIM1 (CT-STIM1; aa 233– 685) was
able to activate SOCE independently of store depletion
(154), several groups truncated CT-STIM1 from both ends
to identify a minimal region that when expressed by itself
evoked constitutive SOCE and ICRAC. This minimal activating region is the CAD domain (amino acids 342– 448; Ref.
284), also known as SOAR (STIM1-Orai activating region;
amino acids 344 – 442; Ref. 434) or Ccb9 (amino acids
339 – 444; Ref. 167) (FIGURE 2A). CAD/SOAR/Ccb9 contains two putative coiled-coil regions (CC2 and CC3) and
appears as a dimer in solution, although a tetrameric form
has also been reported (284, 424). The CAD domain is both
sufficient and necessary for Orai activation. CAD expressed
by itself localizes to the plasma membrane where it binds to
Orai and activates constitutive Ca2⫹ entry. Conversely,
CAD mutations or deletion abrogate Orai1 activity (see
TABLE 2).
Several independent observations indicate that CAD binds
directly to Orai1 rather than through an intermediary:
GST-CAD pulls down affinity-purified Orai1, CAD interacts with Orai1 in a yeast split-ubiquitin assay, and it copurifies with coexpressed Orai1 (284). CAD interacts
strongly with COOH-terminal and weakly with NH2-terminal peptides from Orai1, but not detectably with the
II–III loop (284); binding to the NH2 and COOH termini is
also direct as shown by GST pulldowns using purified
STIM1 and Orai1 fragments (453). While we currently do
not have a definitive picture of how the CAD engages the
COOH-terminal helices of Orai, evidence based on mutagenesis and domain swapping between the CAD of
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The hexameric stoichiometry of the CRAC channel has
been challenged by a report that hexameric hOrai1 concatemers lacked normal Ca2⫹ selectivity (385). However,
a subsequent study found that hexameric concatemers
with longer intersubunit linkers exhibit normal selectivity and channel properties (428). Considering multiple
lines of evidence, including the close correspondence of
the general pore architecture, the agreement between predicted pore-lining residues and the results of cysteine
scanning studies (238, 453), the crosslinking of dOrai
protein in native membranes (153), and the general sequence similarity of Drosophila and human Orai proteins, it seems likely that the hexameric stoichiometry
and overall architecture reported in the dOrai crystal
structure is valid for human Orai1 channels. It is therefore puzzling that studies of concatemers containing four
covalently linked subunits reported channels with apparently normal permeation and gating properties. One possibility is that functional channels with hexameric stoichiometry were formed through the combination of subunits derived from multiple tetramers, but this needs to
be tested experimentally.
C. Activation Gating
STORE-OPERATED CALCIUM CHANNELS
A
B
dOrai dimer
Orai1-C272-292
Orai1-C272-292
L273
L276
L276
C
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CC2
C
Side
CC1distal
distal
90o
N
N
STIM1 A380 A376 L373 A369 K366 Y362
Orai1
L286 Q283 A280 L276 L273
CC2
Top
Orai1
R289 Q285 R281 A277 N274
STIM1
P344 L347 L351 H355 V359
FIGURE 7. A possible structural model for STIM-Orai binding. A: a dimer of subunits from the dOrai crystal
structure (153) is shown at top, with the cytosolic TM4 extension helices in yellow. The bottom two diagrams
show an NMR solution structure of human Orai1 COOH-terminal peptides (yellow) bound to a dimer of human
STIM fragments (amino acids 312–383) containing the distal portion of CC1 (␣3 helix; blue) and the CC2 helix
of CAD (teal) (2MAK.pdb) (368). The orientation of the TM4 extensions (top) is similar to that of the bound
Orai1 COOH-terminal peptides. In the CC1-CC2-Orai structure, the two CC2 helices adopt an antiparallel
orientation that creates a pair of symmetrical binding sites called the STIM-Orai Association Pocket (SOAP).
Binding of the Orai peptide is stabilized by a series of mostly hydrophobic interactions involving the residues
listed at bottom left. Note that the paired binding of L273 to L276 in the Orai structure is disrupted and the
two Orai helices are translated and rotated in opposite directions in the bound structure, potentially creating
a force that could contribute to gating. [Adapted from Stathopulos et al. (368).] B: Surface representations
showing the binding pocket of the SOAP completed with Orai1 COOH-terminal peptides. The middle and bottom
views correspond to the helical representation in A, while the top model is a view from the left end of the SOAP.
Surface electrostatic potential is colored from red (⫺5 kT) to blue (⫹5 kT) (dielectric constant 80).
STIM1 and STIM2 supports an interaction of the CC2 domain of CAD with the cytoplasmic TM4 extension (40,
109, 402). Stathopulos, Ikura, and colleagues (368) have
proposed a plausible model for this interaction based on the
solution NMR structure of a STIM1 fragment including the
distal (␣3) region of CC1 and most of CC2 in complex with
purified Orai1 COOH-terminal helix (FIGURES 2 AND 7). In
this structure, two Orai helices interact via a combination of
polar and nonpolar contacts with CC2 in a pair of grooves
(the STIM-Orai activation pocket, or SOAP) formed by the
antiparallel arrangement of two CC2 domains (FIGURE 7).
It appears from the CC1-CC2 structure that the CC3 domain as depicted in the CAD crystal structure (424) must
dissociate from CC2 to permit CC2-Orai1 binding, and the
authors suggest that three pairs of CC3 helices associate to
form a ring of STIM dimers around the Orai channel, or
that CC3 may be involved in other yet-to-be-defined interactions, for example, with the Orai NH2 terminus.
Although caution is warranted in extrapolating mechanisms from the binding of peptides removed from their full
protein environment, the effects of STIM1 and Orai1 mutations on binding and SOCE support key aspects of the
structure. For example, Orai1 residues L273 and L276 have
long been known to be essential, as L273S/D and L276S
mutations effectively quell STIM1 binding as well as SOCE
(TABLE 2) (205, 256, 260), and these both make hydrophobic interactions with STIM1 side chains in the SOAP (FIG-
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MURALI PRAKRIYA AND RICHARD S. LEWIS
termini (387). It is apparent from the NMR structure that
the L273 and L276 residues that form a paired interaction
between adjacent Orai1 COOH termini are pulled apart
laterally and rotated when bound within the SOAPs (FIGURE 7); thus, based on this structure, the binding of STIM
would be expected to exert torque on the COOH termini
which could contribute to channel gating.
A key unresolved issue is how to account for the striking
differences in the orientations and positions of the STIM
domains in the CAD crystal structure (FIGURE 5D) and the
NMR structures (FIGURE 7). If the CAD crystal structure
represents the inactive state and the NMR complex structure the active state, then several significant reorganizations
of the protein structure need to occur following store depletion. The two CC2 helices in the CAD crystal begin in a
parallel configuration, with CC2-CC3 interactions maintaining each hairpin monomer structure. To attain the
NMR complex structure, the CC2 helices must pivot and
twist to become antiparallel, and CC3 must swing out to
make room for the Orai COOH terminus to bind to the
SOAP. Given that the currently available structural infor-
Table 2. Mutations that inhibit STIM1 activity or Orai1 activity/conduction
Domain
STIM1 SAM
STIM1 CC1
STIM1 CC2
STIM1 CC2-CC3 linker
STIM1 CC3
Orai1 NH2 terminus
Orai1 TM1
Orai1 TM3
Orai1 COOH terminus
Mutation or Deletion
E136X (E128RfsX9 frameshift)
1538-1G>A (splice site)
L347A-Q348A
L347A-W350A-L351A
L347R, L351R, Y361K-Y362K
A376K
K382E-K384E-K385E-K386E (4KE)
F394L/A/H
R426L
R429C
W430A-I433A-L436A
C437G
296
36
434
424
368
64
175
402
255
112, 231
424
284
⌬1–91, ⌬1–85, ⌬73–85, ⌬1–88
L74A–L79A–L81A–L86A
L74S–W76S, L74R–W76R,
L74E–W76E
K85A/E
R91W/F/l
A103E
E106Q/C/A
D112X (A88SfsX25 frameshift)
L194P
⌬267–301, ⌬261–301, ⌬272–279
L273S/D, L276D
R281A, L286S, R289A, (partial
inhib)
D284A–D287A–D291A (partial
inhib)
206, 236, 256, 284, 448
402
80
Naturally occurring human mutations are indicated in bold.
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Reference Nos.
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201, 236
77, 103
234
133, 238, 396
234
234
206, 236, 256
109, 205, 236, 256, 260
368
368
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URE 7A). Likewise, mutations of other Orai1 residues predicted to interact with the SOAP (R281, L286, and R289)
as well as STIM1 residues in the SOAP that interact with
Orai1 (L347, L351, and Y362) are sufficient to disrupt
Orai1-STIM1 interactions and SOCE (368) (TABLE 2). Importantly, mutations of nearby hydrophobic residues in
Orai1 (F279 and L282) that are not predicted to interact
with STIM1 in the structure have no effect on ICRAC or
STIM1-Orai1 FRET (256, 260). An intriguing aspect of the
structure is that the SOAP domains engage two Orai helices
at approximately the same angles shown by the antiparallel
coiled-coil pairs of TM4 extensions in the dOrai structure
(368) (FIGURE 7), suggesting that perhaps the extensions are
maintained in a conformation that is optimal for binding by
a STIM dimer. While this idea needs testing, recent results
using Orai1 concatemers with variable arrangements of defective (L273D) STIM binding sites support the idea that
STIM dimers bind to pairs of TM4 extensions (428). Furthermore, the disruption of STIM1 binding by Cys-crosslinking adjacent Orai1 COOH termini is rapidly reversed
upon disulfide cleavage, consistent with a relatively small
conformational rearrangement of the paired Orai1 COOH
STORE-OPERATED CALCIUM CHANNELS
mation is entirely derived from small STIM fragments, it
will be necessary to use other approaches to establish
whether the reported structures exist naturally in the fulllength protein and to identify conformational intermediates
in the activation process.
Evidence for weak interactions between CAD and Orai1
NH2-terminal peptides (amino acids 73–91) (284, 453) has
led to a two-step model of STIM1 binding, wherein an
initial binding event at the COOH terminus permits subsequent lower-affinity STIM1 interaction with the NH2 terminus and opening of the pore (448). In one version of this
model, Gill and colleagues (402) have proposed that F394,
a critical residue in the CC2-CC3 linker of STIM1, interacts
with exposed leucines in TM1 to open the channel. However, direct binding of STIM1 to the Orai1 NH2 terminus in
the intact channel has not yet been demonstrated, and isolated peptides often exhibit binding properties that are absent in the context of the full-length protein. Orai1 NH2terminal mutations do reduce binding of full-length STIM1
to Orai1 in intact cells, as shown by diminished FRET and
Orai1 puncta formation (80, 236) (TABLE 2). Still, it is not
clear in these studies that the NH2 terminus promotes
STIM1 binding through a direct interaction rather than
through an allosteric coupling mechanism in which channel activation (which requires an intact NH2 terminus)
increases the affinity of STIM1 for the Orai1 COOH
terminus. Thus further studies, especially with full-length
Orai1, will be needed to define more clearly the critical
role of the NH2 terminus for STIM1 binding and gating.
2. Nonlinear stoichiometric requirements for Orai
activation
The activation of Orai channels appears to be highly sensitive to the number of STIMs bound. Increasing the STIMto-Orai heterologous expression ratio was first shown by
Rychkov and colleagues (340) to increase current magnitude as well as enhance CDI, divalent cation selectivity, and
reduce current potentiation by 2-APB. Hoover and Lewis
The highly nonlinear dependence of CRAC current activation on the STIM-to-Orai ratio can be described by a modified Monod-Wyman-Changeux (MWC) kinetic scheme
(147). However, although the MWC formalism offers a
simple way of thinking about the origins of nonlinear gating, Orai channel activation appears to be more complex
than the two-state open/closed mechanism the model assumes. Current noise analysis studies suggest that the slow
activation of CRAC current by store depletion occurs by
stepwise recruitment of single channels from a “silent” state
to a high-open probability (Po) state (⬃0.8 measured during
brief, 200-ms intervals) (299). This process might be envisioned as the binding of channels to STIM1 as they enter
ER-PM junctions. However, the ability of low doses of
2-APB to increase ICRAC by more than threefold implies that
the Po is actually much lower, ⬍0.3. One way to reconcile
these results is that CRAC channels exhibit modal gating,
such that the channel alternates between silent and high-Po
states at a low enough frequency that the transitions evade
detection as noise during brief measurement periods. According to such a scheme, STIM1 binding to Orai1 could
activate the channels by increasing the amount of time spent
in the high-Po mode. It is tempting to speculate that modal
transitions of this sort are related to the high cooperativity
of activation by STIM1 (147) or 2-APB (422).
3. Nature of the activation gate and hinge
The location, disposition, and regulation of the CRAC
channel activation gate has attracted considerable attention. Structural, functional, solvent accessibility analysis as
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The Orai1 NH2 terminus, in particular the region 73–91, is
also a critical determinant of activation gating, as shown by
mutations and deletions that abrogate SOCE and ICRAC
(TABLE 2). While Orai1 remains functional after deletion of
residues 1–73 (206, 284, 448), activation is blocked by
deletion of amino acids 73– 84, a K85E mutation, paired
hydrophilic substitutions for L74 and W76, or alanine substitutions for L74/L79/L81/L86 (80, 201, 236, 284, 402).
Several studies have also noted functional changes in channel chimeras in which NH2-terminal regions were swapped
between the three Orai isoforms (201, 380). While these
experiments all affirm the importance of the NH2-terminal
region in channel activation, its precise role remains unclear, in particular whether it is required for direct interactions with STIM1 rather than with adjacent protein domains within Orai1.
(147) quantified these effects further and found that activation was a highly nonlinear bell-shaped function of the
STIM-to-Orai ratio, as measured by the fluorescence of labeled proteins at ER-PM junctions. In cells expressing similar levels of STIM1, ICRAC increased with Orai1 expression
as long as the STIM-to-Orai ratio was high, but plummeted
when the ratio fell below ⬃2, even as Orai1 expression was
increased; maximal Orai1 channel activation occurred at a
STIM1-to-Orai1 ratio of ⬃2:1 (147). A similar conclusion
was reached by Xu and colleagues (205), who reported that
fusion of a tandem dimer of extended CAD domains (aa
336 – 485) to the Orai1 COOH terminus activates the channel much more effectively than a single domain. The conclusion from these two studies, that a dimer of STIMs binds
to each Orai1 monomer, is apparently at odds with the
model of Stathopulos et al. (368) discussed above, which
shows a STIM dimer engaging a pair of Orai TM4 extensions with a 1:1 STIM-to-Orai stoichiometry. Each approach has shortcomings: the former studies make indirect
inferences about binding, while the latter involve isolated
peptides that may interact differently than the intact proteins. New experimental approaches will be needed to resolve these important questions about the STIM:Orai binding stoichiometry, the structure of the bound state, and their
relationship to gating.
MURALI PRAKRIYA AND RICHARD S. LEWIS
well as disulfide crosslinking approaches have yielded a
complex picture of gating with evidence for at least two
potential gates, located at the extracellular and intracellular
ends of the pore.
In a second gating model, the channel gate is presumed to be
located at the cytoplasmic end of the pore, in TM1 or in the
NH2-terminal cytoplasmic extension of TM1 (325, 445). In
this model, channel opening arises from bending of the
TM1 helices at the conserved G98 residue and from dilation
of the helices in the inner pore, in the region around R91.
G98 was suggested to serve the role of a gating hinge, based
on the constitutive opening of G98D and G98P mutants
(445). R91 was proposed as the physical gate at the cytoplasmic rim of the pore based on the ability of R91C to form
inter-subunit crosslinks that block ion conduction (445).
However given evidence that Cys substitutions at all porelining residues (E106, V102, G98, L95, and R91) can form
intersubunit disulfide bonds (454), the role of R91 as an
inner gate requires further validation. More recently, the
idea of the inner gate has been modified based on the structure of the dOrai crystal structure, which revealed that the
cytoplasmic NH2-terminal helix is contiguous with TM1
(153). Curiously, a prominent ion density attributed to the
binding of anions between the basic residues of the inner
pore (K155, R159 in dOrai) was observed (153) (FIGURE
6C). The triplet of basic residues in the lower pore could
1406
The nature and mechanism of the activation gate in the
CRAC channel thus remains an unsettled issue. The prevailing models come to different conclusions about the location
of the Orai gate. Besides the possibility that there could
indeed be gates at both ends of the pore, these conflicting
models might also be resolved by postulating that gating
occurs across an extended length of the pore due to energetic interactions such as hydrophobic exclusion of water
and ions.
D. Fast Ca2ⴙ-Dependent Inactivation
Ca2⫹ entering through CRAC channels inhibits channel
activity through a process called fast Ca2⫹-dependent inactivation, or CDI (105, 151, 152, 459). CDI is typically
observed as a decline in ICRAC over tens of milliseconds
during hyperpolarizing voltage steps. CDI is specific for
Ca2⫹ over Ba2⫹ or Sr2⫹, and its apparent voltage dependence is accounted for by the voltage dependence of Ca2⫹
entry rather than any intrinsic voltage-dependent inactivation (459). A fast intracellular Ca2⫹ buffer (BAPTA) can
reduce CDI, whereas a slower buffer (EGTA) cannot, consistent with feedback inhibition of channel activity by the
high local [Ca2⫹]i around individual CRAC channels (105,
152, 459). A quantitative analysis of these buffer effects
suggested that CDI is evoked by the binding of at least two
Ca2⫹ ions to a site located several nanometers from the
pore, potentially on the channel or a nearby protein (459).
Interestingly, for endogenous CRAC channels, the extent of
CDI is constant over the time that ICRAC develops and
global [Ca2⫹]i rises, implying that the affinity of the CDI site
is quite low and Ca2⫹ from one channel does not contribute
to inactivation of others (459). This feature is especially
striking, considering that Ca2⫹ from clusters of CRAC
channels would be expected to spread throughout the small
volume of the ER-PM junction.
Structure-function studies have implicated several players
in the CDI mechanism, including STIM1, calmodulin
(CaM), and Orai1. A role for STIM1 was first suggested by
observations that the extent and rate of CDI increase steeply
with the STIM-to-Orai expression ratio (340; see also Ref.
147) and that the isolated CAD domain (STIM1 342– 448)
does not support CDI (284). Residues 470 – 491 were identified as essential for CDI and were named the inactivation
domain of STIM, or IDSTIM (257) (FIGURE 2). Within this
domain, an acidic stretch (475DDVDDMDEE483) appears
to be particularly critical, as neutralization of residues in
this region reduces or eliminates CDI (78, 190, 257). By
analogy to the acidic “Ca2⫹ bowl” sequence of Ca2⫹-activated BK channels (15), a possible role of the acidic region
as a Ca2⫹ sensor for CDI was considered (257). In support
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From the observed differences in the accessibility of thiol
reagents to pore sites in closed versus open channels, McNally et al. (237) concluded that one gate is located towards
the extracellular end of the pore in close proximity to the
selectivity filter formed by E106. Mutational analysis subsequently showed that substitutions of V102 to more polar
residues (Ala, Ser, Thr, or Cys) yielded constitutively open
channels, leading to the proposal of V102 as a hydrophobic
gate (FIGURE 6C) (237). This proposal is congruous with the
dOrai structure, which reveals that the hydrophobic side
chains of the six Val residues are well-packed in the pore
and make extensive van der Waals contacts with each other
(153), thus presenting a large desolvation barrier for ions in
the closed channel (88). Moreover, molecular dynamics
simulations confirm that the (modest) reduction of hydrophobicity caused by the V102A substitution does not appreciably perturb the pore structure, but significantly lowers the energy barrier for entry of water and ions, providing
a possible explanation for constitutive conduction in the
V102A mutant (88). A gate at V102 is also supported by a
recent study that employed changes in the luminescence of
Tb3⫹ bound in the pore, suggesting that STIM1 binding
elicits conformational changes in the vicinity of E106 and
V102 (130). How the native V102 side chains would regulate ion conduction remains unclear; presumably gating
could occur through a rearrangement of V102 side chains,
either by rotation of the TM1 helices or some other mechanism.
thus help stabilize the closed channel either through anion
binding or through electrostatic repulsion of cation conduction in the inner pore.
STORE-OPERATED CALCIUM CHANNELS
CaM has also been implicated as a Ca2⫹ sensor for CDI
(202, 257). This notion was first suggested on the basis of
partial inhibition of native CRAC CDI by overexpression of
a Ca2⫹-nonbinding CaM mutant (CaM1234) and a CaM
inhibitory peptide (202). Later work showed that CaM
binds in a Ca2⫹-dependent manner to solubilized Orai1 and
to an isolated Orai1 NH2-terminal fragment (residues 68 –
91) (257). Consistent with a functional role in CDI, several
mutations that eliminated CaM binding to the peptide
(A73E; W76E, A, or S; Y80E) also suppressed CDI when
introduced into full-length Orai1, and truncations within
the same region in Orai3 produced a parallel loss of CDI
and CaM binding to the NH2-terminal peptide (19). Further validating a role in CDI, a crystal structure of the Orai1
peptide bound to Ca2⫹-CaM confirmed an interaction of
Orai1 W76 and Y80 with CaM, and showed that Orai1
W76 binds strongly within a hydrophobic pocket of CaM
(204).
However, despite strong experimental support, the case for
CaM as the CDI Ca2⫹ sensor also has weaknesses. First,
mutant CaM that cannot bind Ca2⫹ virtually eliminates
CDI of CaV channels where the role of CaM is well established (294), yet only modestly reduces CDI of CRAC channels (202). Second, in the dOrai structure the side chains
that interact with CaM (e.g., W76 and Y80) are predicted
to face the interior of the pore; if this holds for the human
Orai1 channel in the open state, these sites would be inaccessible to CaM without an extreme conformational change
in the pore-lining helices. Finally, the acceleration of CDI by
Y80A and Y80S mutations are not easily explained by effects on CaM binding (257), but rather suggest a role of this
residue in conformational changes in Orai1 leading to CDI.
These issues raise the possibility that Ca2⫹-CaM binding to
the isolated Orai1 NH2-terminal peptide or solubilized
Orai1 may not reflect binding to the native channel and that
NH2-terminal mutations might instead inhibit CDI through
CaM-independent mechanisms. Studies are underway to
address these issues.
The cytoplasmic II–III loop of Orai1 has been suggested to
function as a gate for CDI based on several lines of evidence
(365). Alanine substitutions at four locations within a part
of the II–III intracellular loop (151VSNV154) (FIGURE 3, TABLE 3) abolished fast inactivation while overexpression or
intracellular perfusion of a short peptide encompassing this
region diminished the steady-state ICRAC (365). These results suggested that II–III loop could function as an intracellular blocking particle to inhibit ion conduction and produce inactivation. Such a mechanism is reminiscent of the
hinged-lid inactivation model proposed for voltage-gated
Na⫹ channels in which the intracellular loop connecting
domains III and IV blocks the inner pore to produce fast
inactivation (391). However, it is uncertain whether the
II–III loop peptide functions strictly as a blocker or also
elicits inhibition through allosteric pathways. Unlike in
Na⫹ channels where short peptides containing the so-called
IFM inactivation motif restore time-dependent inactivation
(90), inhibition of ICRAC by a soluble II–III Orai1 loop
peptide is not time-dependent (365). Moreover, increasing
Table 3. Mutations that modulate STIM1 or Orai1 function
Domain
Effect of Mutation
STIM1 NH2 terminus
STIM1 COOH terminus (IDSTIM)
Altered [Ca2⫹]ER sensitivity
Suppression of CDI
Orai1 NH2 terminus
Orai1 TM1
Suppression of CDI
Acceleration of CDI
Reduced Orai1 ion selectivity
Orai1 I–II loop
Orai1 II–III loop
Orai1 TM3
Reduced Orai1 lanthanide sensitivity
Suppression of CDI
Altered Orai1 ion selectivity
Mutation
C56A
DDVDDMDEE483
D, E -⬎A or G
A73E, W76E/A/S, Y80E
Y80A/S
G98D/E
V102C/A/S/T
E106D
D110A-D112A-D114A
V151A-S152A-N153A-V154A
W176C
E190Q
475
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Reference Nos.
138
78, 190, 257
257
257
445
237
297, 396, 429
238, 396, 429
365
366
297, 396
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of such a role, the 470 – 491 fragment bound Ca2⫹ weakly
in a 45Ca overlay, and several partial neutralizations of the
region reduced CDI and 45Ca binding in parallel. However,
one partial neutralization (E482A/E483A) reduced Ca2⫹
binding yet increased the apparent Ca2⫹ sensitivity and accelerated the kinetics of CDI, casting doubt on the acidic
region as a Ca2⫹ sensor for CDI (257). More definitive tests
will be needed to discriminate the possible roles of IDSTIM in
CDI as a potential Ca2⫹ sensor or as a structural element
that acts allosterically to stabilize or promote transitions of
the channel to the inactivated state. These studies were the
first to show that STIM1 was more than just an activating
ligand for Orai1, but serves as an integral part of the channel, allowing it to respond to Ca2⫹ by inactivating; another
example of this integral role, discussed below, is that
STIM1 binding changes the ion selectivity of the Orai channel (237).
MURALI PRAKRIYA AND RICHARD S. LEWIS
the number of intact II–III loops from one to four in tetrameric Orai1 concatemers did not accelerate CDI as might be
expected from simple blockade. Additional kinetic and experimental approaches may help to better define the mechanistic role of the II–III loop in CDI.
The gate for CDI is unknown, but the Orai1 E106D
mutant exhibits little or no fast inactivation, suggesting
that inactivation might be closely linked to conformational changes in the selectivity filter (420). Very rapid
inactivation (␶ ⬍1 ms) was seen in a later study of
Orai1E106D with Ca2⫹ as the current carrier (341); however, this ultrafast inactivation differed from CDI in that
it was insensitive to intracellular BAPTA, did not depend
on the STIM-to-Orai ratio, occurred with CAD as the
stimulus, and was supported by Sr2⫹ as current carrier. A
direct blocking effect of Ca2⫹ has not been ruled out, and
the precise role of E106 in the CDI process remains to be
clarified.
A central remaining question concerns the physiological
role of CRAC channel CDI. By limiting Ca2⫹ entry, CDI
is generally thought to protect against excessive Ca2⫹
elevation, or make Ca2⫹ entry relatively constant over a
range of hyperpolarized potentials (459). However, CDI
is typically only studied under artificial conditions that
maximize its amplitude and speed (10 –20 mM Ca2⫹ and
⫺80 to ⫺120 mV), and there are no data to show
whether it alters Ca2⫹ entry at physiological levels of
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V. CRAC CHANNEL PERMEATION AND
SELECTIVITY
A. Ion Selectivity and Pore Size
CRAC channels select for Ca2⫹ ⬎1,000 times over Na⫹
under physiological conditions, placing them among the
most highly Ca2⫹-selective channels known (152). This is
an important property that enables the channels to conduct
essentially only Ca2⫹, minimizing the depolarizing effects
of cation entry. Biophysical studies indicate that their exquisite Ca2⫹ selectivity is not due to molecular sieving but
arises from ion-ion and ion-pore interactions (13, 148, 152,
191, 299). While CRAC channels readily conduct a variety
of small monovalent ions, including Na⫹, Li⫹, and K⫹ in
the absence of extracellular divalents, micromolar levels of
extracellular Ca2⫹ prevent monovalent permeation by
binding to a site in the pore (Ki ⬃20 ␮M at ⫺100 mV) (13,
152, 191, 299, 301, 378). Ca2⫹ block of Na⫹ flux is only
mildly voltage dependent (299, 420), suggesting that the
binding site is positioned near the external rim of the pore,
consistent with the superficial location of the E106 Ca2⫹
binding site (FIGURE 6C). Another key diagnostic feature of
CRAC channels is their low permeability to the large monovalent Cs⫹ (PCs/PNa ⬃0.1) (191, 301). As discussed further below, this may be related to the narrow dimensions of
the pore.
Early insights into the underlying mechanism of the CRAC
channel’s high Ca2⫹ selectivity came from observations that
mutating conserved acidic residues in transmembrane segments TM1 and TM3 altered ion selectivity. On this basis,
multiple acidic residues including E106 in TM1, E190 in
TM3, and D110/112/114 in the I–II loop were implicated in
regulating Ca2⫹ selectivity (297, 396, 429) (TABLE 3; FIGURE 3). In particular, kinetic measurements revealed that
the E106D substitution diminishes the association rate of
Ca2⫹ binding, thereby lowering Ca2⫹ block affinity, consistent with the idea that the affinity of Ca2⫹ binding at E106
is critical for Ca2⫹ selectivity (420). These findings led to
proposals that residues in both TM1 and TM3 flank the
CRAC channel pore, with the acidic residues within these
segments forming components of the CRAC channel selectivity filter. As described below, this view has been significantly revised by subsequent cysteine scan studies and the
crystal structure of Drosophila Orai that revealed that TM3
does not flank the pore.
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Heterologous Orai3 and Orai2 currents generally inactivate more rapidly than Orai1 currents (110, 190, 200), but
the reasons for this difference are not yet clear. A troublesome complication in many studies is that CDI appears to
be strongly affected by the degree of STIM binding to
Orai (147, 340), raising the possibility that Orai mutations or sequence differences among homologs may alter
CDI indirectly by changing the STIM:Orai binding affinity and stoichiometry, which is not generally known or
controlled. In one study, Orai1 current failed to inactivate, but substituting the Orai3 COOH terminus in
Orai1 restored CDI (190). Neutralization of three glutamates in the COOH terminus of Orai3 (E281, E283, and
E284) inhibited CDI, which was interpreted to indicate a
role of these residues as a CDI Ca2⫹ sensor. However,
mutation of two of the three glutamates in Orai3 significantly increased fast inactivation, as did mutation of all
three analogous residues in Orai2. In view of the role of
analogous glutamate residues in STIM1-Orai1 interactions (39, 40), an alternative explanation is that these
residues affect CDI through effects on STIM1 binding.
Further complexity is indicated by a study of Orai1Orai3 chimeras, suggesting that all three cytosolic domains may interact to determine CDI characteristics
(110) although the nature of these interactions is not yet
clear.
Ca2⫹ and Vm. In the case of CaV channels, much of what
is known about the functions of CDI comes from studies
of channel mutants lacking CDI, such as the Timothy
Syndrome mutation of CaV1.2 (288, 427). Analogous
studies of transgenic animals lacking CRAC channel CDI
may yield important insight into the functions of CDI in
SOCE-mediated events.
STORE-OPERATED CALCIUM CHANNELS
Cysteine scanning studies that followed have broadened
our understanding of what regions in Orai1 regulate ion
conduction. In one study, Prakriya and colleagues (238)
used probes differing in size, charge, and chemistry to probe
the geometry, flexibility, and electrostatic potential of
CRAC channel pore sites. In a second study, Hogan’s group
(453) examined inter-subunit disulfide crosslinks of TM1
residues. These studies revealed that the CRAC channel
pore is formed by the TM1 segments, with a reactivity pattern consistent with E106, V102, G98, L95, and R91 facing
the pore (FIGURE 6C). There was negligible reactivity to
bulky MTS reagents, yet significant coordination by these
residues of the small probe, Cd2⫹, suggesting that the
CRAC channel pore is narrow along much of the length of
TM1 (238). This feature may produce heightened friction
to diffusing ions, thereby accounting for the low permeability of CRAC channels to large cations (⬎3.8 Å) and their
low unitary conductance. Interestingly, the I–II loop segments interact tightly with both large (⬎8 Å) and small (⬍3
Å) probes of different charge, suggesting that the loops form
an outer vestibule with sufficient flexibility to accommodate
ions of different size and charge (238). No significant reactivity was seen at E190 and the other residues of TM3,
indicating that this segment is not pore-lining. Moreover,
individual modification of D110, D112, and D114 in the
I–II loop by Cys or positively charged compounds did not
affect ion selectivity. Thus E190 and the acidic residues in
the I–II loop do not form high-affinity Ca2⫹ binding sites as
was originally thought (297, 396), but rather E106 in the
centrally located TM1 segment solely underlies Ca2⫹ selectivity. Collectively, these studies provided the first step towards building a structural model of the CRAC channel
pore and are quite congruent with the later dOrai crystal
structure (153) which reveals Ca2⫹ and Ba2⫹ densities close
to E178 (E106 in human Orai1) (FIGURE 6C).
How does E106 regulate ion permeation? Biophysical models indicate that a single Ca2⫹ binding site in the pore is
inadequate to describe the movements of Ca2⫹ in CaV and
CRAC channels (68, 334, 422). Rather, multiple closely
spaced ion binding sites are required to accelerate Ca2⫹
conduction by creating electrostatic repulsion between
closely spaced ions in the pore (68, 334, 422). This
knock-on mechanism for Ca2⫹ permeation is supported by
recent crystallographic analysis of a Ca2⫹-selective bacterial NaV channel with multiple Ca2⫹ binding sites in the
selectivity filter (383). However, whether such a structural
model can be to CRAC channels is unclear. The modified
Ca2⫹-selective NaV channel was engineered with two
closely spaced rings of Asp residues in the selectivity filter
(383), yet the available structural evidence in CRAC
channels shows only one obvious high-affinity Ca2⫹
binding site (E106), which is consistent with functional
and mutational effects on Ca2⫹ block (299, 420, 422).
One plausible scenario that could satisfy the requirement
for multiple ion sites demanded by theoretical permeation models is that the side chains of the six Glu residues
at E106 cluster into two groups along the axis of the pore
to form two distinct binding sites. The close proximity of
these sites could yield electrostatic repulsion between
Ca2⫹ ions to enhance permeation, analogous to the scenario proposed for CaV channels (92).
Finally, it is worth noting that despite qualitative similarities in the Ca2⫹ selectivity and permeation between CRAC
and CaV channels, the architecture of the two channel families is markedly different. Whereas CRAC channels display
a long narrow pore flanked by the TM1 segment (153, 238),
CaV channels have a wide pore with an inner vestibule large
enough to accommodate very large thiol reagents and one
or more hydrated ions (451). Moreover, blockade of Na⫹
conduction occurs at substantially lower Ca2⫹ concentrations in CaV channels (0.7 vs. 20 ␮M) (1, 299), suggesting
that high-affinity Ca2⫹ binding to the selectivity filter may
not be the sole mechanism by which CRAC channels
achieve high Ca2⫹ selectivity. Consistent with this possibility, a recent study employing Eyring-rate theory analysis
has postulated that in addition to Ca2⫹ binding at the selectivity filter, CRAC channels acquire high Ca2⫹ selectivity
by restricting the rate of ion flow (for both preferred and
nonpreferred ions) by high entry and exit energy barriers
(422). High-energy barriers for ion flow are likely to be
related to the narrow dimensions of the CRAC channel pore
because conditions that enlarge the pore (mutations or
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Measurements of the permeability of a series of organic
monovalent cations indicate that the narrowest region
across the pore for native CRAC as well as overexpressed
recombinant Orai1 and Orai3 channels is ⬃3.8 Å (299,
420, 422). This is considerably narrower than the dimensions measured using comparable methods for Cs⫹-permeable CaV channels and TRP channels (⬃6 Å) raising the
prospect that steric hindrance to the movement of Cs⫹ (dehydrated diameter ⬃3.4 Å) is responsible for its low permeability in CRAC channels. Consistent with this notion, pore
mutations that increase Cs⫹ permeability also concomitantly widen the pore (420). Moreover, the 2-APB-gated
Orai3 pore, which is readily permeable to Cs⫹ (PCs/PNa
⬃1.0), exhibits a considerably larger minimal diameter of
⬃5.6 Å (422). In general, decreases in Ca2⫹ selectivity of
pore mutants correlate well with increases in Cs⫹ permeability, suggesting that the structural changes that cause pore
widening concurrently affect both Ca2⫹ and Cs⫹ selectivity
in CRAC channels (420). What physical features limit the
pore diameter? In the dOrai crystal structure the distance
between the oxygen atoms of the carboxylates of the Glu
ring, which appears to be the narrowest region of the pore,
is ⬃6 Å, considerably larger than expected from pore sizing
experiments. Thus the structural basis of the limiting pore
diameter as measured by permeation of organic cations and
its precise location along the ion conduction pathway remains to be determined.
MURALI PRAKRIYA AND RICHARD S. LEWIS
channel activation by 2-APB) elicit concomitant increases in
ion flux rates and lower Ca2⫹ selectivity (422).
B. Rectification
The biophysical basis of CRAC channel rectification is
largely unknown. Unlike many K⫹ channels, inward rectification in CRAC channels is not conferred by Mg2⫹ or
polyamine blockade of outward currents (13, 177, 178,
301). Moreover, inwardly rectifying currents are also associated with pore mutants of Orai1 and Orai3 having altered
ion selectivity and with 2-APB-activated Orai channels, especially under divalent-free conditions (236, 420, 422), indicating that the residues in question (such as E106, V102,
R91) are not involved in conferring this biophysical attribute. Two conditions that do appear to alter inward rectification are permeation by ammonium derivatives such as
hydroxyl ammonium or methyl ammonium (299), and the
deletion of the Orai1 NH2 terminus (Orai1 ⌬73– 85
V102C) (236). These observations raise the possibility that
asymmetry of the ion conduction pathway and the interaction of ions with the inner pore may contribute to rectification.
C. Unitary Conductance
One of the most distinctive features of CRAC channels and
one that has been a cause both of fascination and frustration to CRAC channel biophysicists is its extremely low
unitary conductance. CRAC channels produce very small
current fluctuations, and the unitary currents are too small
to resolve by the patch-clamp method. Fluctuation analysis
of whole cell CRAC currents in T cells has yielded unitary
conductance estimates of 9 fS in 2 mM extracellular Ca2⫹
1410
How do these estimates of the unitary conductance compare to other channels? Unitary conductances of most K⫹
and Na⫹ channels are in the 4 –30 pS range, with mammalian Ca2⫹-activated BK channels achieving ⬎200 pS (143).
The closest functional relatives, the Cav L-type channels,
show unitary conductances of 4 –10 pS for Ca2⫹ and 85 pS
for Na⫹ (141), corresponding to ion turnover rates ⬎100fold larger than those of CRAC channels.
Kinetic measurements of Ca2⫹ blockade of the Na⫹-CRAC
currents indicate that the association rate of Ca2⫹ for its
block site is ⬃4 ⫻ 106 M⫺1·s⫺1 (299, 420, 422). This rate,
which is conserved both in native T-cell and in recombinant
STIM1-gated Orai1 and Orai3 channels, is 100-fold slower
than the association rate of Ca2⫹ in L-type Cav channels
(⬃4 ⫻ 108 M⫺1·s⫺1; Ref. 184) and the ion entry rates seen
in K⫹ channels, which are commonly close to the diffusion
limit ⬃109 M⫺1 s⫺1 (143). This striking difference between
CRAC and the other channels indicates the existence of a
significant energy barrier for ion access from the extracellular space to the selectivity filter in CRAC channels, a
feature that could in principle account for their low unitary
conductance. While the molecular/structural basis of this
barrier is currently unknown, one simple possibility is that
it reflects a dehydration step that must occur before Ca2⫹
ions can interact with sites in the CRAC channel.
Curiously, a recent molecular dynamics simulation of ion
permeation in the constitutively active V174A dOrai mutant has suggested that Na⫹ influx at hyperpolarized potentials is coupled to concomitant Cl⫺ efflux (89). Efflux of Cl⫺
ions was postulated to actively promote Na⫹ permeation by
coordinating charge on Na⫹ ions, in effect constituting an
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Inward rectification of the current-voltage relationship is a
consistent feature of CRAC channels. While not as steep as
that of inwardly rectifying K⫹ channels (265), the rectification is nevertheless more prominent than in CaV channels
(143) and is widely employed as an identifier for CRAC
channels. It is worth noting that from an experimental
standpoint, many studies have overemphasized inward rectification by applying voltage ramps (e.g., ⫺100 to ⫹100
mV) directly from a positive holding potential, which can
trigger CDI at negative potentials and distort the currentvoltage relation. Inward rectification is also apparent in the
monovalent CRAC current recorded with symmetrical concentrations of Na⫹ in the internal and external solutions
(13, 299), indicating that it is not simply due to the
⬎10,000-fold concentration gradient of Ca2⫹ across the
plasma membrane. One consequence of inward rectification is that it would be expected to enhance the effect of
hyperpolarizing voltage changes on the rate of Ca2⫹ entry,
which may be important for crosstalk between SOCE and
pathways that influence the membrane potential.
and 24 fS in isotonic Ca2⫹ solution (458). These early measurements were not corrected for the channel Po which subsequent nonstationary analysis showed to be high (Po
⬃0.8), at least under divalent-free conditions when the
channels conduct Na⫹ ions (171, 297, 422). Normalization
to Ca2⫹-conducting solutions is difficult since direct measurements of Po for Ca2⫹-conducting channels are unavailable. However incorporating estimates of channel Po in
Ca2⫹-conducting solutions (corrected for inactivation)
would be expected to increase the unitary conductances to
20 and 33 fS in 2 and 110 mM extracellular Ca2⫹ solutions,
respectively (299). In divalent-free solutions, the unitary
conductance of the Na⫹ current rises to ⬃0.7 pS, consistent
with the increase of the macroscopic Na⫹ CRAC current in
the absence of divalent ions (299). The single-channel Na⫹
current in divalent-free conditions (⬃⫺0.1 pA at ⫺110 mV)
corresponds to an ion turnover rate of 7 ⫻ 105 Na⫹/s (299).
While this is slower than the turnover rates observed for
most other ion channels, the Na⫹ flux rate is faster that any
known carriers, providing the initial evidence that the
CRAC channel operates as a channel rather than a transporter.
STORE-OPERATED CALCIUM CHANNELS
“anion-assisted” permeation mechanism. These simulations present interesting possibilities for the contributions
of the basic residues in the inner hOrai1 pore (e.g., R91,
K87, R83) for counter-ion movements and regulation of
cation permeation. However, at present, the applicability of
these conclusions to CRAC channels is uncertain, given the
lack of experimental evidence for Cl⫺ currents through
CRAC channels.
D. Coupling Between Gating and Permeation
in the CRAC Channel
1. Calcium-dependent potentiation
Although the primary signal for activation of native CRAC
channels is depletion of intracellular Ca2⫹ stores, extracellular Ca2⫹ plays a crucial role in optimizing CRAC channel
activity through a process referred to as calcium-dependent
potentiation (CDP) (54, 299, 378, 461). This phenomenon
is most clearly revealed when Ca2⫹ is readded to cells whose
stores have been depleted in the absence of extracellular
Ca2⫹. Upon the readdition of Ca2⫹, ICRAC appears in two
kinetically different stages. A small fraction of the current
appears instantaneously, representing channels active at the
time of Ca2⫹ readdition (461). This instantaneous current is
followed by a severalfold exponential increase in current
over the following 10 –20 s. Conversely, the removal of
extracellular Ca2⫹ by perfusing divalent-free (DVF) solutions diminishes CRAC channel activity by ⬃80% over tens
of seconds due to reversal of CDP, or depotentiation.
CDP and depotentiation are useful identifiers of native
CRAC channels, but the molecular underpinnings of these
related phenomena are largely unknown. As noted by
Zhang and Cahalan (444), depotentiation of the
Na⫹-ICRAC varies significantly between cells, suggesting
that the process is regulated by components other than the
channel itself, including the relative levels of STIM1 and
Orai1. Noise analysis suggests that CDP occurs through the
2. STIM1 binding affects Orai1 ion selectivity
A second example of the functional coupling between permeation and gating is revealed by the STIM1-mediated alterations in the ion selectivity of constitutively active Orai1
mutant channels. In particular, STIM1 markedly modulates
the ion selectivity of the constitutively open V102X mutant
channels (where X ⫽ C, A, S, or T) (80, 237). STIM1-free
V102C mutant channels exhibit poor Ca2⫹ selectivity and
allow permeation of Na⫹, Cs⫹ and several other large cations that are normally impermeable through CRAC channels (80, 237) (TABLE 3). Interaction of the mutant channels
with STIM1 restores high Ca2⫹ selectivity while significantly narrowing the pore to more closely resemble the
dimensions of wild-type ORAI1 channels. The tuning of
ORAI1 ion selectivity by STIM1 is not unique to the V102X
mutant channels, but is also seen in wild-type ORAI1 channels as the amount of STIM1 bound to ORAI1 is increased
(237), suggesting that the V102X mutations may mimic a
native intermediate channel activation state. Although the
connection between STIM1 binding and the alterations in
ion selectivity remains to be clarified at a structural level,
one possibility is that movement of the NH2 terminus consequent to STIM1 binding allosterically affects the conformation of the selectivity filter located a short distance away
in TM1.
The coupling of permeation and gating in CRAC channels
contradicts conventional assumptions on the separation of
gating and selectivity in ion channels and suggests that there
is much more happening in the vicinity of the selectivity
filter than originally thought. The presence of a gating structure (V102) located in proximity to the selectivity filter
(E106) may enable conformational coupling between the
two structures during gating. From a physiological standpoint, the ability of CRAC channels to conduct Na⫹ under
conditions of low STIM1 occupancy may expand their potential functions when activated by subsaturating concen-
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From a physiological standpoint, the small unitary conductance of CRAC channels coupled with its high Ca2⫹ selectivity implies that CRAC channels function as a highly efficient and selective Ca2⫹ entry mechanism yielding negligible Na⫹ influx. A likely physiological benefit of such a
feature is that it limits membrane depolarization, thereby
diminishing the metabolic demands of pumping out Na⫹
over the long durations in which CRAC currents are generally active, while boosting specificity for activation of particular cellular effector functions. There are numerous examples of functional coupling of CRAC channels to downstream effectors, including c-fos activation (82), NFAT
activation (164, 360), leukotriene production (46), and
PMCA activation (18) driven by highly localized Ca2⫹ elevations that may be attributed to the low unitary conductance of CRAC channels.
recruitment of CRAC channels from a “silent” state to one
of high open probability, analogous to that seen during
channel activation following the depletion of intracellular
stores (299), suggesting that CDP and activation by store
depletion may share common biophysical gating mechanisms. The Ca2⫹ binding site for CDP is probably not intracellular, because intracellular BAPTA and EGTA do not
inhibit the process and extracellular Ni2⫹, a blocker of
CRAC channels, can substitute for Ca2⫹ in keeping the
channels potentiated (54, 461, but see Ref. 378). Several
lines of indirect evidence hint that the CDP site (or a key
regulatory mechanism) lies in the pore: the extent of CDP
increases with hyperpolarization (461), and CDP is profoundly influenced by permeant ions (Prakriya and Lewis,
unpublished data). Moreover, the E106D mutation, which
affects ion selectivity, nearly completely eliminates depotentiation of Na⫹-ICRAC (420).
MURALI PRAKRIYA AND RICHARD S. LEWIS
trations of STIM1 (although their activity under these conditions would likely be greatly reduced).
VI. CRAC CHANNEL MODULATION
In accordance with the multistep nature of the CRAC channel activation process, a panoply of influences regulate the
activity of CRAC channels. Below, we summarize some key
modes of CRAC channel modulation that have attracted
attention, highlighting the molecular mechanisms of the
underlying process wherever known.
During prolonged elevations in [Ca2⫹]i, ICRAC declines
from a combination of deactivation due to store refilling
and a process called Ca2⫹-dependent slow inactivation.
Store refilling triggers deactivation of the channel as STIM1
binds luminal Ca2⫹ and reverts to its resting state. Slow
inactivation is mechanistically distinct from deactivation
and can be observed when store refilling is prevented by
SERCA inhibitors like TG (276, 460). It appears during
whole cell recordings as a decline in current over tens of
seconds as [Ca2⫹] rises to levels above ⬃200 nM. The site of
action of Ca2⫹ is global rather than local to CRAC channels, because slow inactivation is strongly inhibited by slow
intracellular Ca2⫹ buffering conditions that can only capture incoming Ca2⫹ effectively at distances ⬎100 nm from
the channel (460). Thus, while its molecular mechanism is
not known, slow inactivation is easily distinguished from
fast CDI by its slow kinetics and its dependence on global
[Ca2⫹]i, and is distinct from deactivation by its independence of Ca2⫹ store content.
Interestingly, mitochondria can modulate the sensitivity of
CRAC channels to slow inactivation. Conditions that deenergize mitochondria also reduce CRAC channel activity
(149, 150). In whole-cell recordings, slow inactivation can
be attributed to mitochondrial depolarization, as it can be
prevented by supplying the cell with metabolic substrates
and under these conditions be reinstated by mitochondrial
inhibitors (118, 149). The molecular basis of mitochondrial
maintenance of CRAC channel activity is not entirely clear.
However, it is generally thought that, by efficiently buffering Ca2⫹ (320), mitochondria prevent the inactivation of
SOCE by competing with the slow inactivation sites for
intracellular Ca2⫹ (149, 150). In addition, mitochondria
may also facilitate SOCE by releasing pyruvate or ATP (14,
249, 277, 278).
The ability of mitochondria to prolong SOCE may depend
on their proximity to CRAC channels. Hoth and colleagues
(311) have reported that during periods of prolonged Ca2⫹
entry, mitochondria move closer to the PM, and preventing
1412
B. Phosphorylation and Trafficking
An increasing number of studies implicate phosphorylation
as an important inhibitory influence on SOCE (282, 356,
436, 437). Early studies using antagonists of PKC suggested
that PKC-mediated phosphorylation of a SOCE protein inhibits ICRAC (281). Kawasaki et al. (168) subsequently
showed that Orai1 itself can be phosphorylated by PKC in
a reaction that requires calcium, didecanolglycerol, and
phosphatidylserine (168). Alanine substitutions at several
potential phosphorylation sites (S25/S27 and S30/S25 in the
Orai1 NH2 terminus) modestly enhanced steady-state
ICRAC, consistent with an inhibitory action of phosphorylation as well as observations that the sites are phosphorylated both constitutively and following store depletion
(168). PKC-mediated inhibiton of Orai1 may be a widespread mechanism to limit CRAC channel activity through
Ca2⫹-dependent feedback. However, this remains an underexplored question, and in particular, the physiological
conditions under which PKC phosphorylation of Orai1 dynamically regulates CRAC channel activity have not been
identified.
Phosphorylation of STIM1 has also been linked to inhibition of SOCE. Store depletion induces a rapid Tyr
phosphorylation of STIM1 in platelets, and based on
effects of kinase antagonists, Lopez et al. (207) suggested
that this is likely mediated by Src/Abl kinases and may be
central for STIM1-Orai1 association. More recently, Putney and colleagues (356) found that phosphorylation of
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A. Slow Ca2ⴙ-Dependent Inactivation
and Mitochondria
this translocation by nocodazole enhances slow inactivation (311). An attractive mechanism to explain the relocalization of mitochondrial towards sites of active CRAC
channels involves the detachment of the mitochondria-attached kinesin motor from microtubules through its Ca2⫹dependent binding to the EF-hand protein miro (403, 431).
A similar translocation to the immune synapse that forms
between T cells and antigen-presenting cells may enhance
Ca2⫹ signaling during T-cell activation (312, 339). However, the importance for SOCE of mitochondrial motility
and distribution relative to Ca2⫹ influx sites in general has
been questioned in several other studies (108, 176, 259).
While the basis for the different outcomes is not yet clear, a
major complication appears to be that mitochondria modulate SOCE through multiple interacting pathways, none of
which is well understood mechanistically (75). Mitochondria have been reported to increase SOCE by enhancing
IP3-mediated store depletion (117), but they also can promote store refilling (6), and mitochondrial depolarization
may further inhibit SOCE by hindering the translocation of
STIM1 to ER-PM junctions (350). Additionally, mitochondria may indirectly regulate CRAC channel activity through
their ability to produce reactive oxygen species (see below).
Unraveling the basis for these and other effects remains a
major challenge.
STORE-OPERATED CALCIUM CHANNELS
multiple sites in STIM1, in particular S486 and S668,
potently inhibits STIM1 translocation to the ER-PM
junctions and SOCE, which could explain longstanding
observations that SOCE is suppressed during mitosis
(303). Interestingly, the loss of SOCE during mitosis does
not appear to be necessary for normal cell-cycle progression, but the mitosis-specific phosphorylation of STIM1
reduces affinity for EB1 and is needed to release the ER
from the mitotic spindle during cell division (355). The
suppression of SOCE observed during meiosis is thought
to occur through a different mechanism, involving internalization of Orai1, likely through a caveolin- and dynamin-dependent endocytic pathway (436, 437). Thus
changes in both STIM1 and Orai1 proteins are likely
involved in modulating SOCE during the cell cycle.
Cysteine thiol groups make STIM1 and Orai1 sensitive to
multiple modes of redox modulation (28). The ensuing
modulation has implications for a wide range of cellular
effector functions, especially under pathological conditions
such as hypoxia (20, 131, 220, 258). In one mode of regulation, oxidation is reported to activate STIM1 and elicit
Ca2⫹ influx even when stores are replete (138). Activation
was attributed to S-glutathionylation of a Cys residue located close to EF-SAM (Cys 56), resulting in reduction of
the [Ca2⫹]ER sensitivity of the EF-hand and consequent unfolding of this domain. However in the same study, a mutation predicted to eliminate S-glutathionylation (C56A)
was also reported to activate STIM1 (but see Ref. 305).
Further work will be needed to resolve these contradictory
findings; mechanisms of redox modulation are often complex, as they reflect the balance between ROS and antioxidant scavenger levels (317), as well as the potential contributions of additional reactive cysteines located elsewhere in
the protein.
In a second mode of redox-dependent regulation, Niemeyer
and colleagues (29) discovered that Orai1 is directly inhibited by oxidation of a Cys residue (C195 in hOrai1) located
in the extracellular III–IV loop. This residue is absent in
Orai3, making it insensitive to H2O2-induced inhibition.
Interestingly, the differential sensitivity of Orai1 and Orai3
channels to oxidative inhibition may confer resistance to
oxidizing environments as naive T lymphocytes differentiate into effector cells and increase their expression of Orai3
(29). The opposing effects of hypoxia and oxidative stress
on STIM1 activation and Orai1 function suggest that the
overall effect of redox modulation on Ca2⫹ signaling is
likely to be complex and shaped by several feedback and
feedforward loops between oxidant and Ca2⫹ signaling
pathways (425).
Acidification of the extracellular milieu commonly occurs
in normal tissues during episodes of intense metabolic activity, hypoxic insults, and inflammation, as well as during
neoplastic transformation where pH decline is correlated
with tumor aggressiveness (185, 405). Acidification of the
extracellular pH strongly inhibits ICRAC in macrophages
with a pKa of ⬃8.2 (217). CRAC channels are also inhibited
by intracellular acidification with a pKa of 6.8, resulting in
an approximately fivefold inhibition between pH 8.2 and
6.2 without a noticeable change in ion selectivity (169).
Intracellular acidification also speeds the rate and extent of
depotentiation of Na⫹-ICRAC (169). Interestingly, the pKa
of the extracellular and intracellular pH effects are very
similar to those reported for L-type CaV channels, raising
the possibility that the molecular mechanisms of pH regulation could be similar in the two classes of channels. In CaV
channels, the glutamates at the selectivity filter were suggested as a possible molecular target of extracellular pH
regulation (49). Likewise, Scrimgeour et al. (341) discovered that the E106D mutation in Orai1 strikingly reduced
the extracellular pH dependence of Orai1, suggesting that
as in CaV channels, the Glu residues controlling Ca2⫹ selectivity in Orai1 also account for the block by extracellular
protons. Although Glu has a pKa of ⬃4.3 in solution, hydrogen bonding of carboxylates within proteins can elevate
the pKa to as high as 8.8 (106), potentially explaining the
modulation of CRAC channels by high pH. pH modulation
could provide a feedback regulatory mechanism to limit
Ca2⫹ overload during inflammation or hypoxia and control
cell damage under pathological conditions (220).
E. Temperature
It has long been known that the activation of ICRAC is
steeply temperature dependent, displaying an abrupt decline around 21°C (361). This nonlinearity appeared specific to the channel activation process, because it was not
observed once channels were already activated (361). The
nonlinear dependence on temperature was suggested to result from dependence of the activation machinery on the
lipid environment, a premise that remains to be tested.
Based on current knowledge of STIM1 and Orai1 and the
channel activation process, it is possible that cooler temperatures may impede unfolding of STIM1 to the active form
(see above), but once a functional STIM-Orai connection is
made, cooling would be expected to have a smaller effect.
More recently, native CRAC channels and heterologous
STIM1 and Orai1 have been shown to activate in response
to heating without changes in ER Ca2⫹ content (417).
STIM1 appears to be the locus of this effect, as heating cells
above 35°C causes STIM1 clustering in the absence of store
depletion. Interestingly, the heat-induced clusters of STIM1
do not trap or activate Orai1 at ER-PM junctions and Ca2⫹
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C. Redox Modulation of STIM and Orai
Function
D. pH
MURALI PRAKRIYA AND RICHARD S. LEWIS
influx only occurs during a cool-down phase as the temperature is lowered below 37°C. These interesting effects provide new insights into the energetics of STIM1 and Orai1
activation: formation of STIM1 puncta may result from the
unfolding of STIM1, which allows plasma membrane binding through its PBD, but heat induced unfolding may not
activate the CAD domain or high temperature may destabilize CAD-Orai1 binding such that cooling is required for
trapping and activation of Orai1 to occur. Xiao et al. (417)
speculate that this pathway of STIM1 gating may contribute to lymphocyte activation during fever, as lymphocytes
cycle between central areas where they are exposed to the
higher core body temperature and cooler peripheral sites.
Of the many early candidates invoked to explain the link
between store depletion and CRAC channel activation, the
release of a diffusible activator from the ER, termed calcium
influx factor (CIF), attracted the most attention (316). The
existence of CIF has not been formally confirmed, nor has
the activity been purified. Ultimately, the discovery that
STIM1 and Orai1 bind directly to each other (284) and that
this binding is sufficient to activate Orai1 (453) argue
against the necessity for such a messenger. However, a modulatory role for CIF or one that operates in parallel with
direct coupling to potentiate SOCE under physiological
conditions cannot be formally ruled out. A detailed scheme
has been proposed in which STIM1 controls the release or
synthesis of CIF, which would displace CaM from iPLA2,
releasing the enzyme from inhibition and allowing it to
generate lysolipids in the PM that activate CRAC channels
(30, 66, 135, 353). Bolotina (30) has suggested that STIM
and Orai may be sufficient for SOCE only when both are
overexpressed, but that iPLA2 is required for activation at
endogenous levels of expression. One difficulty in testing
this model is that iPLA2 has multiple isoforms that are
involved in many functions, including homeostasis of intracellular organelles like the ER and Golgi (35, 318). A number of fundamental challenges remain to establish a role for
a diffusible messenger such as CIF: isolation and purification of an active substance capable of activating or modulating Orai channels, the demonstration that it is produced
and/or released in response to store depletion, identification
of the enzymatic pathway that generates it, identification of
its biochemical target, and identification of the conditions
under which it is active (i.e., does it work in isolation, or
only in concert with STIM1).
G. ATP
Several early studies showed that CRAC channel activity is
highly dependent on cellular ATP (115, 157, 225). More
recent work by Tepikin and colleagues (56) has extended
upon these findings by tracking the movements of STIM1
1414
H. STIM/Orai-Interacting Proteins
While STIM1 is able to activate Orai1 without additional
proteins in vitro (453), this does not rule out the possibility
that these functional interactions are modulated in vivo by
auxiliary proteins or environmental conditions. An apt
analogy can be made to the membrane fusion process,
which can be driven by SNARE proteins alone in vitro, but
which in vivo involves a large number of additional proteins
that confer essential properties and control (such as high
speed, low noise, Ca2⫹ dependence, etc.) (319). There is
indirect evidence that STIM and Orai interact with other
proteins; for example, store depletion slows the diffusion of
STIM1 even at locations distant from the PM (416), and
Orai1 is excluded from cross-bridged ER-PM junctions
having a gap of 8 –9 nm, which would be expected to otherwise accommodate the extension of the Orai1 cytoplasmic domains (394). An increasing number of STIM- or
Orai-binding proteins have been isolated through tandem
affinity purification, mass spectrometry/proteomics, and
RNAi screens. These include STIM1-binding proteins such
as CRACR2A (364), P100 (412), junctate (363), Golli (97,
98, 401), POST (180), SARAF (274), the ER oxidoreductase ERp57 (305), calnexin (330), and CaM (17, 266).
Orai-binding proteins include CaM (257), CRACR2A
(364), POST (180), and SPCA2 (99). These regulators and
interaction partners are in many cases thought to fine tune
the activity of CRAC channels, but the mechanistic details
have not yet been worked out. For more information, we
refer the reader to the original papers; here we discuss several of the more well-characterized modulator proteins.
Septins were identified as important regulators of SOCE
from a genome-wide siRNA screen for inhibitors of SOCEmediated NFAT signaling in HeLa cells (343). Knockdown
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F. Diffusible Activator
and Orai1 during ATP depletion. They show that as acute
inhibition of mitochondrial and glycolytic ATP production
causes ATP and PIP2 levels in the PM to decline, Ca2⫹ leaks
from the ER, stimulating the accumulation of STIM1 and
Orai1 at ER-PM junctions. As in the earlier studies, the
influx of Ca2⫹ was significantly reduced under ATP-depletion conditions. These results demonstrate that ATP is apparently not required for the events leading up to formation
of STIM-Orai complexes (consistent with the diffusion-trap
mechanism discussed in sect. III) but that the function of the
channel is ATP sensitive in some way, perhaps related to the
loss of PIP2 or other phospholipids from the PM. The identification of the molecular targets of ATP regulation may
not only reveal additional events in the activation of CRAC
channels but also improve our understanding of the biological role of this regulation for cells. ATP regulation of
CRAC channel activity could potentially serve to decrease
metabolic load, in essence providing protection against excessive Ca2⫹ influx under ATP-depleting conditions such as
hypoxia.
STORE-OPERATED CALCIUM CHANNELS
experiments demonstrated that septins 2, 4, and 5 are necessary to maintain the diffuse organization of inactive
Orai1 in the PM when ER stores are full. Septins also facilitated the translocation of STIM1 to ER-PM junctions and
stabilized Orai1 clusters after store depletion, in line with
their requirement for robust SOCE. Interestingly, septins
are also required for the local depletion of PIP2 from
ER-PM junctions that occurs when Orai1 is recruited to
puncta, perhaps playing a role in enhancing stability of the
STIM-Orai complex. Thus septins appear to coordinate the
movements and interaction of STIM1 and Orai1 at several
different stages of SOCE (343).
CRAC regulatory protein 2A (CRACR2A) is a cytoplasmic, EF-hand containing protein that was discovered as
an Orai1 binding partner in a large-scale affinity screen
(364). The protein has been shown to bind directly to
STIM1 and Orai1 NH2 terminus and suggested to form a
ternary complex to stabilize the interaction between
Orai1 and STIM1. Complex formation facilitates clustering of STIM1 and Orai1 at the ER-PM junction and
enhances SOCE (19, 364). Ca2⫹ binding to the EF hand
of CRACR2A promotes its dissociation from STIM1 and
Orai1. Thus CRACR2A may participate in the initial
formation of STIM-Orai complexes, but may dissociate
once channels open and the local [Ca2⫹]i in the junction
rises to high (␮M) levels.
SARAF (SOCE-associated regulatory factor), unlike the three
proteins described above, is thought to promote the disassembly of STIM1-Orai1 complexes upon store refilling. SARAF
was discovered serendipitously from a screen for cDNA candidates that influence mitochondrial Ca2⫹ homeostasis (274).
SARAF is an ER membrane protein that binds to both STIM1
and STIM2 and translocates to ER-PM junctions in a STIMdependent manner following store depletion. Its luminal domain is proposed to sense [Ca2⫹]ER (though the mechanism is
unknown) (274), while its cytosolic region exerts an inhibitory
action on SOCE possibly by binding to the CAD region and
preventing it from binding to and activating Orai1 (161). A
recent study suggests that SARAF binding to STIM is complex
Partner of STIM1 (POST) is a protein with 10 putative transmembrane-spanning segments that was identified by affinity
purification of Orai1 (180). Like STIM1, the majority of
POST protein is located in the ER membrane with 5–10%
residing in plasma membrane. POST is reported to associate
with STIM1 and a number of transporters (SERCA, PMCA,
Na⫹-K⫹-ATPase, and nuclear transporters) following store
depletion and migrate to ER-PM junctions. Although POST is
not required for STIM1-Orai1 interaction and does not influence CRAC channel activation, it appears to inhibit PMCA
activity to some degree, possibly enhancing local [Ca2⫹]i
within the ER-PM junction. An intriguing possible function
based on its association with karyopherins is that POST may
mediate STIM- and [Ca2⫹]ER-dependent modulation of nuclear transport (180).
VII. PHARMACOLOGY
The complexity of the CRAC channel activation process, involving a choreographic sequence of protein-protein and
ER-PM interactions, offers an abundance of potential targets
for pharmacological regulation of channel activity (211). Historically, inhibitors such as SKF96365, La3⫹, and 2-APB were
widely used to identify and assign functions to CRAC channels
in various tissues. However, the selectivity of these SOC inhibitors is generally weak, and with the exception of La3⫹, the
mechanisms by which they affect CRAC channel function are
unclear. Newer reagents have been identified that appear to be
much more selective for CRAC channels, including some purported to be selective for particular isoforms (e.g., Orai1)
(196). Still, the molecular pharmacology of most compounds
is inadequately understood, and the site of action, in most
cases, remains unclear. The pharmacology of CRAC channels
has been reviewed in detail elsewhere (79, 160, 280, 309, 379).
A historical narrative of this topic underscores the obvious but
often overlooked reality that although pharmacology can be
useful for probing ion channel mechanisms, lack of specificity
can undermine utility in probing physiological fuctions. Here
we describe the most widely used compounds and reagents,
focusing on current mechanistic insights into their possible
modes of action (summarized in TABLE 4).
A. Lanthanides
The trivalent ions La3⫹ and Gd3⫹ block endogenous CRAC
currents with high affinity (Ki ⫽ 20 – 60 nM) (9, 324, 430).
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Junctate, a Ca2⫹-binding ER membrane protein, was identified through affinity purification as a STIM1 binding partner with a role in recruiting STIM1 to ER–PM junctions
(363). Overexpression of an EF hand mutant of junctate
with reduced luminal Ca2⫹ binding facilitates STIM1 clustering without store depletion and recruits STIM1 to
ER-PM junctions without phosphoinositide or Orai1 interaction, leading to activation of CRAC channels. Srikanth et
al. (363) suggest that formation of STIM1 clusters via binding to junctate indicates an additional pathway for STIM1
accumulation at the ER-PM junction, and under physiological conditions where Orai1 and STIM1 concentrations are
low, it may be required to ensure efficient assembly and
activation of CRAC channels.
and regulated by a number of other molecules including PIP2,
septins, and the mammalian extended synaptotagmin protein,
E-Syt1 (218). The requirement for PIP2 may restrict SARAF
modulation of STIM1-Orai1 primarily to PIP2-rich domains
(218). These features may explain SARAF’s ability to facilitate
dissociation of STIM1 from the ER-PM junctions upon store
refilling, hence playing a key role in negative regulation of
SOCE and preventing the ER from overloading with Ca2⫹
(274).
MURALI PRAKRIYA AND RICHARD S. LEWIS
Table 4. Pharmacology of CRAC channels
Compound
Effect
La3⫹
Inhibition
Gd3⫹
Inhibition
Mechanism
Pore blockade
Pore blockade
Enhancement of STIMactivated current
Unknown
2-APB
Inhibition
Unknown
2-APB
Activation
Direct channel
activation?
2-APB analogs (DPB162-AE and
DPB163-AE)
ML-9
Inhibition
Unknown
Inhibition
BTP2 (YM58483)
Inhibition
Inhibits STIM
translocation
Unknown
Synta66
Econazole
SK&F96365
GSK-7975A GSK-5503A
RO2959
Inhibition
Inhibition
Inhibition
Inhibition
Inhibition
Unknown
Unknown
Unknown
Unknown
Unknown
AnCoA4
Inhibition
Inhibits Orai1
m2C1.1 (monoclonal Ab)
Anti-Orai1 mAb
Inhibition
Inhibition
Inhibits Orai1
Inhibits Orai1 function (by
endocytosis?)
Reference Nos.
20 nM (SOCE)
33–58 nM, nH⫽1.5
(SOCE)
240 nM, nH⫽1.0
(Orai1)
470 nM, nH⫽1.1
(Orai3)
18–28 nM, nH⫽1.5–2
(SOCE)
46 nM, nH⫽1.0 (ICRAC,
S2 cells)
3 ␮M, nH⫽4 (ICRAC)
9
324
238
422
324
430
300
4 ␮M, nH⫽3 (Orai1)
10 ␮M, nH⫽4 (ICRAC)
8 ␮M, nH⫽3 (Orai1)
24 ␮M, nH⫽8 (Orai3,
ICRAC)
15 ␮M, nH⫽2 (Orai3,
SOCE)
90–170 nM (ICRAC)
290
300
290
422
16 ␮M (SOCE)
354
100–150 nM (SOCE;
acute)
6–12 nM (SOCE; 24 h
preinc)
0.5–2.2 ␮M (ICRAC;
acute)
3 ␮M (ICRAC)
0.6–14 ␮M (ICRAC)
4 ␮M (ICRAC)
4 ␮M (Orai1)
25 nM (Orai1)
530 nM (Orai3)
1–10 ␮M (Orai1)
159
1 nM (Orai1)
⬍1 nM (SOCE)
290
121
456
382
263
55,107
107
81
47
329
196
65
Inhibitory activity measured from endogenous CRAC current (ICRAC) or SOCE, or from heterologous currents
(Orai1, 2, and 3) as indicated.
Although the use of La3⫹ as a selective CRAC channel
blocker is undermined by its ability to also block voltagegated Ca2⫹ (CaV) channels, TRP channels, as well as PMCAs (albeit with much lower affinity) (41, 60, 183), La3⫹
block at low concentrations has proven useful for identifying CRAC channel-mediated Ca2⫹ signals (131, 360) as
well as to reveal the structural features of the CRAC channel pore. For example, La3⫹ and Gd3⫹ blockade in CRAC
channels appears to depend on interactions with acidic residues in the outer mouth of the pore formed by the I–II
loop region (238, 429). A recent report has suggested
that there may be a second, lower affinity lanthanide site
1416
at the selectivity filter that is revealed when the highaffinity lanthanide binding in the outer loops is mutated
(422). Consistent with this notion, when soaked in millimolar concentrations of Gd3⫹, the dOrai crystal structure reveals Gd3⫹ bound by the sextet of glutamate side
chains in the selectivity filter (153). These findings suggest that lanthanides block Orai channels by interfering
with the access of permeant ions to the selectivity filter
and pore, and thus they can reasonably be considered to
be pore blockers of CRAC channels. Finally, it is worth
noting that La3⫹ sensitivity of overexpressed Orai1 and
Orai3 channels (Ki ⫽ 0.2– 0.4 ␮M) is reported to be
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2-APB
IC50
STORE-OPERATED CALCIUM CHANNELS
lower than that found for endogenous CRAC currents
(238, 422). This difference may be related to the higher
extracellular Ca2⫹ concentration used in the Orai measurements (20 mM rather than 2 mM Ca2⫹) which could
compete more effectively with La3⫹ for access to its
blocking site in the pore.
B. 2-APB
2-APB is relatively nonselective, as it activates or inhibits
many classes of ion channels and receptors including IP3Rs,
TRPC, TRPM, and TRPV channels, and mitochondrial
Ca2⫹ efflux (156, 195, 300, 399). While this limits its usefulness in identifying activity of endogenous CRAC channels in native cells, it is nonetheless useful for probing gating
and permeation mechanisms for heterologously expressed
Orai channels (335, 422, 444).
2-APB inhibits ICRAC with an IC50 of ⬃10 ␮M and reduces
fast Ca2⫹-dependent inactivation (CDI) of ICRAC in parallel
with inhibition of the current amplitude (300). The precise
mechanisms by which 2-APB inhibits CRAC channel activity is unclear. 2-APB could affect STIM1, Orai1, or the
coupling between the two proteins. Based on observations
that high doses of 2-APB dissociate STIM1 puncta, some
reports have suggested that 2-APB inhibition of the storeoperated current is mediated by disruption of STIM1-Orai1
coupling (73, 290). However, reversal of STIM1 puncta is
observed only when STIM1 is overexpressed alone; when
Orai1 and STIM1 proteins are coexpressed, puncta are unaffected by 2-APB even as ICRAC is inhibited (73, 260).
Furthermore, FRET measurements indicate that the STIMOrai interaction is maintained even though the current is
strongly inhibited (260, 421). Thus, although 2-APB clearly
affects STIM1, the mechanism of ICRAC inhibition is unlikely to be related to its ability to inhibit STIM1 translocation into the ER-PM junctions. Still, there are hints that
2-APB affects the functional coupling of STIM1 on the
CRAC channel. For example, as noted above, 2-APB eliminates CDI (300, 421). Because CDI requires the presence of
STIM1, the inhibition of CDI by 2-APB may indicate the
2-APB enhances STIM-activated ICRAC with an EC50 of 3– 4
␮M. The underlying mechanism is not known but may
involve increased binding of STIM1 to CRAC channels.
Such a mechanism is qualitatively consistent with findings
that 2-APB modestly increases STIM1-Orai1 FRET (260)
and enhances FRET between the cytoplasmic domain of
STIM1 (CT-STIM1) and Orai1 (404). In addition, high
doses of 2-APB can transiently activate mutants of STIM
and Orai1 that are otherwise poorly responsive (402).
Noise analysis indicates that potentiation by 2-APB and
activation by store depletion both reflect the recruitment of
CRAC channels from a silent state to one of high Po (299),
as if both modes of regulation share common mechanisms,
either increased binding of STIM1 to Orai or enhanced
coupling between STIM1 binding and channel opening. Interestingly, studies using overexpressed proteins indicate
that the degree of potentiation is larger in cells with low
STIM-to-Orai ratios compared with those with high
STIM1-to-Orai1 ratios (340). Thus the degree of 2-APB
potentiation may be related to the extent of initial channel
activation by STIM1: lower STIM1-Orai function stoichiometry leading to stronger 2-APB potentiation possibly due
to the lower initial channel Po at this condition. Furthermore, because STIM1-Orai1 puncta are unaffected by
2-APB, these effects may occur through allosteric modulation of Orai1 gating.
Finally, 2-APB can also directly activate Orai3 (and to a
lesser extent, Orai1) independently of ER Ca2⫹ depletion
and STIM1 (73, 290, 335, 422, 444). These 2-APB-evoked
currents are only seen when Orai3 is overexpressed and are
abolished by Orai3 TM1 mutations such as R66W and
G73A (5, 335), indicating that they are not due to 2-APB
stimulation of endogenous TRP or other channels. Interestingly, 2-APB-activated Orai3 currents exhibit strikingly different biophysical properties than STIM1-activated currents, including low Ca2⫹ selectivity and ability to conduct
Cs⫹ and large monovalents normally impermeable through
STIM1-activated Orai3 channels, indicative of a wider pore
(335, 422).
How 2-APB gates Orai3 channels is unknown. Zhang and
colleagues (444) used chimeras of Orai1 and Orai3 to determine that an extended region spanning TM2 through
TM3 is critical for 2-APB gating of Orai3 channels. Moreover, examination of gating in a G158C Orai3 mutant suggests that G158 in TM3 moves close to C101 in TM2,
thereby trapping Orai3 channels in a distinct intermediate
open state (4). A key issue is whether 2-APB and STIM1
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2-APB, a noncompetitive antagonist of IP3 receptors (226),
is a widely used SOCE/ICRAC inhibitor. Its ability to antagonize IP3 receptors (IP3Rs) was used initially to probe the
possible involvement of IP3Rs in the gating of SOCs. Based
on the inhibition of SOCE elicited by 2-APB, one early
study concluded that gating of SOCs occurred through conformational coupling with IP3Rs (215). However, subsequent work using DT40 pre-B cells deficient in IP3R expression found that the effects of 2-APB on SOCE are unrelated
to IP3Rs (216, 300). Moreover, these studies also uncovered
a dose-dependent bimodal effect of the compound on
SOCE, with strong enhancement of ICRAC and SOCE at low
doses (⬍5 ␮M) and transient enhancement followed by
inhibition at high concentrations (⬎20 ␮M).
loss of functional coupling between STIM1 and Orai1
(421). One possibility is that 2-APB could either competitively or allosterically displace STIM1 from the NH2 terminus of Orai channels, thereby inhibiting store-operated gating without interrupting tight STIM1 binding at the COOH
terminus.
MURALI PRAKRIYA AND RICHARD S. LEWIS
dently of global ATP concentrations (56), suggesting that
this is not the most straightforward explanation. Thus, although its target and mechanism of action are unknown,
ML-9 is the only inhibitor so far to inhibit SOCE by interference with STIM1 localization. Further work to define its
mechanism of action, as well as to identify related SOCE
inhibitors that lack effects on MLCK, may cast new light on
the mechanisms of STIM function, including clustering at
the ER-PM junctions.
Despite its weak selectivity for CRAC channels, the ability
of 2-APB to potentiate and inhibit ICRAC has generated
interest in using 2-APB as a basis for future drug development. From the parent chemotype, two related stereoisomers, DPB-162AE and DPB-163AE, were constructed as
dimers of 2-APB (121). These compounds are over 100-fold
more potent than 2-APB itself in inhibiting SOCE (IC50 of
⬃0.6 ␮M), but do not affect IP3R function at these concentrations (121). DPB-162AE elicits bimodal modulation of
SOCE, potentiating at low doses and inhibiting at high
doses, whereas DPB-163AE only inhibits. The myriad nonspecific effects of 2-APB on other channels and its many
effects on the CRAC channel itself present formidable challenges as a platform for developing specific CRAC channel
therapeutics. Still, the ability of 2-APB to modulate key
aspects of CRAC channel behavior such as permeation and
inactivation is likely to be useful in understanding the molecular mechanisms underlying these properties.
D. Imidazoles
C. ML-9
ML-9 [1-(5-chloronaphthalene-1-sulfonyl)homopiperazine
HCl] is a potent inhibitor of MLCK, and early evidence of
its ability to inhibit SOCE suggested a role for MLCK in the
SOCE mechanism, prior to the discovery of STIM and Orai
(for references, see Ref. 354). In store-depleted cells expressing exogenous STIM1, ML-9 was found to disperse
STIM1 puncta at doses comparable to those that inhibited
SOCE, and slightly preceding the reduction in SOCE (354).
These results led Putney and colleagues (354) to propose
that ML-9 inhibits SOCE by preventing the accumulation
of STIM1 in puncta. Inhibition of STIM1 translocation appears to be independent of MLCK inhibition, as it persists
in cells in which MLCK is knocked down, and MLCK inhibition by wortmannin did not affect SOCE (354). Interestingly overexpression of STIM1 reduces the sensitivity of
SOCE to inhibition by ML-9 (354), and ML-9 is essentially
ineffective when Orai1 is also coexpressed (73); the reasons
for this are unclear, but could potentially be related to the
increased stability of STIM1 at the ER-PM junctions caused
by STIM1-Orai1 overexpression. Because ML-9 inhibits
MLCK by competing with ATP binding, one possibility is
that its effects on SOCE are related to the requirement for
ATP in the induction of CRAC channel activation (157,
225). However, STIM1 puncta formation occurs indepen-
1418
The imidazole compound SKF96365 and related antimycotic compounds including econazole, miconazole, and clotrimazole inhibit all SOCs with IC50 values in the range of
0.6 –14 ␮M (55, 107). However, these compounds also
independently block TRP channels (31), voltage-gated
Ca2⫹ channels (241, 351), and K⫹ channels (338), as well
as the cytochrome P-450 enzyme (55). It is worth noting
that although some reports raised the possibility that P-450
may be involved in the control of SOCE based on the inhibition of both P-450 and SOCE (188), Christian et al. (55)
showed that application of econazole through the recording
pipette does not inhibit ICRAC while a less membrane-permeant derivative applied extracellularly blocked ICRAC
with normal efficacy and kinetics, suggesting that econazole
works through an extracellular interaction as a true CRAC
channel inhibitor (55). SKF96365 has been used in many
cell-based and in vivo studies (432, 457), but the inadequate
specificity, slow kinetics, and incomplete reversibility of
these compounds limits their usefulness. The effects of these
compounds on recombinant, overexpressed STIM1 and
Orai1 have not been reported, and the mechanism by which
these compounds exert their inhibitory effects is unknown.
E. BTP2
Another class of agents that inhibits CRAC channel activity
are the bis(trifluoromethyl)pyrazoles (BTPs) which potently
inhibit cytokine release from human lymphocytes and suppress T-cell proliferation (52, 159, 390). The best-studied
member of this group is the compound BTP2 (also called
YM-58483), which inhibits TG-evoked Ca2⫹ influx and
ICRAC in Jurkat T cells (159, 382, 456). BTP2 inhibits ICRAC
in Jurkat T cells with a KD of ⬃10 nM following overnight
incubation, and CRAC current inhibition is essentially irreversible when preapplied (456). However, the specificity
and effectiveness of BTP2 for ICRAC has been challenged by
findings showing that the compound’s potency is ⬃100fold lower when applied acutely and that it potently activates the Na⫹-permeable channel TRPM4 at low nanomolar concentrations (382). Based on these findings,
Takezawa et al. (382) have proposed that a key mechanism
of BTP2 inhibition of Ca2⫹ influx and cytokine release is
related to its ability to depolarize the cell membrane via
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gating mechanisms occur through similar or distinct pathways. Noise analysis has indicated that both gating modes
appear to involve the stepwise recruitment of closed channels to a long-lasting, high Po state (422). Furthermore, the
STIM1- and 2-APB-dependent modes of Orai3 activation
appear to be mutually exclusive: Orai channels bound to
STIM1 resist 2-APB gating, and 2-APB antagonizes STIM1
gating (421). These findings suggest that despite different
pore structures, the two modes of channel activation may
share common gating steps.
STORE-OPERATED CALCIUM CHANNELS
that the compounds act as Orai1 pore blockers, the slow
onset of inhibition argues against such a mechanism, and
other possibilities including stabilization of closed channels
and allosteric effects on STIM1 function cannot be ruled
out. Interestingly, along with Orai inhibition, these compounds also inhibit TRPV6 channels, an effect that was
attributed to possible structural similarities between CRAC
and TRPV6 channels in the target site (81). More studies
are clearly needed to define the mechanism and site of antagonism and selectivity of these compounds before they
can be employed as tools to probe CRAC channel mechanisms and functions.
F. Synta 66
H. RO2959
Synta 66 [3-fluoropyridine-4-carboxylic acid (2=,5=-dimethoxybiphenyl-4-yl)amide, Synta Pharmaceuticals, Lexington, MA] is structurally similar to BTP2 but contains
a biphenyl group rather than the pyrazole ring in BTP2.
The compound is reported to inhibit ICRAC in RBL cells
with an IC50 of ⬃ 3 ␮M and appears to be selective at
least in so far as it does not inhibit K⫹ channels or Ca2⫹
pumps (263). As with BTP2 and the GSK compounds (see
below), the speed of inhibition by Synta 66 is slow, requiring cells to be preincubated for long periods (⬎1 h),
and its effects are poorly reversible, if at all, making a
pore blocking mechanism unlikely. Synta66 has no effects on STIM1 puncta formation, suggesting that it does
not inhibit the early steps of STIM1 activation and its
translocation to junctional ER sites (194). Despite limited information on its mechanism of CRAC channel inhibition, however, a growing number of studies have
employed this compound to probe the physiological contributions for CRAC channels for effector function, including antigen-induced signaling, cytokine production,
and transcriptional regulation (7, 85, 331).
RO2959 (Roche) is another recently developed compound
with functional properties analogous to BTP2, the GSK
compounds, and Synta66 in that it requires preincubation
for suppression of SOCE and exhibits a modest affinity for
suppression of ICRAC (47). However, a key notable feature
of this compound is its apparent selectivity for Orai1 channels over Orai2 or Orai3 channels (IC50 values for recombinant Orai1 and Orai3 channels were 25 and 530 nM,
respectively). SOCE in CD4 T cells is highly sensitive to
RO2959 (IC50 ⬃40 nM), and the compound potently inhibits a variety of effector functions including gene expression, cytokine production, and T-cell proliferation (47).
Puzzlingly, however, currents in RBL cells exhibited a much
a lower sensitivity (IC50 ⬃400 nM), raising the possibility
that the CRAC channels in RBL cells have a somewhat
different molecular composition (Orai1-Orai2 or Orai1Orai3 heteromers), or that the compound acts at a different
target site that is only indirectly coupled to CRAC channel
activation. RO2959 does not antagonize a variety of other
channels, including TRPM2, TRPM4, TRPC1, CaV1.2, and
various KV channels (47). The molecular basis of drug action, including whether it affects the function and choreography of STIM1, remains unclear.
G. GSK-7975A and GSK-5503A
Romanin and colleagues (81) have recently described two
pyrazole derivatives, GSK-5503A [2,6-difluoro-N-(1-(2phenoxybenzyl)-1H-pyrazol-3-yl)benzamide] and GSK7975A [2,6-difluoro-N-(1-(4-hydroxy-2-(trifluoromethyl)
benzyl)-1Hpyrazol-3-yl)benzamide], that inhibit recombinant Orai1 and Orai3 currents with an IC50 of ⬃4 ␮M (81).
As with many other inhibitors described above, the inhibition is slow to develop, requiring several minutes of preincubation. FRET studies show no inhibition of STIM1Orai1 coupling, suggesting that the compounds do not affect the proximal steps of the CRAC channel activation
process. The IC50 for inhibition increased 10-fold in the
E106D Orai1 mutant and in 2-APB-activated Orai3 channels (81), both of which are poorly selective for Ca2⫹ and
which exhibit wider pores than the wild-type Orai1 channel
(420, 422). Although these results were interpreted to imply
I. AnCoA4
Sadaghiani et al. (329) employed a novel strategy to enhance drug specificity for the Orai1 and STIM1 proteins by
screening for compounds that bound to peptide fragments
of Orai1 and/or STIM1 immobilized in microarrays. The
resulting library screen yielded several promising hits, one
of which (AnCoA4) was characterized in detail in biophysical and functional tests. AnCoA4 inhibits CRAC channels
at concentrations in the low micromolar range and attenuates T-cell activation in in vitro and in vivo assays. The
identification of AnCoA4 represents a different methodology of screening compounds based on mechanism–in this
case, the coupling of STIM1 to Orai1. The drug reduces the
recruitment of Orai1 into puncta and also directly inhibits
the activity of the constitutively active Orai1 V102C chan-
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TRPM4 activation, thereby reducing the driving force for
Ca2⫹ entry. BTP2 exhibits specificity for CRAC channels
over K⫹ channels (456), CaV channels (159), and TRPV6
channels (139) but inhibits overexpressed TRPC3 and
TRPC5 channels (139), raising the question of whether inhibition of both classes of channels occurs through a common mechanism. Although the compound’s effects have
been extensively characterized in many immune-based cellular and even animal models of autoimmune diseases for
asthma and delayed-type hypersensitivity (47, 267, 268,
433), the precise mechanisms by which the BTP compounds
inhibit Ca2⫹ influx are not fully understood.
MURALI PRAKRIYA AND RICHARD S. LEWIS
partial suppression achieved by the administration of anti-CRAC channel mAbs. These unforeseen results highlight the growing need for a better understanding of the
effects of partial inhibition of CRAC channels and the
pitfalls of extrapolating expected results from the phenotypes of human SCID patients with complete loss-offunction mutations in Orai1.
J. Monoclonal Antibodies
K. Miscellaneous
As an alternative to small-molecule antagonists, Lin et al.
(196) employed a novel approach to generate high-affinity fully human monoclonal antibodies (mAbs) against
human Orai1 (hOrai1). These recombinant mAbs exhibited strong and specific binding to human Orai1 with Kd
values of 20 –100 pM. Analysis of binding selectivity to
the human and rodent homologs revealed high specificity
to human Orai1. Epitope mapping revealed amino acid
residues 210 –217 in the second extracellular loop of
Orai1 as the interaction site for the antibody. The recombinant mAbs inhibited ICRAC in Jurkat T cells as well as
HEK293 cells stably expressing Orai1 and STIM1, and
one mAb in particular (2C1.1) suppressed SOCE in Jurkat T cells with high potency (KD ⬃1 nM). Functional
assays indicated that the mAbs strongly inhibited NFATdependent gene transcription in Jurkat cells and TG- and
ionomycin-induced cytokine secretion from human T
cells.
In addition to the compounds described above, CRAC
channels are also inhibited by endogenous regulators of cell
growth and proliferation such as sphingosine, ceramide,
and related analogs at micromolar concentrations (IC50 values ⬃1–10 ␮M) (229). Sphingosine and ceramide are bioactive lipid metabolites generated by the breakdown of the
widespread lipid sphingomyelin and can directly bind proteins, activate signaling pathways, and affect a variety of
cellular responses. Ceramide and sphingosine are generated
in T cells by cross-linking of Fas (CD95) on the cell surface.
Thus inhibition of CRAC channels by these lipid mediators
may represent a mechanism for conferring immunosuppression by tumor cells expressing high levels of Fas ligand,
enabling these cells to evade immune surveillance in vivo.
Although sphingosine has been suggested to inhibit CRAC
channels in mast cells through a “direct” effect (229), the
precise mechanism of sphingosine inhibition remains unknown.
Taking a similar approach, Cox et al. (65) reported that a
specific anti-Orai1 monoclonal antibody targeting the second extracellular loop inhibited the proliferation of T cells
and cytokine production in vitro and in vivo. Their analysis
indicated that the inhibition of T-cell effector functions occurred through the internalization of Orai1. Examination
of Orai1 expression on subsets of immune cells from patients with rheumatoid arthritis (RA) suggested strong upregulation of Orai1 expression in RA patients, and the mAb
was found to be effective in inhibiting T cell-mediated graftversus-host disease in a mouse model. Collectively, these
antibody-based approaches support Orai1 as a potential
target for the treatment of autoimmunity and other inflammatory immune diseases.
The biotechnology firm CalciMedica has described two
structurally related inhibitors, CM2489 and CM3457, that
inhibit CRAC current in lymphocytes and T cell-derived
cytokine production (314). They report that CM2489 has
completed phase 1 clinical trials for the treatment of moderate-to-severe plaque psoriasis. This is the first CRAC
channel inhibitor to be tested on humans and represents a
promising lead for the development of novel therapeutics
for allergic autoimmune disorders.
The findings of these carefully executed studies validate
anti-CRAC channel antibodies for therapeutic applications
for immune diseases, in particular for autoimmune diseases
to regulate runaway activation of immune cells. It is worth
noting, however, that attempts to translate the therapeutic
potential of these mAbs in vivo have not yet succeeded. In
vivo tests in cynomologus monkeys injected with the 2C1.1
mAb revealed unexpected generation of antigen-specific antibodies, despite robust inhibition of T-cell cytokine production (IL-2) ex vivo (114). This result was interpreted to
indicate that full inhibition of the CRAC channel is required to suppress the antibody response, rather than the
1420
Some compounds that have attracted wide attention for
their therapeutic and/or beneficial effects on human health
have been found to potently inhibit CRAC channel activity.
For example, diethylstilbestrol, a synthetic estrogen and
endocrine disruptor that was widely manufactured and
clinically prescribed until 1971 to pregnant women (when it
was banned), was found to inhibit ICRAC in RBL cells (IC50
⬃0.5 ␮M) and SOCE in several other cell types through an
extracellular mechanism (440). Likewise, the plant-derived
polyphenol, curcumin, which has drawn attention for potential therapeutic effects based on its anti-inflammatory
properties, reportedly inhibits ICRAC in Jurkat T cells (IC50
⬃ 6 ␮M) and recombinant ICRAC in HEK293 cells overexpressing STIM1 and Orai1 (IC50 ⬃0.5 ␮M) (346). This
effect was suggested to contribute to curcumin’s reported
anti-inflammatory effects. The mechanism(s) underlying
the inhibitory effects of these compounds are unknown.
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nels independently of STIM1, suggesting that binding to the
COOH terminus of Orai1 inhibits STIM1-Orai1 binding
and also delivers an inhibitory signal to Orai. This novel
drug screening strategy may yet prove useful to arrive at
different classes of drugs based on a highly focused step of
the activation mechanism rather than the entire target protein or the end result of target activation.
STORE-OPERATED CALCIUM CHANNELS
VIII. PHYSIOLOGICAL FUNCTIONS AND
DISEASE
In light of the widespread tissue distribution of Orai and
STIM proteins, many specific roles are to be expected for
CRAC channels in different organ systems. Studies in human patients and genetically engineered mice with loss- or
gain-of-function STIM1 and Orai1 genes have revealed serious functional abnormalities in several systems including
the immune system, musculature, and the skin, supporting
the concept that STIM1 and Orai1 are important mediators
of many cellular functions and pathological events. Several
excellent reviews (63, 84, 100, 101, 304, 326) have described a diverse set of cell behaviors that are regulated by
STIM1/Orai1. In this section, we present an overview of key
functions attributed to CRAC channels in a narrow range of
cells and tissues, with the expectation that many more functions are likely to be recognized in the future. In particular,
the roles of the noncanonical homologs, STIM2, Orai2, and
Orai3, remain largely unknown. Identification of human
mutations in these proteins should further our understanding of their physiological and pathophysiological roles.
Mouse knockout models for these less studied homologs
will also be informative, but if the immune system is a guide,
the usage of particular STIM and Orai isoforms is likely to
differ between humans and mice, which may complicate
attempts to extrapolate to their roles in human physiology.
A. Spatiotemporal Control of Gene
Expression
A critical and widespread function of SOCE is to control
gene expression through Ca2⫹-sensitive transcription factors such as NFAT (102). Ca2⫹ activates calcineurin which
dephosphorylates NFAT, exposing a nuclear localization
sequence that enables its translocation into the nucleus
where it combines with other transcription factors to control a wide variety of genes in many cells (145a). Although
[Ca2⫹]i oscillations most often occur through repetitive release of Ca2⫹ from the ER (21), in T lymphocytes global
[Ca2⫹]i oscillations also arise through cyclical activation
and deactivation of CRAC channels (87, 193). STIM1
movements toward the PM in HEK cells also fluctuate in
synchrony with [Ca2⫹]i oscillations, consistent with the oscillatory SOCE seen in T cells (27). [Ca2⫹]i oscillations have
been shown to increase both the efficiency and the specificity of transcriptional activation among NFAT, NF␬B, and
Oct1-OAP pathways (86). Interestingly, Parekh and colleagues (164) have reported that activation of NFAT in
HEK cells occurs preferentially at ER-PM junctions, based
on the inability of slow Ca2⫹ buffers (which reduce global
but not local [Ca2⫹]i) to inhibit NFAT translocation to the
nucleus. Similarly, local Ca2⫹ near CRAC channels activates NFAT in neural stem cells (360). Thus one attractive
idea is that pulsatile Ca2⫹ influx through CRAC channels,
which may be too small to contribute significantly to global
[Ca2⫹]i, may nonetheless serve to effectively and selectively
drive NFAT activation by elevating [Ca2⫹]i in the restricted
volume of the junctional cleft. The molecular basis for local
NFAT activation may involve the scaffold AKAP79 that
brings calcineurin to the vicinity of CaM bound to Orai1 at
ER-PM junctions (165). Related mechanisms may underlie
the ability of Orai channels to activate Fos (264), leukotriene C4 production (46), and TRPC1 insertion (48) and to
modulate enzymes like adenylyl cyclase 8 (410) and the
plasma membrane Ca2⫹-ATPase 4b (18) through local elevation of [Ca2⫹]i.
B. Immune System
Human and mouse studies by Feske and colleagues show
that Orai1 deficiency or loss-of-function diminishes ICRAC
in T cells and impairs the production of key cytokines including interleukin (IL)-2, IL-4, interferon (IFN)-␥, and
IL-10 (102, 134) (reviewed in Refs. 100 and 101). Likewise,
STIM1 knockout mice exhibit severely reduced SOCE and
ICRAC in various immune cells including T cells, mast cells,
B cells, and macrophages (12, 25, 33, 269). These effects on
CRAC channel function are accompanied by striking defects in effector function including impaired degranulation
and cytokine production in mast cells and production of
cytokines such as IL-2, IFN-␥, and IL-4 by T cells (12, 25,
33, 269). Proliferation of B cells in response to antigenic
stimulation is also suppressed (134). These impairments
collectively lead to the loss of host defense pathways and a
devastating immunodeficiency in human patients (101).
However, the same reduction in T-cell effector function
may in some cases be beneficial in ameliorating pathological
responses associated with graft rejection, multiple sclerosis,
and inflammatory bowel disease (233, 337).
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Additionally, Sirolimus (rapamycin), a bacterial byproduct
that blocks T- and B-cell activation through inhibition of
mTOR (mammalian target of rapamycin), is widely used to
reduce transplant rejection. A recent report finds that rapamycin inhibits both recombinant STIM1/Orai1-mediated
currents as well as SOCE in human arterial smooth muscle
at clinically relevant concentrations (179). This finding
raises the interesting possibility that its immunosuppressive
effects may be mediated at least in part through inhibition
of effector functions driven by CRAC channels (gene expression and cytokine production). Thus rapamycin may
represent the first CRAC channel inhibitor to modulate immune functions in human patients. Finally, N-arachidonoyl
glycine (NAGly), a derivative of the endocannabinoid
anandamide that functions as a ligand for the cannabinoid
receptors CB1 and CB2, inhibits SOCE by interfering with
STIM1-Orai1 association (71). This finding raises the interesting notion that the well-known pleiotropic effects of endocannabinoids might be mediated at least in part through
modulation of SOCE in the brain.
MURALI PRAKRIYA AND RICHARD S. LEWIS
Given the immunosuppressive effects described above, it is
somewhat surprising that mice (and humans) lacking critical CRAC channel proteins also exhibit autoimmunity,
characterized by hemolytic anemia, thrombocytopenia, and
lymphoproliferative disease (296). Studies in STIM1/
STIM2 double knockout mice indicate that this phenotype
may arise from a reduction in the number of regulatory T
cells (Tregs) (269), a subpopulation of T cells with suppressor functions required for maintaining tolerance to selfantigens. The autoimmunity is surprising because mice (and
humans) without functional SOC proteins should lack immune effector cell function. The paradox suggests that low
levels of T-cell effector function may be retained even in the
absence of SOC proteins, and when left unchecked by
Tregs, lead to autoimmunity. However, autoimmunity and
Treg deficiencies are not always seen in SOCE-deficient animal models, for example, in the Orai1⫺/⫺ or STIM1⫺/⫺
mice where Treg numbers are in the normal range (233,
269). These phenomena reveal the complexity of STIM and
Orai function in immune cells and deserve further study.
The Orai isoforms may be differentially regulated during
development. It has been reported that Orai2 is expressed in
mouse naive T cells and may underlie the seemingly normal
T-cell effector responses and CRAC currents seen in the
naive T cells of Orai1-deficient mice (233, 397). Given that
mature T cells lacking Orai1 also lack ICRAC and functional
responses, these data point to a switch from Orai2 to Orai1
expression during T-cell maturation in mice. The prevalence of such phenotypic switching and its functional relevance for Ca2⫹ signaling remain largely unknown.
An unexpected result from the knockout studies is that loss
of Orai1, STIM1, and STIM2 does not grossly impair the
development and differentiation of T cells (CD4 and CD8),
B cells, and mast cells (12, 25, 134, 269). These observations are consistent with the normal levels of CD4 and CD8
1422
T cells seen in Orai1-and STIM1-deficient patients (100)
and indicate that the CRAC channel machinery is not critical for the development of immune cells in the bone marrow and thymus. However, a recent study has found that
pharmacological suppression of CRAC channel function or
deletion of Orai1 impairs the differentiation of a subclass of
proinflammatory CD4 T cells, the Th17 cells (172). This
finding is reminiscent of the lack of Tregs in STIM1/STIM2
DKO mice (269) and highlights the complexity of SOCE
contributions to the development of subpopulations of T
cells. Thus it would appear that in thymocytes, Ca2⫹ influx
pathways unrelated to SOCE are responsible for stimulation of the calcineurin-NFAT pathway, which has long been
implicated in effector T-cell development.
An intriguing finding is that STIM and Orai localize to the
immune synapse that forms between T cells and antigenpresenting cells during the immune response (16, 197). At
later times following contact, STIM and Orai are also reported to accumulate at the distal pole of the cell (16).
STIM-Orai FRET suggests a close interaction between the
proteins at both locations (16), while Ca2⫹ influx appears
to be concentrated at the synapse (197). These finding raise
a number of important questions: what mechanisms determine the redistribution of STIM and Orai to these locations,
and what are their functions there (181)?
C. Platelets
STIM1 and Orai1 are reported to be the primary Ca2⫹
influx pathway for agonist-evoked Ca2⫹ influx (thrombin
and ADP-induced signaling) in platelets, and STIM1-deficient mice exhibit impaired agonist-stimulated platelet
SOCE and impaired thrombus formation, resulting in a
mild increase in bleeding time following injury (393). These
mice are significantly protected from arterial thrombosis
and ischemic brain infarction. Likewise, Orai1-deficient
mice exhibit defective SOCE in platelets, impaired thrombus formation, and resistance to downstream consequences
including pulmonary thromboembolism, arterial thrombosis, and ischemic brain infarction (34). A key role for CRAC
channels in platelet function is further highlighted by recent
reports identifying missense gain-of-function mutations in
Orai1 (P245L) and STIM1 (R304W). These mutations
boost Ca2⫹ signals including resting Ca2⫹ levels by activating Orai1 and STIM1 through mechanisms that are likely
related to destabilization of their closed or inactive states
(250, 262, 275). The ensuing defect causes thrombocytopenia characterized by preactivation of platelets, coagulation,
and fibrinolysis (224, 248, 250, 262). Thus CRAC channels
appear to be critical regulators of platelet effector function,
raising the possibility that modulation of CRAC channel
activity could be beneficial for the treatment of diseases
associated with vascular thrombosis including cerebrovascular disorders.
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Although deficiency in STIM2 does not significantly impair
CRAC channel function and SOCE in T cells when measured immediately following store depletion, a marked deficit in Ca2⫹ levels and activation of the Ca2⫹-dependent
transcriptional regulator NFAT was noted at longer times
(⬎1 h) following stimulation, suggesting that STIM1 alone
is not sufficient to maintain prolonged Ca2⫹ signals (269).
The basis for this phenotype is not entirely clear, but as
noted above, STIM2 exhibits a lower affinity for Ca2⫹ than
STIM1 and as a consequence is activated by lower levels of
store depletion near the resting [Ca2⫹]ER (32). Thus one
possibility is that as IP3 levels decline and stores slowly refill
during the late stages of the prolonged stimulation, STIM2
knockout T cells are unable to generate the minimal level of
Ca2⫹ entry that is required for cytokine induction. STIM2
deficiency also results in a reduction in effector T-cell function and cytokine production, although to a milder extent
than in STIM1 KO cells (269).
STORE-OPERATED CALCIUM CHANNELS
D. Skeletal Muscle
STIM1 is localized to the muscle SR at triadic junctions
where Ca2⫹ is released by RyR1, and it appears that STIM1
senses the gradual depletion of SR stores during tetanic
activity, thereby stimulating SOCE and ultimately store refilling to maintain the SR Ca2⫹ content (373). Skeletal muscle expresses high levels of both STIM1 and Orai1 and the
muscle defects seen in the KO mice are consistent with the
congenital myopathy observed in patients deficient in Orai1
and STIM1. More recent work from Dirksen and colleagues
(406) indicates that in skeletal muscle store depletion triggers SOCE via rapid conformational coupling between
STIM1 luminal Ca2⫹ sensor proteins located in the SR and
Orai1 channels present in the transverse (t)-tubule membrane. Interestingly, unlike in nonexcitable cells, STIM1
and Orai1 proteins appear to be prelocalized in close proximity to each other within the triad junction in skeletal
muscle under resting conditions, thus permitting extremely
fast activation and deactivation of SOCE and presumably,
efficient trans-sarcolemmal Ca2⫹ influx during SR depletion (91). Depletion of SR Ca2⫹ stores and accompanying
activation of SOCE under conditions of repetitive stimulation has, however, not been measured directly. Launikonis
and colleagues (91, 186) have argued that the level of store
depletion required for activation of SOCE in skeletal muscle
is far greater than in nonmuscle cells, raising questions
about precisely how the Ca2⫹ concentration in the SR
changes and how this relates to CRAC channel activation
E. Cell Migration and Cancer
STIM1 and Orai1 have emerged as potential new targets for
treatments of several types of cancers. In fact, before the
discovery that STIM1 is the ER Ca2⫹ sensor, one study
implicated STIM1 in cancer metastasis and suggested that
STIM1 could be a tumor suppressor (377). In several types
of cancer, SOCE has been associated with tumorigenesis,
tumor growth, and metastasis. One study found that Orai1
and STIM1 are essential for breast tumor cell migration in
vitro and tumor metastasis in mice (423), with pharmacological inhibition or knockdown of Orai1 or STIM1 reducing metastasis. These protective effects were attributed to
blockade of the assembly and disassembly of focal adhesions, which are crucial for cellular migration. Another
study (232) reported that human breast cancer lines displayed increased levels of Orai1, and microarray analysis of
295 breast cancers showed that women with a transcriptional profile characterized by high STIM1 and low STIM2
expression had the poorest prognosis. Interestingly, tumorigenesis is reportedly accompanied by a switch in expression of the Orai isoform, from Orai1 to Orai3 functioning
in a store-independent fashion (252). Whether the switch to
Orai3 is the cause or consequence of enhanced tumorogenesis is not clear. Along the same lines, there is evidence that
tumorigenesis in breast cancer cells may be driven by constitutive activation of Orai1 channels through a mechanism
that involves the Golgi Ca2⫹-ATPase SPCA2. Aberrant expression of SPCA2 in breast cancer cells in the ER was
postulated to constitutively activate plasma membrane
Orai1 channels through a direct interaction, resulting in
enhanced Ca2⫹ entry and promoting tumorigenesis by increasing the expression of cell cycle proteins (99).
STIM1-dependent Ca2⫹ signaling is reported to be important for cervical cancer cell proliferation, migration, and
angiogenesis (50, 51). The level of STIM1 expression was
associated with the risk of metastasis and survival. Analogous to the findings in breast cancer cell lines described
above, STIM1 expression was found to regulate focal adhesion dynamics, through modulation of the activity of the
protease calpain and the kinase Pyk2 (50), which regulates
focal adhesion turnover. Another recent study has found
that Ca2⫹ oscillations driven by STIM1 and Orai1 promoted melanoma metastasis by driving the assembly of in-
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An important physiological role for SOCE in skeletal
muscle was historically considered unlikely because Ca2⫹
entry is not directly required for contraction, being instead mediated by Ca2⫹ release through RyRs in the SR
that are mechanically coupled to CaV L-type channels in
the T tubules. Several studies, however, identified an
SOCE mechanism of Ca2⫹ entry in skeletal muscle fibers
that requires the canonical STIM1 and Orai1 proteins
(373, 406). STIM1 appears to be essential for myotube
development; loss of STIM1 causes defects in muscle differentiation both in vitro and in vivo, and has been proposed to underlie the high perinatal mortality of STIM1deficient mice (373). In humans, loss of STIM1 or Orai1
expression is associated with a congenital nonprogressive
myopathy attributed to loss of the fast twitch type II
muscle fibers (101, 234). In addition, mouse studies have
revealed that loss of STIM1 increases the propensity for
rapid muscle fatigue during prolonged stimulation (373).
Interestingly, patients with gain-of-function mutations in
STIM1 and Orai1 (e.g., R304W in STIM1 and G98S,
L138F, and P245L in Orai1) exhibit significant muscle
abnormalities, including tubular aggregate myopathy,
which results in muscle weakness and propensity for fatigue (93a, 224, 250, 262). These studies indicate that the
proper regulation of SOCE is required to support the
normal development and homeostatic function of skeletal muscle.
during tetanus. Regardless of their possible role in acute
muscle contractility, it is becoming clear that the storeoperated and excitation-coupled Ca2⫹ pathways reflect two
distinct molecular channel complexes within the triad junction that may enable trans-sarcolemmal Ca2⫹ entry across a
wide range of transmembrane voltages. These results have
challenged preexisting notions about the importance of
SOCE for skeletal muscle function and indicate the importance of SOCE for the development and function of skeletal
muscle fibers (173).
MURALI PRAKRIYA AND RICHARD S. LEWIS
vadopodia and degradation of the extracellualr matrix
through a Src kinase mechanism (375). Collectively, the
growing literature on the involvement of CRAC channels in
cancer metastasis and proliferation indicates that the Orai
and STIM proteins are likely to represent major therapeutic
targets in the quest for developing novel cancer therapies.
F. Brain Development and Function
Consistent with the above expression studies, functional
studies implicate SOCE in several neuronal cellular functions, including neurotransmitter release (93), synaptic
plasticity (10), Ca2⫹ oscillations (349), and gene expression
(182, 360). Moreover, aberrant SOCE has been implicated
in hypoxia-mediated neuronal death (20), epilepsy (372),
and the response to axonal injury (116). More recently,
Konnerth and colleagues (136) have reported that STIM1 is
a key regulator of mGluR1-dependent intracellular Ca2⫹
signals and synaptic potentials. Ablation of STIM1 suppressed mGluR1 evoked slow synaptic potentials and impaired motor coordination, indicating a role for STIM1 in
cerebellar homeostasis and function. This finding is consistent with reports that intact SOCE is vital for the firing of
flight motorneurons in Drosophila (395). On the postsynaptic side, Sun et al. (376) found that SOCE activated by
STIM2 and the ensuing activation of Ca2⫹/calmodulin-dependent kinase II (CAMKII) is a key regulator of spine
maturation. They further reported that familial Alzheimer’s
disease mutations compromised the SOC activation, resulting in impaired spine maturation, a defect that could be
rescued by overexpression of STIM2. These interesting results suggest that impairments in SOCE in the brain may be
linked to neurodegenerative diseases and impaired neuronal function.
In the developing nervous system, CRAC channels may
have a particularly important role for neurogenesis and
proliferation given the neuroepithelial origin and nonexcitable nature of neural stem cells. Indeed, CRAC chan-
1424
IX. FUTURE DIRECTIONS
Over the past 30 years the study of SOCE has progressed
from a physiological era (Ca2⫹ imaging and electrophysiological studies) to a molecular era (the choreography of
SOCE, knockout studies of function) to the beginning of a
structural era. During this time, numerous important advances have been made in understanding both the underlying mechanisms and physiological functions of SOCE. The
properties of the CRAC channel have been described in
detail, including its dependence on [Ca2⫹]ER and its unique
ion selectivity and conductance properties. The major proteins underlying CRAC channel function have been identified as the ER Ca2⫹ sensor STIM and the CRAC channel
subunit Orai, and a mechanism has been described whereby
store depletion causes them to accumulate at ER-PM junctions, where STIM binds to Orai to open the channel. The
structural basis for these events is beginning to unfold as
well. The identification of STIM and Orai, along with development of knockout models and human gene mutations,
has led to a rapid increase in awareness of their physiological functions and the pathological consequences of dysfunction.
As with any rapidly evolving field, many areas are only
beginning to be understood, and a number of controversies
remain unresolved. One general challenge will be to relate
the mechanisms that have been revealed under nonphysiological conditions (heterologous overexpression, massive
and irreversible store depletion, nonphysiological temperature) to the physiological responses of cells expressing endogenous levels of native proteins and after exposure to
physiological agonists. A short list of more specific challenges that lie ahead can be grouped into mechanistic and
functional categories.
In terms of mechanism, we need to know more about the
conformational transitions of STIM proteins following unbinding of Ca2⫹ from the cEF hand. What controls these
transitions, and how does the stoichiometry of STIM complexes change during activation? How does STIM bind to
Orai, where is the channel gate, and how does STIM binding lead to its opening? How are ER-PM junctions formed,
maintained, and disassembled, and what factors regulate
these critical processes? Finally, how do accessory proteins
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A potential role for CRAC channels in regulating effector
functions in the brain is of particular interest since Ca2⫹
regulates critical neurobiological processes including
neuronal excitability, synaptic transmission, and neuronal development (322). Although voltage- and ligandgated Ca2⫹ channels have been widely studied for contributions to these functions, the role of CRAC channels
for neuronal physiology remains poorly understood. Expression studies show high levels of Orai2 and Orai3 in
the brain and moderate levels of Orai11 (128, 132, 133).
mRNA and immunohistochemistry studies have also
shown the presence of STIM1 and STIM2 in several regions of the brain, including the hippocampus, dentate
gyrus, and cerebellum as well as in proliferating regions
such as the subventricular zone and the olfactory bulb
(20, 127, 352).
nels composed of Orai1 and STIM1 appear to be a major
route of Ca2⫹ entry in neural stem/progenitor cells and
regulate key effector functions including gene expression
and proliferation (360). Because recent findings indicate
that neurogenesis persists into adulthood (240) and may
assume special importance for recovery following brain
injuries, the regulation of NPC proliferation by CRAC
channels may have implications for manipulating neurogenesis after brain injury and the treatment of neurodegenerative diseases.
STORE-OPERATED CALCIUM CHANNELS
shape the SOCE response under different conditions and in
specific cell types to optimize it for particular functions?
6. Arnaudeau S, Kelley WL, Walsh JV, Demaurex N. Mitochondria recycle Ca2⫹ to the
endoplasmic reticulum and prevent the depletion of neighboring endoplasmic reticulum regions. J Biol Chem 276: 29430 –29439, 2001.
There are also many functional questions to address, including the functions of Orai2 and Orai3, and how their
unique properties may contribute to tissue- or cell-specific
responses. How are the unique features of the CRAC channel adapted for physiological responses, and can these be
revealed by engineering animal models with altered function rather than simple loss-of-function channel mutations?
To what extent does SOCE signaling occur locally at the
ER-PM junction rather than globally throughout the cell,
and does STIM have additional functions besides the control of store-operated channels? If the pace of progress over
the past several years is any indication, we should expect
answers to these questions in the not so distant future.
7. Ashmole I, Duffy SM, Leyland ML, Morrison VS, Begg M, Bradding P. CRACM/Orai ion
channel expression and function in human lung mast cells. J Allergy Clin Immunol 129:
1628 –1635, 2012.
We thank Ann Shim for help with preparing FIGURE 6 and
members of the Lewis lab for helpful comments on the
manuscript.
Address for reprint requests and other correspondence: R. S.
Lewis, Dept. of Molecular and Cellular Physiology, Beckman
Center B-121A, 279 Campus Dr., Stanford, CA 94305 (email: [email protected]) or M. Prakriya, Dept of Pharmacology, Northwestern University, Searle 8-510, Chicago, IL
60611 (e-mail: [email protected]).
GRANTS
Research in the authors’ laboratories is supported by National Institutes of Health Grants NS057499 (to M.
Prakriya) and GM45374 (to R. S. Lewis) and by the
Mathers Charitable Foundation (to R. S. Lewis).
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared
by the authors.
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