STIM1 couples to ORAI1 via an intramolecular transition into an extended conformation.

Abstract

Stromal interaction molecule (STIM1) and ORAI1 are key components of the Ca(2+) release-activated Ca(2+) (CRAC) current having an important role in T-cell activation and mast cell degranulation. CRAC channel activation occurs via physical interaction of ORAI1 with STIM1 when endoplasmic reticulum Ca(2+) stores are depleted. Here we show, utilizing a novel STIM1-derived Förster resonance energy transfer sensor, that the ORAI1 activating small fragment (OASF) undergoes a C-terminal, intramolecular transition into an extended conformation when activating ORAI1. The C-terminal rearrangement of STIM1 does not require a functional CRAC channel, suggesting interaction with ORAI1 as sufficient for this conformational switch. Extended conformations were also engineered by mutations within the first and third coiled-coil domains in the cytosolic portion of STIM1 revealing the involvement of hydrophobic residues in the intramolecular transition. Corresponding full-length STIM1 mutants exhibited enhanced interaction with ORAI1 inducing constitutive CRAC currents, even in the absence of store depletion. We suggest that these mutant STIM1 proteins imitate a physiological activated state, which mimics the intramolecular transition that occurs in native STIM1 upon store depletion.

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STIM1 couples to ORAI1 via an intramolecular
transition into an extended conformation
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Martin Muik1,4, Marc Fahrner1,4,
Rainer Schindl1,4, Peter Stathopulos2,
Irene Frischauf1, Isabella Derler1,
Peter Plenk1, Barbara Lackner1,
Klaus Groschner3, Mitsuhiko Ikura2
and Christoph Romanin1,*
1Institute of Biophysics, University of Linz, Linz, Austria,2Department
of Medical Biophysics, University of Toronto, Toronto, Ontario, Canada
and3Institute of Pharmaceutical Sciences, University of Graz, Graz,
Austria
Stromal interaction molecule (STIM1) and ORAI1 are key
components of the Ca2þrelease-activated Ca2þ(CRAC)
current having an important role in T-cell activation and
mast cell degranulation. CRAC channel activation occurs
via physical interaction of ORAI1 with STIM1 when endo-
plasmic reticulum Ca2þstores are depleted. Here we
show, utilizing a novel STIM1-derived Fo ¨rster resonance
energy transfer sensor, that the ORAI1 activating small
fragment (OASF) undergoes a C-terminal, intramolecular
transition into an extended conformation when activating
ORAI1. The C-terminal rearrangement of STIM1 does not
require a functional CRAC channel, suggesting interaction
with ORAI1 as sufficient for this conformational switch.
Extended conformations were also engineered by muta-
tions within the first and third coiled-coil domains in the
cytosolic portion of STIM1 revealing the involvement of
hydrophobic residues in the intramolecular transition.
Correspondingfull-length
enhanced interaction with ORAI1 inducing constitutive
CRAC currents, even in the absence of store depletion.
We suggest that these mutant STIM1 proteins imitate a
physiological activated state, which mimics the intramo-
lecular transition that occurs in native STIM1 upon store
depletion.
The EMBO Journal (2011) 30, 1678–1689. doi:10.1038/
emboj.2011.79; Published online 22 March 2011
Subject Categories: membranes & transport; signal trans-
duction
Keywords: FRET sensor; intramolecular transition; ORAI1;
STIM1
STIM1mutantsexhibited
Introduction
Calcium signalling in the cytosol of excitable and non-
excitable cells is of crucial importance. It triggers both
short-term responses like secretion, muscle contraction or
metabolism and also long-term regulation including tran-
scription, cell growth and apoptosis (Berridge et al, 2003).
A major calcium pathway is mediated by store-operated
channels (SOCs). The endoplasmic reticulum (ER) calcium
stores are depleted upon binding of inositol-1,4,5-triphos-
phate (IP3) to their receptors, resulting in the activation of
the plasma membrane Ca2þrelease-activated Ca2þ(CRAC)
channels (Parekh and Putney, 2005). CRAC channels are
characterized by a high Ca2þselectivity and very low single
channel conductance (Hoth and Penner, 1992; Parekh and
Putney, 2005). Their activation enhances cytosolic Ca2þ
levels and thereby stimulates gene expression via the nuclear
factor of activated T cells, resulting in cytokine secretion in
the early stages of immune responses (Feske, 2007; Oh-hora
and Rao, 2008).
A systematic genetic screen by RNA interference has
discovered the stromal interaction molecule (STIM1) and
ORAI1 (also termed CRACM1) as the main molecular com-
ponents of CRAC channels (Liou et al, 2005; Roos et al, 2005;
Feske et al, 2006; Zhang et al, 2006; Vig et al, 2006b; Hogan
et al, 2010). STIM1 has been identified as the ER-located Ca2þ
sensor (Liou et al, 2005; Roos et al, 2005), which senses the
luminal Ca2þcontent by its N-terminal EF hand. ORAI1 is a
Ca2þselective channel located in the plasma membrane with
four transmembrane segments and cytosolic N- and C-term-
inal strands (Prakriya et al, 2006; Yeromin et al, 2006; Vig
et al, 2006b; Schindl et al, 2008). A mutation within ORAI1,
that is R91W, leading to a non-functional channel has been
directly linked to severe combined immune deficiency (SCID)
(Feske et al, 2006).
At resting state, STIM1 is uniformly distributed throughout
the ER membrane. Store depletion triggers STIM1 multimer
formation. These aggregates then translocate into puncta
close to the plasma membrane (Liou et al, 2005, 2007;
Zhang et al, 2005; Baba et al, 2006; Luik et al, 2006; Mercer
et al, 2006; Soboloff et al, 2006; Wu et al, 2006; Xu et al,
2006), thereby activating ORAI1/CRAC channels. Besides an
EF-hand pair, the luminal N-terminus of STIM1 further
contains a sterile-a motif, all these domains are required for
multimerization (Stathopulos et al, 2008, 2009). Following a
single transmembrane helix, the cytoplasmic C terminus
includes three coiled-coil domains (Hogan et al, 2010),
which overlap with an ezrin–radixin–moesin (ERM)-like
domain, a serine/proline- and a lysine-rich segment (Liou
et al, 2005; Baba et al, 2006; Huang et al, 2006; Smyth et al,
2006) (see also Supplementary Figure S4A). The CAD/SOAR
Received: 9 December 2010; accepted: 25 February 2011; published
online: 22 March 2011
*Corresponding author. Institute of Biophysics, University of Linz, 4040
Linz, Austria. Tel.: þ43 732 2468 9272; Fax: þ43 732 2468 9280;
E-mail: christoph.romanin@jku.at
4These authors contributed equally to this work
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domain encompasses roughly aa 340–448 including the
second and third coiled-coil domain and represents the
smallest STIM1 C-terminal fragment that couples to and
activates ORAI1 channels (Park et al, 2009; Yuan et al,
2009). An interaction between CAD and both the N- and
C-terminal regions of ORAI1 has been determined by in vitro
pull-down experiments (Park et al, 2009).
We and others have visualized coupling between STIM1
and ORAI1 in store-depleted cells by Fo ¨rster resonance en-
ergy transfer (FRET) microscopy (Barr et al, 2008; Muik et al,
2008; Navarro-Borelly et al, 2008). The cytosolic C-terminal
portion of ORAI1 that contains a putative coiled-coil domain
couples with STIM1 (Muik et al, 2008; Frischauf et al, 2009).
In this study, we designed a STIM1 conformational sensor
that demonstrates an intramolecular transition into an exten-
ded conformation when binding to ORAI1. Engineering
extended conformations via mutations in coiled-coil domains
facilitated STIM1 coupling to ORAI1 probably by alterations
of coiled-coil intramolecular interactions. The STIM1 confor-
mational sensor further revealed novel characteristics of the
SCID-linked ORAI1–R91W mutant and the CRAC modifier
2-aminoethoxy-diphenyl-borate (2-APB) in their interactions
with STIM1.
Results
A STIM1-derived conformational sensor
The conformational choreography that evokes activation of
ORAI channels via their interaction with STIM1 is unclear.
While intermolecular FRET measurements within an assem-
bly of CFP- and YFP-tagged ORAI1 proteins have been carried
out (Navarro-Borelly et al, 2008), intramolecular FRETwithin
one ORAI1 protein might provide a more direct read-out of
conformational rearrangements that occur upon coupling
with STIM1. However, N- and C-terminally double-labelled
ORAI1 proteins were not applicable by reason of significantly
reduced plasma membrane localization (data not shown).
Therefore, we focused on the development of a STIM1-
derived conformational sensor that might allow for monitor-
ing of intramolecular rearrangements within the STIM1
cytosolic portion when interacting with ORAI1. As the STIM1
C-terminus acts as a surrogate for full-length STIM1 (Huang
et al, 2006; Muik et al, 2008), we initially started to generate
a variety of double-labelled STIM1 C-terminal constructs
of decreasing length (Figure 1) in an attempt to optimize
sensor features for reporting conformational rearrangements
relevant for the STIM1/ORAI coupling machinery. Focus was
placed on constructs that (i) contained at least the minimal
regions previously identified for the interaction with ORAI1
(Muik et al, 2009; Park et al, 2009; Yuan et al, 2009) and
(ii) were still functional both for the coupling to as well as
activation of ORAI1 channels. All constructs included the first
coiled-coil region in addition to the CAD/SOAR domain.
Expression of these double-labelled constructs in HEK 293
cells revealed a range of FRET values between B0.9 and
B0.2 (Figure 1A). While showing a remarkably high maxi-
mum (B0.9) around our previously reported ORAI1 activat-
ing small fragment (OASF; aa 233–474), C-terminal extension
up to the wild-type length (aa 685) resulted in a gradual
decrease of FRET. Interestingly, fragments shorter than OASF
also exhibited an attenuation of FRET (Figure 1A), indicating
that construct length is not the only factor determining FRET.
Double-labelled fragments aa 233–420/430 are most likely
inactive (Zhang et al, 2008; Muik et al, 2009; Park et al, 2009;
Yuan et al, 2009), while 233–450 or 233–474 (OASF) and
larger fragments are proposed as sufficient for interaction and
ORAI1 current activation (Muik et al, 2009). In this study
we focused on the double-labelled aa 233–474 fragment, as
the former tends to form large clusters without ORAI1
co-expression (Muik et al, 2009; Yuan et al, 2009). Indeed,
double-labelled YFP–STIM1–233–474–CFP (termed YFP–
OASF–CFP) allowed for robust constitutive activation of
ORAI1-derived currents with CRAC-like biophysical charac-
teristics (Figure 1B). The current densities obtained with the
double-labelled OASF sensor were somewhat smaller than
those of its single-labelled form (Muik et al, 2009) pointing
to a slightly reduced activation capacity and/or affinity
for ORAI1. The functionality of this conformational STIM1
sensor constitutes a powerful tool to monitor intramolecular
rearrangements within OASF upon binding to ORAI1 (see
below).
This double-labelled YFP–OASF–CFP construct when
expressed alone in HEK 293 cells mainly exhibited a uniform,
cytosolic distribution and yielded remarkably high FRET
(Figure 1A and C), reaching B0.9, much higher than B0.2
typically found in our previous experiments with single-
labelled constructs detecting OASF oligomerization (Muik
et al, 2009). YFP–OASF–CFP proteins were detected by an
anti-GFP antibody as a single band in western blot corres-
ponding to the complete sensor form without any smaller,
cleaved fragments (Figure 1D). The high FRET value of
YFP–OASF–CFP might result from both intramolecular and
intermolecular proximity of fluorophores, representing a
head-to-tail orientation and dimerization/oligomerization
(Muik et al, 2009), respectively (Figure 1E).
In an attempt to roughly estimate intermolecular FRET, we
generated additional constructs that carried identical labels
(either CFP or YFP) on both N- and C-termini giving rise only
to intermolecular but not intramolecular FRET (Figure 1E).
Co-expressed CFP–OASF–CFP and YFP–OASF–YFP revealed
smaller FRET values of B0.18 (Figure 1E). Thus, the high
FRETobserved with the YFP–OASF–CFP conformational sen-
sor is suggested to reflect primarily intramolecular rather
than intermolecular interactions and assumedly arises from
a head-to-tail proximity of fluorophores within OASF (see
Figure 1E).
Conformational coupling of OASF sensor to ORAI1
The cytosolic portion of STIM1 interacts with both ORAI1
N- and C-termini (Park et al, 2009). The multiple interaction
sites leading to ORAI1 activation might involve a conforma-
tional rearrangement within STIM1. The YFP–OASF–CFP
conformational sensor enabled us for the first time to address
this question. Its expression together with unlabelled ORAI1
in HEK293 cells led to a clear redistribution of the OASF
sensor with partial plasma membrane as well as cytosolic
targeting. The OASF FRET sensor exhibited clearly stronger
membrane targeting than the double-labelled whole STIM1
C-terminus (233–685) consistent with its higher affinity to
ORAI1, which was abolished with the shorter 233–430 frag-
ment as evident from density profiles (Supplementary Figure
S1). YFP–OASF–CFP yielded higher FRET in the cytosol,
while significantly lower FRETwas obtained with the fraction
of the sensor that was targeted close to the plasma membrane
Conformational switch upon STIM1–ORAI1 coupling
M Muik et al
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where ORAI1 is located (Figure 2A). This decrease of FRET
was not due to a diminished intermolecular FRET, as dimeri-
zation/oligomerization of OASF when coupled to ORAI1
slightly enhances FRET as previously shown (Muik et al,
2009) for single-labelled and uniformly double-labelled con-
structs (Supplementary Figure S2A and B). Hence, our data
suggest a reduction of intramolecular FRET by a rearrange-
ment within YFP–OASF–CFP into an extended head-to-tail
configuration upon its coupling to ORAI1, although an addi-
tional change in fluorophore orientation affecting FRET
cannot be excluded. This decrease of FRET from the OASF
conformational sensor was similarly observed upon co-
expression with the non-conducting (Prakriya et al, 2006;
Yeromin et al, 2006; Vig et al, 2006a) ORAI1–E106Q mutant
(Figure 2B), indicating that it was not directly linked to Ca2þ
entry or caused by increases in submembrane intracellular
Ca2þconcentrations. Thus, the intramolecular transition to
the extended conformation apparently resulted from OASF
coupling with ORAI1 upstream of CRAC channel opening
and Ca2þentry. The ORAI1–L273S mutant that exhibits
disrupted communication with STIM1 (Muik et al, 2008)
consistently failed to interact with OASF conformational
sensor, displaying a rather uniform, high FRET that is similar
both in cytosolic and plasma membrane adjacent regions
(Figure 2C). The extended conformation of OASF when
coupled to ORAI1 may reflect a specific, intramolecular
transition possibly exposing the minimal region, that is
CAD/SOAR (Park et al, 2009; Yuan et al, 2009), essential
for this interaction with and/or gating of the ORAI1 channel
(Park et al, 2009).
Engineering head-to-tail proximity of OASF
STIM1 encodes three putative coiled-coil domains (Hogan
et al, 2010) within the cytosolic portion (Supplementary
Figure S2A) that might contribute to the OASF confor-
mation via intramolecular interactions. In general, coiled-coil
domains are well known for mediating intermolecular as well
as intramolecular protein associations via both hydro-
phobic and electrostatic interactions (Steinmetz et al, 2007;
Grigoryan and Keating, 2008; Parry et al, 2008). In an attempt
to engineer OASF in its extended conformation, we initially
decreased OASF length from its N-terminal side, as CAD/
SOAR is primarily devoid of the first coiled-coil domain (aa
233–342). We further mutated various hydrophobic leucines
CA
1.0
n=28
n=19
n=49
n=74
n=35
n=45
n=24
0.4
0.6
0.8
NFRET
0.0
0.2
(OASF)
YFP–233–474–CFP
YFP–233–430–CFP
YFP–233–450–CFPYFP–233–85–CFP
YFP–233–535–CFP
YFP–233–685–CFPYFP–233–420–CFP
YFP–OASF–CFP
0
ORAI1 +
YFP–OASF–CFP (n=10)
EDB
–10
–5
I (pA/pF)
+ La3+
Intra
Inter
Intra
Inter
0
Time (s)
ORAI1 +
YFP–OASF–CFP
–5
0
I (pA/pF)
–90
–10
V (mV)
1.0
YFP–233–420–CFP
YFPNFRETCFP
0
YFP–233–474 (OASF)–CFP
CFP YFP
1.0
0
NFRET
1.0
YFP–233–535–CFP
YFPNFRETCFP
0
IntraIntra
50100 150
0.0
0.2
0.4
NFRET
YEP–OASF–CFP
CFP–OASF–CEP
+
YEP–OASF–YFP
0.6
0.8
1.0
n=74
n=44
30600–30 –60
0
1.0
YFP–233–685–CFP
YFP
NFRET CFP
20
Y 233–474 C
25
37
50
75
100
150
250
12% SDS
MW (kDa)
Figure 1 Designing a STIM1-derived conformational sensor. (A) Block diagram summarizing intermolecular/intramolecular NFRET of
double-labelled YFP–STIM1–CFP fragments: 233–420, 233–430, 233–450, 233–474 (OASF), 233–485, 233–535 and 233–685 (complete
STIM1 C-terminus). (B) Time course of constitutive whole-cell inward currents at ?86mV of HEK293 cells expressing YFP–OASF–CFP with
ORAI1 (upper panel) and respective I/V curve taken at t¼0s (lower panel). (C) Localization and calculated NFRET life cell image series of
selected STIM1 fragments: 233–420, 233–474 (OASF), 233–535 and 233–685. Calibration bar is 5mm throughout. (D) The YFP–OASF–CFP
FRETsensor detected by western blot with an anti-GFPantibody when expressed in HEK293 cells. (E) A cartoon indicating the intramolecular/
intermolecular FRETof OASF labelled either with YFP/CFP (left) or CFP/CFP, YFP/YFP (right). Block diagram comparing intermolecular with
intramolecular NFRETof double-labelled STIM1 OASF fragments as depicted in the upper panel.
Conformational switch upon STIM1–ORAI1 coupling
M Muik et al
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at position a or d within a heptad repeat (Woolfson, 2005)
that are highly conserved between various species (Supple-
mentary Figure S3), in an attempt to locally interfere with
the putative first coiled-coil structure. Among several
constructs (Supplementary Figure S4B), the very N-terminal
portion (aa 233–251) appeared most interesting, as its dele-
tion or L to S point mutations therein (L248S, L251S) led to
constructs with a substantial reduction of FRET compared
with the wild-type sensor (Figure 3A and C; Supplementary
Figure S4B). To circumvent the impact of N-terminal trun-
cations on FRET, we focused (below) on the OASF L251S
point mutant, which assumed an extended conformation
independent of interaction with ORAI1. Several other L to
S mutations within the first coiled-coil downstream to
aa L251 led to smaller or almost no reduction of FRET
(Supplementary Figure S4B) underscoring the importance
of the N-terminal region (aa 233–251) of OASF to this con-
formational transition. Thus, the first, putative coiled-coil
domain likely has a role in intramolecular coiled-coil associa-
tions within OASF.
To reveal an involvement of the second coiled-coil domain
in the head-to-tail proximity of OASF, we engineered L373S,
L373S A376S, A376K hydrophobic mutations (Frischauf et al,
2009; Covington et al, 2010) to analogously interfere with the
putative coiled-coil structure. Additionally, this region has
been suggested (Frischauf et al, 2009; Calloway et al, 2010;
Covington et al, 2010) to encompass the STIM1-binding site
for ORAI1, as the above mutations interfered with STIM1
coupling to ORAI1 (Frischauf et al, 2009; Covington et al,
2010). All of these YFP–OASF–CFP sensor mutants exhibited
a significant reduction of FRETcompared with wild type, with
the A376K mutant being most pronounced (Supplementary
Figure S4B; Figure 3A and C). These data suggest a contribu-
tion of the second coiled-coil domain in controlling intramo-
lecular transitions in addition to its role as potential binding
site for ORAI1.
0.8
YFP–OASF–CFP
+ ORAI1
n=32
A
0.2
0.4
0.6
NFRET
Cytosolic
region
0.0
Membrane
region
YFP–OASF–CFP
+ORAI1 E106Q
n=20
0.4
0.6
0.8
NFRET
B
0.0
0.2
Cytosolic
region
Membrane
region
YFP–OASF–CFP + ORAI1L273S
CFP YFP
Membrane
NFRET
1.0
0
Cytosol
NFRET
Cytosolic
region
Membrane
region
C
0.8
1.0
YFP–OASF–CFP
+ ORAI1 L273S
n=11
0.2
0.4
0.6
0.0
YFP–OASF–CFP + ORAI1
CFP
Membrane

NFRET
1.0
0
Cytosol
YFP
YFP–OASF–CFP + ORAI1E106Q
CFP
Membrane

NFRET
1.0
0
Cytosol
YFP
Figure 2 OASF sensor coupling to ORAI1. (Right panel) Localization and calculated NFRET life cell image series of HEK293 cells expressing
YFP–OASF–CFP and (A) ORAI1, (B) ORAI1E106Q(C) ORAI1L273S. Calibration bar is 5mm throughout. Magnified section as indicated by the
white box highlights the decrease of FRET in regions of the plasma membrane compared with the cytosol in (A, B). (Left panel) Respective
block diagram of separately calculated NFRET for regions including the plasma membrane (within the two yellow borders) and the cytosol
(within white border). The ‘plasma membrane’ was assumed as 1.5–2mm of the edge of the cell image.
Conformational switch upon STIM1–ORAI1 coupling
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The C-terminal segment of OASFaround aa 420–430 might
also have a role in the head-to-tail proximity based on
the substantial reduction of FRET upon its deletion (see
Figure 1A). In an attempt to alternatively affect head-to-tail
proximity of OASF, which might be governed by intra-
molecular coiled-coil interactions, we aimed to potentially
disrupt (L416S, V419S, L423S) or enhance (R426L) the third
putative coiled-coil domain (Hogan et al, 2010) by intro-
ducing L/V to S or R to L mutations in this region (Gruber
et al, 2006). All four residues are highly conserved between
various species (Supplementary Figure S3). The former
mutants indeed showed a significant attenuation of FRET
(Supplementary Figure S4B) with the YFP–OASF L416S
L423S–CFP double mutant revealing the most pronounced
reduction, while the R426L OASF sensor mutant displayed a
significantly higher FRET than wild type (Figure 3A and C;
Supplementary Figure S4), implicating the third coiled-coil
region in mediating OASF head-to-tail proximity.
In summary, the L251S, A376K and L416/423S OASF
sensor mutants showed substantial FRET reduction by B0.4
compared with the OASF wild-type form, which likely
resulted from a pronounced decrease of head-to-tail proxi-
mity. Intermolecular FRET measurements suggested compar-
able degree of dimerization/oligomerization of OASF and
mutants (Figure 4A).
In vitro analyses of purified OASF wild-type and mutant
forms provided further evidence for distinct conformations
with no change in the oligomerization state (Figure 4B and
C). An extended version of OASF (OASF-ext), encompassing
aa 234–491 was used in the in vitro analyses because this
protein was less susceptible to degradation in Escherichia coli
cells. Wild-type, L251S, L416S and L423S OASF-ext proteins
were attainable at 495% purity (Figure 4B, inset). All four of
the recombinant proteins showed a high a-helicity, assessed
by far-UV circular dichroism (CD), typical of coiled-coil
motifs. Interestingly, all three mutant forms exhibited more
pronounced negative ellipticity (i.e. see 208 and 222nm)
compared with wild type (Figure 4B). These spectral changes
probably reflected a conformational change rather than an
increased a-helical content, since the thermal melts of the
mutant proteins, measured at 222nm, did not show an
enhanced stability expected to accompany increased levels
of secondary structure compared with wild type (Supple-
mentary Figure S5). A similar phenomenon occurs with
calmodulin, which displays enhanced negative ellipticity in
the far-UV CD spectra in response to Ca2þbinding (Martin
and Bayley, 1986) without a change in helical content, but
rather a conformational rearrangement of the secondary
structure elements in three-dimensional space (Finn et al,
1995). Consistent with a mutation-induced conformational
YFP–OASF–CFP
YFP
NFRET CFP
1.0
n=32
n=91
n=20
n=25
n=32
ACB
ORAI1 + YFP–OASF–CFP
CFP
YFP
NFRET
1.0
1.0
0.4
0.6
0.8
NFRET
0
0.0
0.2
YFP–OASF A376K–CFP
YFP–OASF L251S–CFP
YFP–OASF L416/423S–CFP
YFP–OASF R426L–CFP
D
YFP–OASF–CFP
YFP–OASF L251S–CFP
CFP
YFP


NFRET
ORAI1 + YFP–OASF L251S–CFP

CFP
YFP
NFRET
1.0
0
1.0
1.0
R426L (n=5)
L416S
L423S(n=8)
wt (n=5)
A376K (n=6)
YFP–OASF A376K–CFP
CFP
YFP

NFRET
ORAI1 + YFP–OASF A376K–CFP
CFP
YFP
0
1.0
0
0.0
0.5
Intensity
L251S (n=7)
NFRET
1.0
0
654
3
2
Length (µm)
10
YFP–OASF L416S L423S–CFP
CFP
YFP
NFRET
ORAI1 + YFP–OASF L416S L423S–CFP
CFP
YFP
NFRET
1.0
0
1.0
1.0
A376K (n=6)
R426L (n=5)
YFP–OASF–CFP + ORAI1
ORAI1 + YFP–OASF R426L–CFP
YFPCFPNFRET
YFP–OASF R426L–CFP
CFP
YFP
NFRET
0
0
0.5
L416S
L423S
(n=8)
Intensity
wt (n=5)
1.0
0
1.0
0
0.0
L251S (n=7)
Length (µm)
0123456
YFP–OASF–CFP
Figure 3 Engineering OASF head-to-tail proximity by mutations. (A, B) Localization and calculated NFRET life cell image series of YFP–OASF–
CFP wild-type and mutants without (A) or with (B) ORAI1 co-expressed. Calibration bar is 5mm throughout. (C) Block diagram summarizing
NFRET of double-labelled OASF mutants: YFP–OASF–CFP (wild type), YFP–OASF L251S–CFP, YFP–OASF A376K–CFP, YFP–OASF L416S
L423S–CFP and YFP–OASF R426L–CFP. (D) Intensity plots representing localization of YFP–OASF–CFP wild-type and mutants without (upper
panel) and with (lower panel) ORAI1 in regions close to the plasma membrane as indicated by the dashed line.
Conformational switch upon STIM1–ORAI1 coupling
M Muik et al
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transition, all three mutant proteins exhibited a shorter
elution time than the wild-type form in size exclusion
chromatography (SEC) experiments (Figure 4C). The shorter
elution times are not due to a significant change in quatern-
ary structure, since the in-line multiangle light scattering
(MALS) analyses suggested dimer molecular weights for
mutant and wild-type proteins (Figure 4C). The shorter
elution times suggested an extended conformation for the
OASF-ext mutants compared with the wild-type form in line
with the FRET measurements (Figure 3A).
Thus, based on our mutation data, all three coil-coiled
regions within the cytosolic portion of STIM1 have a
role in mediating the intramolecular head-to-tail proximity
of OASF.
Coupling of OASF mutants to ORAI1
A decreased YFP–OASF–CFP FRET might reflect an intra-
molecular transition to an extended conformation with a
potential exposure of the CAD/SOAR domain. This concept
was tested by co-expressing ORAI1 with L251S, A376K or
L416/423S YFP–OASF–CFP mutants that exhibited the most
pronounced reduction of FRET compared with the wild-type
sensor in the absence of ORAI1. Co-expression of ORAI1
together with the L251S and L416/423S OASF mutants
(Figure 3B) revealed clearly stronger membrane localization
than that obtained with wild-type OASF sensor as evident
from respective intensity profiles (Figure 3D). Hence, a more
extended OASF conformation might allow for enhanced
binding to ORAI1. The A376K OASF mutant behaved differ-
ently, however, exhibiting a reduced FRET but failing to
interact with ORAI1 (Figure 3B–D). Previous studies focused
on the second coiled-coil domain as a potential site for
interaction with ORAI1 C-terminus (Frischauf et al, 2009;
Calloway et al, 2010; Covington et al, 2010). This interaction
may have been impaired by the A376K mutation despite the
extended OASF conformation. The R426L OASF mutant failed
to interact with ORAI1 (Figure 3C and D), suggesting that a
sequentially more canonical third coiled-coil domain inter-
fered with the extended conformation potentially required for
coupling to ORAI1. Hence, mutations within the first and
third coiled-coil domains designed to potentially destabilize
intramolecular coiled-coil interactions promoted switching
of OASF into an extended conformation, this in turn facili-
tated interaction with ORAI1 probably by enhanced exposure
of the CAD/SOAR domain.
Coupling of OASF to ORAI1–R91W
The ORAI1–R91W mutant linked to SCID represents a non-
functional CRAC channel due to a defect in gating/permea-
tion rather than in interaction with full-length STIM1
(Navarro-Borelly et al, 2008; Derler et al, 2009). However,
previous FRET microscopy studies show that while the
STIM1–ORAI1–R91W interaction is preserved, it is somewhat
attenuated (Muik et al, 2008). Surprisingly, co-expression of
ORAI1–R91W with wild-type YFP–OASF–CFP revealed no
clear evidence for an interaction (Figure 5A). On the other
hand, the OASF L251S and L416/423S mutants that showed
enhanced interaction with wild-type ORAI1 were capable of
coupling to ORAI1–R91W (Figure 5B and C). As expected,
they failed to induce an ORAI1–R91W current (data not
shown), consistent with a profound ORAI1 gating defect.
The density profiles of OASF L251S and L416/423S mutants
co-expressed with ORAI1–R91W (Figure 5B and C) suggested
a decrease in the affinity for their interaction when compared
with profiles of the OASF mutants with wild-type ORAI1
(Figure 3D), consistent with the reduced but detectable
interaction with full-length STIM1 (Derler et al, 2009).
Hence, the ORAI1–R91W mutant appears deficient in
the ability to switch OASF into its extended conformation
via interaction. To evaluate the degree of this deficiency,
we tested the V419S OASF mutant that showed only
slight attenuation of FRET compared with wild-type OASF
when expressed in the absence of ORAI1 (Supplementary
Figure S4B). The V419S sensor mutant was indeed able to
0.8
1.0
n=9
n=63
n=24
n=15
A
0.0
0.2
0.4
0.6
NFRET
YFP–OASF–YFP
CFP–OASF–CFP
+
YFP–OASF L416S L423S–YFP
CFP–OASF L416S L423S–CFP
+
YFP–OASF L251S–YFP
CFP–OASF L251S–CFP
+
YFP–OASF R426L–YFP
CFP–OASF R426L–CFP
+
20
M
20
15
30
50
kDa
wt
L251S L416S L423S
OASF-ext (aa234–491)
20 °C
B
–10
5
10
Mean residue ellipticity ×10–3
(deg cm2 dmol–1)
Wild-type
L251S
L416S
L423S
–25
220200210240 230
Wavelength (nm)
C
1.2
80
100
OASF-ext (aa234–491)
4
4 °C
Superdex 200 10/30
0.6
0.9
40
60
MALS molecular weight (kDa)
wt
L251S
65±7kDa
L416S
63±7kDa
L423S
60±4kDa
61±1 kDa
Monomer mass
Dimer mass
0
0.3
10
10
20
Elution volume (mI)
Relative UV absorbance
(280nm)
141312 11
Figure 4 Dimerization/oligomerization and conformation of OASF
and mutants. (A) Block diagram summarizing intermolecular
NFRET between double-labelled CFP/CFP and YFP/YFP OASF
forms: OASF (wild type), OASF L251S, OASF L416S L423S and
OASF R426L. (B) Purity and far-UV CD spectra of OASF-ext (aa
234–491) mutant and wild-type forms. Spectra were acquired at
201C in 20mM Tris, 200mM NaCl, 2mM DTT, pH 8 using protein
concentrations ranging from 0.14 to 0.35mgml?1. Protein purity
was confirmed using Coomassie-stained SDS–PAGE (inset). (C) SEC
with in-line MALS analyses of OASF-ext (aa 234–491) mutant and
wild-type forms. SEC experiments were performed at 41C in 20mM
Tris, 100mM NaCl, 50mM L-Arg/L-Glu, 2mM DTT, pH 8 using
0.85–2.0mgml?1protein.
Conformational switch upon STIM1–ORAI1 coupling
M Muik et al
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Page 7

interact with the ORAI1–R91W mutant, and this interaction
was accompanied by a substantial decrease of FRET close
to the plasma membrane (Figure 5D), consistent with full-
length STIM1 observations (Muik et al, 2008; Derler et al,
2009). The ability of the ORAI1–R91W mutant to drive the
V419S OASF mutant into a more extended conformation
might imply that the intramolecular transition within
OASF is coupled to the interaction with rather than the
gating/permeation of ORAI1.
Mutations in full-length STIM1
To validate these findings derived from the OASF conforma-
tional sensor, we introduced some selected mutations within
the full-length STIM1 (Figure 6) and an ER-located STIM1
fragment (aa 199–535; Supplementary Figure S6). The latter
construct lacking the luminal N-terminal region and compris-
ing only the ER membrane sequence (aa 199–232) together
with the ERM domain (aa 233–535) allowed the elimination
of store-dependent effects and the contribution of the
polybasic cluster to puncta formation (Liou et al, 2007).
While wild-type STIM1 (Figure 6A) only co-localized with
and activated ORAI1 following store depletion by the Ca2þ-
ATPase inhibitor 2,50-di(tert-butyl)-1,4-benzohydroquinone
(BHQ), boththeL251S (Figure
(Figure 6C) STIM1 mutants co-clustered with ORAI1 before
6B)andL416/423S
store
currents. Similarly, the ER-localized mutant STIM1 fragments
(Supplementary Figure S6B, C and E) clustered with ORAI1,
producing constitutive currents without store depletion.
These inward currents were significantly reduced (Supple-
mentary Figure S6E) or absent (Figure 6A) in experiments
using the wild-type STIM1 forms.
Store depletion only slightly altered co-clustering of ORAI1
and full-length STIM1 with mutations introduced in the first
or third coiled-coil domain. Hence, these STIM1 mutants
without store depletion might have led to a conformational
exposure of the ORAI1 interaction site(s) within the STIM1
C-terminal region thereby outweighing most of the inhibitory
effect exerted by the full Ca2þstores via the N-terminal
region of STIM1. Further, the ER-located fragments with the
L251S and the L416/423S coiled-coil mutations resulted in
substantially more co-clustering with ORAI1 and larger
ORAI1 currents compared with the wild-type form (Supple-
mentary Figure S6). Consistent with R426L OASF that
resisted a conformational transition, the R426L full-length
STIM1 mutant (Figure 6D) and the corresponding ER-located
mutant STIM1fragment(Supplementary
and E) exhibited minimal (Figure 6D) or lack of (Supple-
mentary Figure S6D) co-clustering with ORAI1 after store
depletion. Consequently, these constructs yielded only minor
depletion,leading toconstitutiveORAI1-derived
FigureS6D
1.0
n=6
0.0
0.5
Intensity
Length (µm)
76543210
Intensity
1.0
Length (µm)
0.0
0.5
n=9
7
6
5
4
3
210
Length (µm)
7
6
5
4
3
210
1.0
0.0
0.5
n=9
Intensity
Length (µm)
76543210
Intensity
0.5
1.0
0.0
n=7
B
CFP
YFP
1.0
0
YFP–OASF L251S–CFP + ORAl1 R91W
1.0
0
C
YFP–OASF L416S L423S–CFP + ORAl1 R91W
CFP
YFP
D
1.0
0
YFP–OASF V419S–CFP + ORAl1 R91W
CFP
YFP
A
1.0
0
CFPYFP
YFP–OASF–CFP + ORAl1 R91W
NFRET
NFRET
NFRET
NFRET
Figure 5 OASF sensor coupling to ORAI1–R91W. (Left panel) Localization and calculated NFRET life cell image series of HEK293 cells
expressing ORAI1R91Wand (A) YFP–OASF–CFP, (B) YFP–OASF L251S–CFP,(C) YFP–OASF L416S L423S–CFP, (D) YFP–OASF V419S–CFP.
Calibration bar is 5mm throughout. (Right panel) Corresponding intensity plots representing localization of YFP–OASF–CFP and mutants in
regions close to the cell membrane as indicated by the dashed line.
Conformational switch upon STIM1–ORAI1 coupling
M Muik et al
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Page 8

(Figure 6D) or no (Supplementary Figure S6E) ORAI1
currents. Noteworthy, the interaction as well as stimulatory
capability of the R426L full-length STIM1 mutant on
ORAI1 was much more impaired than its ability to form
puncta in response to store depletion. Hence, STIM1 oligo-
merization is not sufficient for its conformational coupling
with ORAI1 unless an extended C-terminal conformation
is adopted.
STIM1-derived FRET sensor as tool for drug screening
We investigated whether the OASF conformational sensor
might be utilized for screening of STIM1/ORAI1 as drug
targets. As proof of principle, 2-APB was used, a powerful
modifier of the STIM1/ORAI machinery with several, com-
plex target sites. 2-APB stimulates ORAI3 channels (Lis et al,
2007; Schindl et al, 2008), disrupts STIM1 clusters (DeHaven
et al, 2008; Peinelt et al, 2008; Tamarina et al, 2008) and
Overlay
CFP–ORAI1
YFP–STIM1
A
–4
–6
–2
0
2
CFP–ORAI1 +
YFP–STIM1
n=19
– BHQ
B
0
–12
–10
–8
Time (s)
I (pA/pF)
+ La3+
+ BHQ
Overlay
CFP–ORAI1
YFP–STIM1
L251S
– BHQ
+ BHQ
Overlay
CFP–ORAI1
YFP–STIM1
L416 423S
C
– BHQ
+ BHQ
CFP–ORAI1
YFP–STIM1
R426L
D
Overlay
– BHQ
+ BHQ
250 200150 10050
–4
–2
0
2
I (pA/pF)
CFP–ORAI1 +
YFP–STIM1–L251S
n=12
–8
–6
Time (s)
+ La3+
0
250
200
150
100
50
–6
–8
–4
–2
0
2
I (pA/pF)
CFP–ORAI1 +
YFP–STIM1–L416S–L423S
n=13
–12
–10
Time (s)
+ La3+
0250
200
150
100
50
–4
–6
–2
0
2
n=7
CFP–ORAI1 +
YFP–STIM1–R426L
–12
–10
–8
I (pA/pF)
Time (s)
+ La3+
0 250
200
150
100
50
Figure 6 Controlling full-length STIM1 clustering and coupling efficiency with ORAI1. (Left panel) Life cell image series showing localization
and overlay from HEK293 cells expressing CFP–ORAI1 and (A) YFP–STIM1, (B) YFP–STIM1 L251S (C) YFP–STIM1 L416S L423S (D) YFP–
STIM1 R426L under resting cell conditions (upper panel) and following 5min store depletion with 60mM BHQ in nominally free extracellular
Ca2þsolutions (lower panel). (Right panel) Respective time courses of whole-cell inward currents at ?86mV activated by passive store
depletion and blocked by 10mM La3þat t¼200s.
Conformational switch upon STIM1–ORAI1 coupling
M Muik et al
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Page 9

ORAI1–STIM1 complexes with canonical transient receptor
potential channels or IP3 receptors (Hong et al, 2011).
In contrast, the C-terminus of STIM1 or a shorter fragment
(aa 235–505) has been reported to rapidly associate with
ORAI1 when 2-APB is administered (Wang et al, 2009). In
our experiments, treatment of the OASF sensor with 75mM
2-APB induced a slight FRET decrease, suggesting a confor-
mational rearrangement towards an extended conformation
(Supplementary Figure S7A and B). In the presence of ORAI1,
the OASF sensor exhibited further redistribution close to the
plasma membrane following 2-APB addition, together with
a further decreased FRET (Supplementary Figure S7C and D).
A similar 2-APB driven interaction of STIM1 C-terminal
fragments with ORAI1 has previously been described as an
ORAI1-mediated process, resulting in a current increase
(Wang et al, 2009). Hence, the OASF sensor indeed allowed
to detect an effect of 2-APB via FRETand suggested enhanced
interaction of the slightly extended conformation of OASF as
potential mechanism for strong coupling to and activation of
ORAI1 currents. However, the OASF C-terminal fragment
seems to behave distinct to the full-length STIM1 form
where 2-APB exerts complex, distinct actions as previously
mentioned.
Discussion
Here, we presented a STIM1-derived conformational sensor
that allowed for probing of intramolecular transitions within
its cytosolic domains. This region of STIM1 switched from a
tight into an extended conformation either by interaction
with ORAI1 or via mutations introduced in the first or third
putative coiled-coil domain. These engineered STIM1 con-
structs with extended conformations exhibited an enhanced
interaction with both wild-type and the SCID-linked R91W
mutant ORAI1. The mutation-induced extended STIM1
C-terminal conformation reflects an intramolecular transi-
tion, which likely exposes the CAD/SOAR domain and
promotes interaction of full-length STIM1 with ORAI1, even
in the absence of store depletion. We suggest that these
mutant full-length STIM1 proteins that interact with and
constitutively activate ORAI1 channels imitate a physio-
logical activated state, which mimics the conformational
change that occurs in native STIM1 upon store depletion.
The transition is mediated by a change in the intramolecular
associations and/or orientations of the three putative coiled-
coil domains within the STIM1 C-terminus.
CAD/SOAR (Park et al, 2009; Yuan et al, 2009) represents
the minimal cytosolic region within STIM1 that is sufficient
for its homomerization, interaction with and activation
of ORAI1. It comprises the second (aa 364–389) and third
(aa 399–423) coiled-coil domains together with an extended
stretch (aa 423–448) contributing
(Muik et al, 2009). Here, we presented evidence that the
OASF FRET sensor that encompasses the first coiled-coil
region (aa 238–342) and the CAD/SOAR domain adopts a
tight conformation that is extended upon coupling to ORAI1.
As this required intact N- and C-termini of ORAI1 (Figures 2C
and 5A), it is tempting to speculate that the extended con-
formation reflects bridging of OASF between ORAI1 cytosolic
termini, which is facilitated by the OASF coiled-coil mutants
(L251S, L416, 423S) pre-locked in the extended conformation.
Accordingly, N-terminal deletions (D233–251) or the L251S
tohomomerization
mutation located in the N-terminal region of the first putative
coiled-coil domain set OASF in an extended conformation
that promoted enhanced interaction with ORAI1. As the
essential ORAI1-binding site within OASF is suggested within
the second rather than the first coiled-coil domain (Frischauf
et al, 2009; Calloway et al, 2010), these manipulations that
locally disturbed the putative first coiled-coil structure of
OASF probably removed its masking effect on the CAD/
SOAR domain thereby facilitating an interaction with
ORAI1. A similar phenomenon was observed with mutations
within the third coiled-coil domain in that variations that
locally destabilize (L416, 423S) or stabilize (R426L) the
putativecoiled-coilstructure
coupling to ORAI1, respectively. Thus, the OASF conforma-
tion defined by the precise intramolecular coiled-coil arrange-
ments regulates the affinity of STIM1:ORAI1 coupling
requisite for channel gating.
We and others have recently provided data (Frischauf et al,
2009; Calloway et al, 2010) that suggest the second coiled-coil
domain encompasses the potential interaction site for cou-
pling to a putative coiled-coil domain on the C-terminus of
ORAI1. Destabilizing the putative second coiled-coil domain
by L373S and A376S as well as A376K mutations inhibited
the interaction with ORAI1 (Frischauf et al, 2009; Covington
et al, 2010), and switched OASF in an extended conformation.
Recently, data from the Balla laboratory (Korzeniowski
et al, 2010) suggest a basic segment within the second
coiled-coil domain to interact with an acidic cluster in the
first coiled-coil region (see Supplementary Figure S3) in
resting STIM1. Following store depletion, this interaction
may be disrupted by an intramolecular switching mechanism
that enables the basic segment in the second coiled coil of
STIM1 to couple to acidic residues within the C-terminal
coiled-coil domain of ORAI1 (Korzeniowski et al, 2010).
The OASF FRET sensor presented here allowed for the first
time measurement of the conformational change accompany-
ing the intramolecular switch upon STIM1 C-terminus inter-
action with ORAI1. Introduction of neutralizing mutations
(Korzeniowski et al, 2010) within the acidic cluster (E318/
319/320/322A, ¼4EA) in the OASF FRET sensor signif-
icantly reduced FRET compatible with a decrease of the
proposed intramolecular interaction with the basic segment
(Supplementary Figure S4). In addition to the acidic cluster in
the first coiled-coil region, we further identified hydrophobic
amino acids in the first and the third coiled-coil domains that
allowed for switching STIM1 C-terminal fragments from a
tight into an extended conformation as determined by FRET
and MALS. These mutations (L251S, L416S and L423S)
introduced into full-length STIM1 apparently locked a physio-
logically active state resulting in co-clustering with and
activation of ORAI1 currents without store depletion.
These observations suggest that the STIM1 C-terminal
intramolecular transition is controlled by a complex interplay
between the first, second and third coiled-coil domains,
together controlling the exposure of CAD/SOAR domain
and regulating the affinity of the potential ORAI1-interacting
site. In a model combining Balla’s work (Korzeniowski et al,
2010) and results from this study, we suggest that both
hydrophobic as shown here together with electrostatic inter-
actions contribute to the intramolecular coiled-coil transi-
tions within the STIM1 C-terminal portion. In a straight-
forward scenario, oligomerization of STIM1 likely helps to
enhancedorattenuated
Conformational switch upon STIM1–ORAI1 coupling
M Muik et al
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extend its C-terminal conformation controlled by domains
within the first and third coiled-coil regions. Particularly, the
R426L mutation in the latter that allowed for clustering of
full-length STIM1 upon store depletion but failed to markedly
interact with ORAI1 revealed the requisite for a balanced
interplay culminating in CAD/SOAR exposure. The precise
mechanism as to how oligomerization of STIM1 induced via
store depletion (Luik et al, 2008) achieves this intramolecular
transition requires further studies (Wang et al, 2010).
Our OASF conformational sensor additionally served as a
sensitive tool to resolve deficiencies in the SCID-linked
ORAI1–R91W mutant (Feske et al, 2006) aside from the
profound gating defect. This non-functional ORAI1–R91W
mutant, in contrast to wild-type ORAI1, was unable to inter-
act with the OASF conformational sensor. While the latter
was able to induce an extended OASF conformation, the
R91W mutant could not promote the transition. Further, an
interaction with the R91W mutant was only observed when
the OASF sensor was in a slightly or fully extended form by
mutation in the first or third coiled-coil domain. Thus, the
ORAI1–R91W mutant has an attenuated ability to induce an
extended STIM1 conformation. None of the interacting OASF
mutants were able to recover the channel function of the
ORAI1–R91W mutant consistent with a severe impairment
of gating/permeation (Derler et al, 2009).
The OASF conformational sensor may be used to study the
action of small molecules on the principal molecular compo-
nents of SOC. For example, incubation of 2-APB with the OASF
conformational sensor induced a slightly extended conforma-
tion reflected by a decrease of FRET. Thus, interaction of 2-APB
with OASF might initiate an intramolecular transition that
facilitates coupling with ORAI1, although the structural basis
might be distinct to that obtained by coiled-coil mutations.
In conclusion, our STIM1-derived conformational sensor is
applicable as a general tool not only for further dissecting the
nature of STIM1 to ORAI1 coupling, but also for characteri-
zing STIM1 interactions with drugs and other proteins such
as CRACR2A (Srikanth et al, 2010) or the L-type Ca2þ
channel (Park et al, 2010).
Materials and methods
Molecular cloning and mutagenesis
Human ORAI1 (ORAI1; accession number NM_032790) was kindly
provided by A Rao’s lab (Harvard Medical School). N-terminally
tagged ORAI1 constructs were cloned via SalI and SmaI restriction
sites of pECFP-C1 and pEYFP-C1 expression vectors (Clontech)
and C-terminally tagged ORAI1 constructs were cloned using the
XhoI and BamHI sites of the vectors pECFP-N1 and pEYFP-N1.
All ORAI1 point mutants (R91W; E106Q; L273S) were produced
using the QuikChange XL site-directed mutagenesis kit (Stratagene).
For untagged ORAI1 constructs, stop codons were introduced
in ORAI1–YFP, ORAI1–YFP–R91W, ORAI1–YFP–E106Q and ORAI1–
YFP–L273S directly after the ORAI1-coding sequence.
Human STIM1 (STIM1;accession
N-terminally ECFP- and EYFP-tagged was kindly provided by
T Meyer’s Lab, Stanford University. For double-tagged STIM1
constructs, CFP was cloned into pEYFP-C2 via SacII and Xba1 and
the respective STIM1 fragments were introduced via EcoRI and
SacII (233–420, 233–430, 233–450, 233–474, 233–485, 233–535,
233–685). The same procedure was used for construction of YFP–
233–474–YFP and CFP–233–474–CFP. All STIM1 point mutants
(L251S; A376K, L416S L423S; R426L) were generated using the
QuikChange XL site-directed mutagenesis kit (Stratagene).
The integrity of all resulting clones was confirmed by sequence
analysis.
numberNM_003156)
Electrophysiology and cell transfection
Cells transfected (Transfectin, Bio-Rad) with 1mg DNA of ORAI1
and STIM1 constructs were identified by CFP/YFP fluorescence.
Electrophysiological experiments were performed after 12–48h,
using the patch-clamp technique in whole-cell recording configu-
rations at 21–251C. For reducing cell density, cells were some-
times reseeded 47h before the experiments started. An Ag/AgCl
electrode was used as reference electrode. Voltage ramps were
applied every 5s from a holding potential of 0mV, covering a range
of ?90 to 90mV over 1s. For passive store depletion the internal
pipette solution included (in mM): 145 Cs methane sulphonate, 20
EGTA, 10 HEPES, 8 NaCl, 3.5 MgCl2, pH 7.2. Standard extracellular
solution consisted of 145 NaCl, 10 HEPES, 10 CaCl2, 10 glucose,
5 CsCl, 1 MgCl2, pH 7.4. A liquid junction correction of þ12mV
resulted from a Cl?based bath solution and a sulphonate-based
pipette solution. Current traces were leak corrected by subtracting
the remaining currents after 10mM La3þapplication at the end of
the experiment.
Fluorescence microscopy
Confocal FRET microscopy was performed as previously described
(Singh et al, 2006). In brief, a QLC100 Real-Time Confocal System
(VisiTech Int., UK) was used for recording fluorescence images
connectedtotwoPhotometrics
cameras (Roper Scientific) and a dual port adapter (dichroic:
505lp; cyan emission filter: 485/30; yellow emission filter: 535/50;
Chroma Technology Corp.). This system was attached to an
Axiovert 200M microscope (Zeiss, Germany) in conjunction with
an argon ion multiwavelength (457, 488 and 514nm) laser (Spectra
Physics). The wavelengths were selected by an Acousto Optical
Tuneable Filter (VisiTech Int., UK). Image acquisition and control of
the confocal system was performed with MetaMorph 5.0 software
(Universal Imaging Corp.). CFP, FRET and YFP images were
typically illuminated over 900–1500ms and consecutively recorded
with a minimum delay. Image correction due to cross-talk and
cross-excitation were performed prior to the calculation. Therefore,
appropriate cross-talk calibration factors were determined for each
construct on every day of the FRET experiment. After threshold
determination and background subtraction, the corrected FRET
image (NFRET) was calculated on a pixel-to-pixel basis with a
custom-made software (Derler et al, 2006) integrated in MatLab
7.0.4 according to the method published (Xia and Liu, 2001). The
local CFP to YFP ratio might vary due to different localizations of
diverse protein constructs, which could result in the calculation of
false FRET values (Berney and Danuser, 2003). Accordingly, the
analysis was limited to pixels with a CFP:YFP molar ratio within
1:10 to 10:1 to assure reliable results (Berney and Danuser, 2003).
Line-scans were extracted from individual images as indicated by
the dashed lines with a total scan width of 25 pixels corresponding
to a length of 5mm. Intensity values along each individual scan
were normalized to the highest value in the respective scan. Then,
normalized line-scans were aligned for averaging either at the peak
maximum or along the increasing slope representing the cell edge
when no clear peak was present.
CoolSNAPHQmonochrome
Cloning and recombinant expression of OASF-ext
Human STIM1 cDNAwas from Origene (Origene Technologies, Inc).
The cytosolic region encompassing residues Asn234 to Gln491 (aa
234–491) was subcloned into a pET-28a vector (Novagen, Inc.) and
expressed with an N-terminal His6-tag in BL21(DE3) E. coli cells.
The protein was extracted out of inclusions using guanidine,
isolated using Ni-NTA resin (Qiagen, Inc.) and refolded into
20mM Tris, 100mM NaCl, 50mM L-Arg/L-Glu, 2mM DTT (pH 8).
After thrombin digestion of the His6-tag, the protein was further
purified by SEC on a Superdex 200 PG 10/60 column. The identity
of the protein was confirmed by positive electrospray ionization
mass spectrometry. Protein concentration was estimated using
e280nm¼0.95 (mgml?1)?1cm?1.
Far-UV CD
Far-UV CD spectra were recorded on a Jasco J-815 CD Spectrometer
(Jasco, Inc.). Data were collected in 1nm increments using 0.1cm
ES quartz cuvette pathlengths. Wavelength scan rates were at 20nm
per minute, with a response time of 8s and bandwidth of 1nm at
each wavelength. Thermal melts were acquired in 11C increments at
a scan rate of 11C per minute, with a response time of 8s at each
Conformational switch upon STIM1–ORAI1 coupling
M Muik et al
&2011 European Molecular Biology Organization The EMBO JournalVOL 30|NO 9|2011 1687

Page 11

temperature through 0.1–0.2cm pathlengths. Protein concentration
ranged from 0.14 to 0.35mgml?1.
Analytical SEC with MALS
SEC was performed on Superdex 200 10/300 GL columns (GE
Healthcare Biosciences Corp.) at 41C. MALS analyses were
performed in-line, using the three angle (451, 901 and 1351)
miniDawn static light scattering instrument with a 690-nm laser
(Wyatt Technologies, Inc.) and an Optilab rEX differential refract-
ometer (Wyatt Technologies, Inc.). Molecular weight was calculated
from the ASTRA software (Wyatt Technologies, Inc.) based on
Zimm plot analysis using a dndc?1¼0.185lg?1.
Western blot
HEK cells transiently transfected with YFP–OASF–CFP were lysed
with 1ml high salt lysis buffer (20mM Tris/100mM NaCl/2mM
EDTA/10% glycerol/1% nonidet P-40/protease inhibitor cocktail).
After centrifugation, supernatants were mixed with laemmli buffer
and lysates were separated by 10% SDS–PAGE. After western
blotting, Y–OASF–C was detected by an anti-GFP antibody (Roche)
and the correct size of the protein (YFP/CFP-tags: B26kDa, OASF:
B27kDa) was verified by a suitable protein Standard (Bio-Rad).
Supplementary data
Supplementary data are available at The EMBO Journal Online
(http://www.embojournal.org).
Acknowledgements
We thank S Buchegger for excellent technical assistance. Irene
Frischauf (T442) and Isabella Derler (T466) are Hertha-Firnberg
scholarship holders. Barbara Lackner is a recipient of a scholarship
holder from the Austrian Academy of Sciences. This work was
supported by the Canadian Institutes of Health Research (CIHR) and
Heart and Stroke Foundation of Ontario (HSFO) to MI. This work
was also supported by the Austrian Science Foundation (FWF):
project P22747 to RS, project P21925 to KG, and project P21118 as
well as P22565 to CR.
Conflict of interest
The authors declare that they have no conflict of interest.
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Conformational switch upon STIM1–ORAI1 coupling
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