Supplementary Information
Supplementary Figures
Supplementary Figure 1. Orai1 is a plasma membrane protein with intracellular
N- and C-termini.
a, HEK293 cells expressing HA-tagged Orai1, myc-tagged Orai1, or GFP alone were
stained with anti-HA antibody without permeabilization. Staining of the HA-tagged
protein shows that Orai1 is a plasma membrane protein. The other proteins serve as
negative controls for the staining procedure.
b, Fibroblasts from one SCID patient were transduced with N-terminally (panels A, A’,
B) and C-terminally (panels C, C’, D) myc-tagged Orai1, fixed, permeabilized (A, A’, C,
C’) or left unpermeabilized (B, D) followed by staining with anti-myc antibodies. No
staining was observed in the large majority of non-permeabilized cells, indicating that
both the N- and C-terminus are inaccessible to the antibody applied to the outside of the
cell. GFP expression is from the same bicistronic vector as myc-Orai1.
*Note that the legend to Supplementary Figure 1a was updated on 24 August 2006 to
correct a minor error.
Supplementary Figure 2. Alignment of protein sequences of Drosophila Orai and
its three mammalian homologues Orai1, Orai2 and Orai3.
Alignment was performed using ClustalW (http://www.ebi.ac.uk/clustalw/)
30
. The
transmembrane segments predicted for Orai1 by TMpred
(http://www.ch.embnet.org/software/TMPRED_form.html are indicated in blue, the two
conserved glutamate residues that are the focus of this study (E106 and E190 in human
Orai1) are shown in red, and the N-glycosylation site (N223) is shown in green.
Supplementary Figure 3. Expression of mutants in S2 cells and selective
depletion of endogenous dOrai by dsRNA treatment.
a, S2R+ cells were stably transfected with C-terminally V5-tagged wild-type or mutant
dOrai. In the mutant dOrais, E178, E221, E245 and E262 were individually replaced with
glutamine, respectively. Expression levels were evaluated by immunoblotting with
antibody to the V5 tag.
b, RNAi against the 3’-UTR of endogenous dOrai does not significantly affect expression
of ectopically expressed dOrai lacking the 3’-UTR. S2R+ cells stably transfected with Cterminally V5-tagged wild-type dOrai lacking the 3’-UTR were incubated with the
indicated dsRNAs. The V5-tagged recombinant protein was detected by immunoblotting
with antibody to the V5 tag. Depletion of V5-tagged dOrai is accomplished by dsRNA
against the coding region of dOrai (Orai), but not by dsRNAs against the 3’-UTR (UTR)
or an irrelevant control sequence (GFP).
Supplementary Figure 4. Evolutionarily conserved E106 in Orai1 is required for
Orai1-mediated Ca2+ influx.
a, Ca2+ influx-deficient fibroblasts from the SCID patients were stably transduced with a
panel of myc-tagged Orai1 mutants in which glutamates (E) at positions 106 and 190
were individually replaced with alanine (E106A, E190A), glutamine (E106Q) or aspartate
(E106D). Ca2+ influx was compared in GFP+ (Orai1-expressing) cells in response to
perfusion with 20 mM extracellular Ca2+ following store-depletion with 1 M thapsigargin
(TG). Left panels: Mutant E106A did not restore Ca2+ influx in the SCID cells whereas a
partial restoration was observed with the E106D and E106Q mutants. Mutant E190A
reconstituted normal peak Ca2+ levels. Right panels: Residual Ca2+ influx in the E106D,
E106Q and E190A mutants was sensitive to inhibition by 2 M La3+. A moderate
potentiation of Ca2+ influx with low doses of 2-APB (2 M) was seen in the E190A mutant
but not in the unresponsive E106A mutant.
b, Summary of peak Ca2+ responses (top panel) and initial rates of Ca2+ influx (bottom
panel) from experiments similar to those shown in a. Averages are from two to five
independent experiments per mutant.
Supplementary Figure 5. Orai1 mutant proteins are expressed in the plasma
membrane.
HEK293 cells were transfected with mutant forms of Orai1 containing an HA-tag in the
predicted second extracellular loop. Cells were left unpermeabilized, fixed, and stained
on ice with anti-HA antibodies. GFP expression is from an IRES-site in the same
expression vector. Note that all mutants of Orai1 are expressed in the plasma
membrane and show staining that is comparable to the staining of wild-type Orai1 (see
Fig. 1).
Supplementary Figure 6. Ca2+ influx in SCID T cells expressing glutamate mutants
of Orai1 is sensitive to inhibition by La3+.
T cells from CRAC-deficient SCID patients were stably transduced with myc-tagged wildtype Orai1 or Orai1 mutants in which glutamates (E) in positions 106 and 190 were
individually replaced with alanine (E>A), glutamine (E>Q) or aspartate (E>D). Ca2+ influx
was compared in GFP+ (Orai1-expressing) cells following store depletion with
3+
shortly after the peak of the
Ca2+ response in 2 mM Ca2+. Experiments shown are representative of at least three
similar experiments.
Supplementary Figure 7. Leak-corrected current in control cells is negligible.
I-V curves obtained after leak correction with La3+ from an untransfected SCID T cell
(GFP-) taken from one of the same experiments in which GFP-positive cells showed
reconstitution of CRAC current, and from a SCID T cell expressing a nonfunctional
mutant Orai1 (R91W). The lack of evident inward or outward current in these cells
validates the conclusion that the leak-corrected currents in cells expressing E106D,
E190D, and E190Q Orai1 proteins represent altered CRAC channel currents.
Supplementary Figure 8. Concentration dependence of the block of monovalent
ion current by Ca2+.
A further representative experiment illustrating the concentration dependence of Na+ICRAC block by extracellular Ca2+ in cells expressing E106D Orai1. Standard Ringer
solution containing 20 mM Ca2+ was replaced by solutions with [Ca2+]o as indicated.
DVF indicates divalent ion-free solution.
Supplementary Methods
Cell lines. Human T cell lines from one control individual and SCID patient 2 were
immortalized by transformation with herpes virus saimiri (HVS) as described 31. Foreskin
fibroblasts from the newborn SCID patient 2 and a healthy newborn (Hs27 cell line,
ATCC, Manassas, VA) were immortalized by retroviral transduction with a telomerase
expression plasmid (hTERT, S. Lessnick, DFCI, Boston, MA). T cells and fibroblasts
were grown as described 32. The macrophage-hemocyte-like Drosophila cell line S2R+
was grown in Schneider’s medium with 10% fetal calf serum (Invitrogen) according to
standard protocols.
Plasmids, retro- and lentiviral transductions. Full-length cDNA for Orai1 (BC015369)
was purchased from OpenBiosystems (Huntsville, AL) and subcloned into pENTR11
(“Gateway” system, Invitrogen, Carlsbad, CA) for use in retroviral transductions. For all
myc-epitope tagged vectors, the c-myc sequence (peptide: MEQKLISEEDL; nucleotide:
ATG GAA CAA AAA CTT ATT TCT GAA GAA GAT CTG) was placed in frame
immediately in front of (N-terminal myc-tag) or behind (C-terminal myc-tag) the coding
sequence of Orai1. In case of the N-terminal myc-tag, the transcription start site (ATG)
was placed at the beginning of the myc sequence deleting the endogenous start codon;
in case of the C-terminal myc-tag, the endogenous stop codon was replaced with GAG
encoding the first E of the myc-tag; at the end of the myc sequence a TAG codon was
added to terminate translation. For mutagenesis of Orai1, a standard 2-step PCR
approach with pairs of overlapping degenerate primers spanning the mutation site was
used. Tagged Orai1 cDNA was then transferred from pENTR11 to the bicistronic
retroviral expression vector pMSCV-CITE-eGFP-PGK-Puro (M. Ohora, CBRI, Boston),
which allows for simultaneous expression of Orai1, GFP and a puromycin resistance
gene. C-terminally FLAG-tagged Orai1 was subcloned into XhoI-EcoRI site of pMSCVCITE-eGFP-PGK-Puro. The endogenous termination codon was substituted with the
FLAG-tag coding sequence (AADYKDDDDK) followed by a TAG termination codon. For
mutation of the N-glycosylation site (N223) in Orai1, AAC (Asn) was replaced with GCC
(Ala). For lentiviral transduction, N-terminally myc-tagged versions of Orai1 were directly
cloned NotI-BamHI into the bicistronic pHAGE-UBC-eGFP-IRES-eGFP vector (R.
Mulligan, Harvard Medical School), replacing the first eGFP with myc-Orai1. For the
introduction of an HA-tag into the second extracellular loop of Orai1, an XbaI-XhoI site
was introduced between amino acid positions 207 and 208 or 214 and 217 (in the latter
case deleting amino acids 215 and 216) followed by ligation of a double-stranded
oligonucleotide encoding the HA peptide flanked by a glycine-serine-glycine-serine
linker on either side (5’ CT AGA GGA AGC GGA AGC TAT CCA TAT GAC GTC CCA
GAC TAT GCC GGA AGC GGA AGC C; 3’ TC GAG GCT TCC GCT TCC GGC ATA
GTC TGG GAC GTC ATA TGG ATA GCT TCC GCT TCC T). For retroviral
transductions, HEK293FT (Invitrogen) cells were co-transfected with plasmids encoding
Orai proteins, gag-pol and env to produce amphotropic, replication-incompetent
retrovirus; virus-containing supernatant was collected 1, 2 and 3d after transfection and
or lentiviral
transductions, HEK293FT cells were co-transfected with plasmids encoding Orai
proteins, gag-pol, rev, tat and VSVG proteins, virus was collected 1, 2 and 3d after
transfection, filtered and concentrated by centrifugation at 40,000 x g for 3h.
Transduction efficiencies were evaluated by GFP expression using flow cytometry and
myc-Orai1 expression using immunoblotting and immunocytochemistry.
Western blots, immunoprecipitation and surface biotinylation. HEK293 cells were
stably or transiently transfected with mammalian expression vectors. In transient
expression studies, cells were harvested in Triton lysis buffer (1.0 % TritonX100, 20 mM
Tris pH 7.4, 150 mM NaCl, 1 mM EDTA, 20 mM -glycerol-phosphate, 10 mM sodium
pyrophosphate, 0.1 mM sodium orthovanadate, 10 mM NaF, 1 mM phenylmethylsulfonyl
fluoride (PMSF), 10 g/ml aprotinin, 10 g/ml leupeptin) 48 h post tranfection. Lysates
precleared for 2 h at 4oC with 25 L packed protein G sepharose were
immunoprecipitated overnight at 4oC with anti-FLAG resin (Sigma). Following four
washes in lysis buffer, immunoprecipitates were resolved by 10% SDS-PAGE followed
by immunoblotting using anti-FLAG antibodies. For surface biotinylation, 5x107 HEK293
cells were treated with tunicamycin (2 g/ml) or left untreated for 18 h, then placed on
ice. Cells were biotinylated with 0.5 mg/ml sulfo-NHS-biotin/PBS for 2 h at 4oC. After
quenching, cells were lysed with Triton lysis buffer, the lysates were precleared for 2 h
with protein G sepharose and precipitated with anti-FLAG antibody conjugated resin
overnight. Proteins were eluted with 0.2 mg/ml FLAG peptide (Sigma), 2X SDS-PAGE
denaturing sample buffer was added for electrophoresis and membranes probed with
anti-FLAG antibody or HRP-conjugated streptavidin. For peptide N-glycosidase F
digestion, anti-FLAG immunoprecipitates from FLAG-Orai1 transfected HEK293 cells
were treated with PNG-F (5,000 U/ml) for 2 h at 37C.
Immunocytochemistry and confocal imaging. Immunocytochemistry for Orai1 was
carried out as described 32. Briefly, retrovirally-transduced T cells and fibroblasts were
fixed with 3% paraformaldehyde, left unpermeabilized or permeabilized with wash buffer
containing 0.5% NP-40, incubated with anti-myc (9E10) or anti-HA (12CA5) antibodies
and Cy3-labeled secondary antibodies. For Figure 1c, d, immunofluorescence was
analyzed by epifluorescence microscopy using an Axiovert 200 epifluorescence
microscope (Zeiss) and a Plan Apochromat 63x oil DIC objective with a N.A. of 1.4. Zstacks of immunofluorescence images were acquired with a step-interval of 0.3 m and
deconvolved using a two nearest neighbor algorhythm in the deconvolution module of
OpenLab imaging software (version 3.1.2., Improvision). For Supplementary Figure 1,
immunofluorescence was analyzed by confocal imaging using a Radiance 2000 Laserscanning confocal system (Bio-Rad Laboratories) on a BX50BWI Olympus microscope
using a 63x water immersion objective.
Generation of dOrai mutant S2R+ cell lines and Ca2+ measurements. S2R+ were
stably transfected with wild-type or mutant dOrai (E178Q, E221Q, E245Q and E262Q)
subcloned into the EcoRI and XhoI site of the expression plasmid pAc5.1 (Invitrogen).
The endogenous termination codon (TAG) was substituted with a V5 epitope or
polyhistidine, followed by a termination codon. For mutagenesis, the coding sequence
for glutamate residues (GAG) was replaced with the coding sequences for glutamine
(CAG). S2R+ were co-transfected with pAc5.1 dOrai and a hygromycin resistance gene
under the control of a constitutively active promoter (pCoHygro, Invitrogen) at a ratio of
19:1. Cells were selected for 3-4 weeks with 300 g/ml hygromycin. Expression levels of
dOrai were evaluated by immunoblotting with antibody to the V5 tag. For RNAi, dsRNA
(5 g) against coding sequence or 3’-untranslated region of dOrai mRNA was
transfected into S2R+ cells and after 4d of incubation, Ca2+ influx was measured by flow
cytometry. S2R+ cells were detached from the dish with trypsin (CellGro, Herndon, VA),
loaded with the Ca2+ indicator dyes Fluo4-AM and Fura-Red (2 M each, Molecular
Probes, Eugene, OR) for 45 min at room temperature and then resuspended in loading
medium (Schneider’s medium + 10% FCS). Immediately before flow cytometric Ca2+
measurements, cells were resuspended in Ringer solution containing 0 mM Ca2+ and
analyzed for basal Ca2+ levels on a FACSCalibur (BD Biosciences, San Jose, CA). After
30 s, thapsigargin (3 M) was added to deplete Ca2+ stores. After 180 s, twice the
volume of Ringer solution containing 4 mM Ca2+ was added to the samples (final [Ca2+]o
2 mM) and intracellular Ca2+ levels were monitored until 300 s. The ratio of Fluo-4 and
Fura-Red emission was analyzed using FlowJo software (Tree Star, Inc., Ashland, OR).
Single-cell Ca2+ imaging. T cells were loaded at 1x106 cells/ml with 1 M fura-2/AM
(Molecular Probes) in loading medium (RPMI + 10% FBS) for 30 min at 22-25oC,
resuspended in loading medium and attached to poly-L-lysine–coated coverslips for 15
min. Fibroblasts were grown directly on UV-sterilized coverslips and loaded with 3 M
fura-2/AM for 45 min at 22-25oC. For [Ca2+]i measurements, cells were mounted in a
RC-20 closed-bath flow chamber (Warner Instrument Corp., Hamden, CT) and analyzed
on an Axiovert S200 epifluorescence microscope (Zeiss) with OpenLab imaging
software (Improvision). Cells were perfused in Ca2+-free Ringer solution and Ca2+ stores
were passively depleted with 1 M thapsigargin. Were indicated, cells were perfused
with 0.5 M La3+ shortly after the peak of the Ca2+ response in 2 mM Ca2+. Fura-2
emission was detected at 510 nm with excitation at 340 and 380 nm and Fura-2
emission ratios (340/380) were calculated for each 5-s interval after subtraction of
background. For each experiment, 50 - 100 individual cells were analyzed for 340/380
ratios using Igor Pro (Wavemetrics, Lake Oswego, OR) analysis software. For
determination of intracellular Ca2+ concentrations, GFP/Orai positive cells were
selected and [Ca2+]i was estimated from the relation [Ca2+]i = K* (R-Rmin)/(Rmax-R). K*,
Rmin, and Rmax were measured in control human T cells in situ as previously described33.
Ca2+ influx rates were inferred from the maximal rate of rise in Ca2+ concentrations
(d[Ca2+]i/ dt) immediately after readdition of Ca2+o.
Patch-clamp measurements. Patch-clamp recordings were performed using an
Axopatch 200 amplifier (Axon Instruments, Foster City, CA) interfaced to an ITC-18
input/output board (Instrutech, Port Washington, NY) and an iMac G5 computer.
Currents were filtered at 1 kHz with a 4-pole Bessel filter and sampled at 5 kHz.
Recording electrodes were pulled from 100-µl pipettes, coated with Sylgard, and firepolished to a final resistance of 2-5 M. Stimulation and data acquisition and analysis
were performed using routines developed on the Igor Pro platform (Wavemetrics, Lake
Oswego, OR) by R.S. Lewis. Data are corrected for the liquid junction potential of the
pipette solution relative to Ringer’s in the bath (-10 mV) and for the bath DVF solution
relative to Ringer’s in the bath-ground agar bridge (+5 mV). Currents were leak
subtracted by exposing the cell to 20 mM [Ca2+]o + 20 µM La3+ to eliminate ICRAC and
using this remaining current as “leak”. The standard extracellular Ringer’s solution
contained (in mM): 120 NaCl, 4.5 KCl, 20 CaCl2, 1 MgCl2, 10 D-glucose, and 5 NaHepes (pH 7.4). In some experiments, 2 mM CaCl2 was used in the standard
extracellular solution and the NaCl concentration was raised to 150 mM. The standard
divalent-free (DVF) Ringer’s solutions contained (in mM): 155 Na methanesulfonate, 10
HEDTA, 1 EDTA and 10 Hepes (pH 7.4). For experiments examining block of Na+-ICRAC
by Ca2+, MgCl2 was omitted from the standard extracellular solution and CaCl2 was
added to the indicated concentration. In some experiments an NMDG-based solution
was employed containing 150 NMDG-Cl, 10 D-glucose, and 5 Hepes. When Ca2+ was
included, the composition of this solution was modified to 120 NMDG-Cl, 20 CaCl2, 10 Dglucose, and 5 Hepes. 25 nM charybdotoxin (Sigma) was added to all extracellular
solutions to eliminate contamination from Kv1.3 channels. The standard internal solution
contained (in mM): 150 Cs aspartate, 8 mM MgCl2, 10 BAPTA, and 10 Cs-Hepes (pH
7.2). For the experiment shown in Fig. 3b, Cs+ was replaced with Na+.
Data analysis. Averaged results are presented as the mean value ± s.e.m. Curve fitting
was done by least-squares methods using built-in functions in Igor Pro 5.0. Relative
permeability of Cs (PCs/PNa) was calculated from the biionic reversal potential using the
relation:
PCs [Na]o Erev F RT

e
PNa [Cs]i
where R is the gas constant (8.314 J K-1 mol-1), T is the absolute temperature and F is

the Faraday
constant (96480 C mol-1), and PCs and PNa are the permeabilities of the Cs+
and Na+, respectively, [Cs]i and [Na]o are the ionic concentrations, and Erev is the
reversal potential.
For calculations of the relative calcium permeability, the Goldman-Hodgkin-Katz
equation34,35 has been used in its extended form that allows comparison of divalent ion
and monovalent ion permeabilities36,37. Several restrictive assumptions are made in the
derivation of the equation for this case: that ions transiting the membrane do not interact;
that each ionic species has a constant mobility in the membrane; and that the electrical
potential experienced by a permeating ion changes linearly with distance through the
membrane. These assumptions do not in fact hold for Ca2+-selective channels, including
the CRAC channel. For example, the first assumption is explicitly at variance with
findings showing ion-ion interactions (saturation of Ca2+ currents and pore block of
monovalent ions by divalents), and the second and third assumptions are inconsistent
with the existence of a Ca2+-binding site in the permeation pathway. As a result, the
ratios PCa/PNa obtained from the extended Goldman-Hodgkin-Katz equation reflect the
properties of the channels only indirectly, and need not remain constant when
membrane potential or ionic composition of the internal and external solutions are
changed. Nevertheless, the PCa/PNa values derived for other channels show a general
correlation with the ability of those channels to conduct Ca2+, and are cited in the
literature on Ca2+ permeability. The following calculations allow for comparison with that
literature.
At the reversal potential, Erev,
INa  ICa  ICs  0
where INa, ICa and ICs reflect the inward Na+ and Ca2+ currents and outward Cs+ current
through the CRAC channel, respectively. Substituting the values for the currents in
terms of theirpermeabilities and solving for the reversal potential at which the net ionic
flux is zero, and ignoring the distinction between ionic concentrations and ionic activities,
we get 38,39
Vrev 
RT b  (b 2  4ac)1/ 2
ln
F
2a
where,

a  [Cs]i
b  [Cs]i 
c 
PNa
[Na]o
PCs
PNa
P
[Na]o  4 Ca [Ca]o
PCs
PCs
From the observed reversal potentials for E106D currents (36 mV in 20 mM [Ca2+]o and
2+
13 mV
 in 2 mM [Ca ]o), and assuming that PCs=PNa for E106D currents, these relations
yield PCa/PNa values of 28 ([Ca2+]o=20 mM) and 32 ([Ca2+]o=2 mM). By contrast, for the
E190D Orai1 currents, the calculated PCa/PNa is >950 assuming a reversal potential of
+80 mV with internal Na+ as the charge carrier (Fig. 3b).
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