Pore architecture of the ORAI1 store-operated ... - Semantic Scholar

Pore architecture of the ORAI1 store-operated calcium channel Yubin Zhoua,b, Sweta Ramachandrana, Masatsugu Oh-horaa,b, Anjana Raoa,b, and Patrick G. Hogana,1 a

Immune Disease Institute and Program in Cellular and Molecular Medicine, Children’s Hospital Boston, 200 Longwood Avenue, Boston, MA 02115; and Department of Pathology, Harvard Medical School, 200 Longwood Avenue, Boston, MA 02115

b

Contributed by Anjana Rao, January 29, 2010 (sent for review January 6, 2010)

ORAI1 is the pore-forming subunit of the calcium release-activated calcium (CRAC) channel, a store-operated channel that is central to Ca2þ signaling in mammalian cells. Electrophysiological data have shown that the acidic residues E106 in transmembrane helix 1 (TM1) and E190 in TM3 contribute to the high selectivity of ORAI1 channels for Ca2þ. We have examined the pore architecture of the ORAI1 channel using ORAI1 proteins engineered to contain either one or two cysteine residues. Disulfide cross-linking shows that ORAI1 assembles as a tetramer or a higher oligomer with TM1 centrally located. Cysteine side chains projecting from TM1 at position 88, 95, 102, or 106 cross-link efficiently to the corresponding side chain in a second ORAI1 monomer. Cysteine residues at position 190 or at surrounding positions in TM3 do not cross-link. We conclude that E106 residues in wild-type ORAI1 are positioned to form a Ca2þ binding site in the channel pore and that E190 interacts less directly with ions traversing the pore. The cross-linking data further identify a relatively rigid segment of TM1 adjacent to E106 that is likely to contribute to the selectivity filter. cross-linking ∣ membrane protein ∣ store-operated calcium entry ∣ stromal interaction molecule ∣ structure

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he electrophysiologically defined calcium release-activated calcium (CRAC) channel is responsible for physiological Ca2þ influx in T cells and mast cells and for store-operated Ca2þ entry in other cells (1). CRAC currents in human T cells require the protein ORAI1 (2–4), which has been shown to be a plasma membrane Ca2þ channel protein [(5–8); reviewed in refs. 9–11]. The severe combined immunodeficiencies that result from rare mutations in the human ORAI1 gene and the functional deficits of Orai1−∕− mice dramatize the crucial signaling role played by ORAI1 in Tcells and mast cells (2, 12–14). The hallmarks of native CRAC channels and of recombinant ORAI1 channels are that they open in response to a reduction of Ca2þ concentration in the endoplasmic reticulum (ER) lumen, that they are highly selective for Ca2þ under physiological conditions, and that they carry only very small single-channel currents (1, 11). The sensitivity of the channel to the level of ER Ca2þ stores has been traced to the ER-resident Ca2þ sensor protein STIM1 (15–20), but the basis for other channel properties is not understood in any detail. Point mutations introduced into the transmembrane helices of ORAI1 or its Drosophila orthologue alter ion selectivity (5–7). The replacements E106D or E190Q in human ORAI1 sharply reduce the ability of the channel to discriminate between Ca2þ and Naþ under physiological conditions and reduce the decided preference of the channel for Naþ over Csþ under conditions where the channel conducts monovalent ions (6, 7). However, although the electrophysiological analysis shows that E106 and E190 influence ion movements within the channel pore, it does not establish whether these residues do so directly by coordinating ions as they traverse the pore or indirectly by maintaining the geometry of the ion conduction pathway. In this study we have examined the pore architecture of the ORAI1 channel by oxidative cross-linking of cysteine residues placed in TM1 or TM3. The cross-linking approach rests on the ability of aqueous iodine or Cu2þ -phenanthroline (CuP) at 4896–4901 ∣ PNAS ∣ March 16, 2010 ∣ vol. 107 ∣ no. 11

low concentrations to cause disulfide bond formation between native or introduced cysteine residues that are in close proximity and thereby to map protein structure (21–23). Our analysis affords an initial view of ORAI1 channel structure and assigns distinct roles to E106 and E190. Results Cysteineless ORAI1. Wild-type ORAI1 in membranes can cross-link

spontaneously to dimers and oligomers when incubated under nonreducing conditions. To facilitate gel electrophoretic analysis of the cross-linking pattern of ORAI1, we engineered a human ORAI1 cDNA with the three naturally occurring cysteine codons changed to valine codons and with the glycosylation site N223 mutated to alanine. In addition, the nonconserved N-terminal region of ORAI1, residues 1–64, was removed (Fig. 1A). Neither glycosylation nor the nonconserved region ORAI1(1–64) is required for CRAC channel function (6, 8, 24). To assess the ability of cysteineless (C > V) ORAI1 to assemble into CRAC channels, we expressed wild-type and cysteineless ORAI1 in primary CD4þ T cells isolated from Orai1−∕− mice. Immunocytochemistry showed that FLAG-tagged cysteineless ORAI1, like wild-type ORAI1 (2, 6), localizes at or near the plasma membrane (Fig. S1). As reported previously (13), storeoperated Ca2þ entry into CD4þ T cells obtained from Orai1−∕− mice is compromised (Fig. 1B); the residual Ca2þ entry has been ascribed tentatively to the ORAI-family proteins ORAI2 and ORAI3 (13, 14). Expression of wild-type ORAI1 in Orai1−∕− T cells restored store-operated Ca2þ entry, but expression of cysteineless ORAI1 failed to reconstitute store-operated Ca2þ entry, and, in fact, diminished the small residual Ca2þ entry (Fig. 1B). Furthermore, overexpression of cysteineless ORAI1 suppressed store-operated Ca2þ entry into wild-type T cells (Fig. 1B). The dominant-interfering effects of cysteineless ORAI1 are reminiscent of those of the pore mutant ORAI1(E106Q) and indicate that cysteineless ORAI1 assembles with wild-type ORAI1, and probably with ORAI2 or ORAI3, into nonfunctional channels. This experiment indicates that oligomerization of cysteineless (C > V) ORAI1 is unimpaired, despite the inability of the mutated protein to function as a store-operated Ca2þ channel. Cross-Linking of TM1 Residues. To probe the structural context of E106, a residue critically involved in Ca2þ flux through the channel, we carried out cysteine substitution analysis of TM1 in the cysteineless ORAI1. Twenty variants of FLAG-tagged ORAI1, with cysteine replacing individually the residues A88 to V107 in TM1, were expressed in HEK293 cells. Membranes were isolated and incubated under oxidizing conditions, either with CuP or with aqueous iodine, to promote disulfide bond formation between adjacent cysteine side chains. Remaining free sulfhydryl Author contributions: Y.Z. and P.G.H. designed research; Y.Z., S.R., and M.O.-h. performed research; Y.Z., A.R., and P.G.H. analyzed data; and Y.Z. and P.G.H. wrote the paper. The authors declare no conflict of interest. 1

To whom correspondence should be addressed. E-mail: [email protected]

This article contains supporting information online at www.pnas.org/cgi/content/full/ 1001169107/DCSupplemental.

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incubation to 30 min resulted in substantial cross-linking at every position in the N-terminal half of TM1 (Fig. 3). This indiscriminate cross-linking is reminiscent of findings with other protein complexes after prolonged exposure to CuP and has been attributed to rotational mobility or flexibility of the protein backbone (25, 26). In contrast, the increase in cross-linking in the C-terminal half of TM1 was relatively selective for positions 98, 101, and 105, reinforcing the evidence of a helical repeat, and the fractional cross-linking at residues 99, 100, 103, and 104 remained very low (Fig. 3). The retention of a helical pattern even after extensive oxidation indicates that the region between residues 99 and 104 is more rigid than other parts of TM1 or is partially inaccessible due to structural constraints.

Fig. 1. Cysteineless ORAI1 used for the cross-linking studies. (A) Schematic depiction of the topology and primary sequence of cysteineless ORAI1. Nterminal residues 1–64 were replaced by a FLAG epitope, and the three native cysteine residues were replaced by valine residues (C126V/C143V/C195V, yellow circles). The glycosylation site was eliminated by an N223A substitution (pink circle). Residues E106 in TM1 and E190 in TM3, which have been linked to the Ca2þ selectivity of the channel, are highlighted in red. (B) Single-cell ½Ca2þ i measurements in primary CD4þ T cells. Orai1−∕− T cells (KO) had been infected with empty vector (magenta), expression vector for wild-type ORAI1 (green), or expression vector for cysteineless ORAI1 (red). T cells from a wildtype littermate (WT) received empty vector (black) or expression vector for cysteineless ORAI1 (blue). Intracellular Ca2þ stores were depleted by treatment with 1 μM thapsigargin (TG) in a nominally Ca2þ -free buffer, and Ca2þ influx was monitored by the increase in cytoplasmic Ca2þ following readdition of extracellular Ca2þ. Data are plotted as mean SEM.

groups were then blocked by treatment with N-ethylmaleimide, and samples were analyzed by SDS-polyacrylamide gel electrophoresis under nonreducing conditions. For membranes incubated in buffer containing 0.3 mM DTT, each ORAI1 protein migrated as a single band corresponding to ORAI1 monomer, whereas for membranes incubated in buffer supplemented with CuP or aqueous iodine, certain ORAI1 proteins displayed a second band, indicating cross-linking (Fig. S2). The CuP-catalyzed cross-linking could be reversed by addition of DTT (Fig. S3), indicating that cross-linking was due to disulfide bond formation. Treatment on ice for 10 min with 1 mM CuP or with dilute aqueous iodine led to more than 60% disulfide bond formation at A88C, L95C, and V102C, and to ∼40% cross-linking at E106C (Fig. 2A–C). The efficient formation of disulfides at these positions is strong evidence that the specified side chains in two ORAI1 monomers are in close proximity. The cross-linking at positions 88, 95, 102, and 106, all on one predicted helical face of TM1 (Fig. 2D), demonstrates that TM1 is centrally located between or among ORAI1 subunits and also provides confirmation of the helical secondary structure of TM1. Interestingly, 1 mM CuP also resulted in weak cross-linking, 5–30%, at several other positions in the N-terminal (inner) half of TM1 (S90C, T92C, S93C, A94C, and L96C), but not in the C-terminal (outer) half of TM1 (Fig. 2B and D). Increasing the concentration of CuP to 10 mM and extending the time of Zhou et al.

ORAI1 Assembles as a Multimer. The efficient cross-linking at specific positions along TM1 provided an opportunity to probe the oligomeric state of human ORAI1 in the membrane. We engineered cDNAs encoding ORAI1 proteins with cysteine residues at pairs of cross-linkable positions in TM1 (A88C/L95C, L95C/ V102C, and A88C/V102C), expressed the proteins, and performed CuP oxidation reactions. Since the reaction mixture contained 0.3 mM DTT, no cross-linking was detected at low CuP concentrations. With 0.5 mM CuP, distinct dimer, trimer, and tetramer bands were visible for A88C/V102C (Fig. 5A). Formation of cross-linked oligomers occurred within 1 min in the case of A88C/V102C, and neither the overall band pattern nor the relative intensity of the oligomeric bands changed with an increase in CuP concentration or an increase in elapsed time (Fig. 5A and B). Oligomer bands also appeared within 1 min for A88C/L95C and L95C/V102C, but for these proteins there was a clear increase in the oligomer bands with time (Fig. 5B). We take the presence of cross-linked tetramer as evidence that ORAI1 assembles as a tetramer or larger oligomer in the plasma membrane. The most likely reason for the limited amount of tetramer is that once the first cross-links are established, they constrain the possible rotations of TM1 and its movements relative to other ORAI1 monomers and thereby constrain the formation of additional cross-links.

Discussion We have probed the pore structure of the ORAI1 channel by cross-linking cysteine residues engineered into specific sites in transmembrane helices 1 and 3. The principal conclusions are that TM1 helices are centrally placed in the assembled ORAI1 channel multimer and that inward-facing E106 side chains of separate monomers are in a position to form a binding site or binding sites for Ca2þ within the ORAI1 channel pore. Given the high sequence conservation in the transmembrane helices, and in particular the sequence identity in TM1 (Fig. S4), these conclusions are very likely to apply to the other human ORAIfamily channels. The cross-linking data indicate that the ORAI1 channel is at least a tetramer. An ORAI1 tetramer is supported by other lines of evidence, including electrophysiological studies of concatenated ORAI1 monomers (27) and single-molecule photobleaching of channels assembled from ORAI1-EGFP or GFPPNAS ∣ March 16, 2010 ∣

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Cross-Linking of TM3 Residues. A second negatively charged residue that has been linked to Ca2þ selectivity of the ORAI1 channel is E190 in TM3. Under the conditions used for TM1 cross-linking, pairs of E190C residues failed to cross-link (Fig. 4A). Cysteine residues inserted at the neighboring positions 185–195 in TM3, covering three turns of the predicted helix, did not cross-link (Fig. 4B), ruling out the possibility that E190C side chains are juxtaposed but in an orientation that does not favor cross-linking. Thus there is no biochemical support for a close physical contact between E190 or nearby residues and the corresponding residues in a separate ORAI1 monomer.

Fig. 2. E106C and TM1 are at an interface between ORAI1 monomers. (A–C) Membranes isolated from HEK293 cells expressing ORAI1 with a single cysteine residue in place of the TM1 residue indicated were incubated on ice for 10 min (A) in a buffer containing 0.3 mM DTT, (B) in a buffer supplemented with 1 mM CuP, or (C) in a buffer supplemented with 2 mM aqueous iodine (I2 ). Samples were separated by SDS-PAGE under nonreducing conditions, and ORAI1 was detected by staining with anti-FLAG monoclonal antibody. (D Left) Cross-linking efficiency at each position is the fraction of total anti-FLAG staining in the dimer band. The range of the means in two experiments is plotted, except for A88C, L95C, and E106C, where the mean SEM for three experiments is given. (D Right) The cross-linking patterns of TM1 in incubations with CuP or with aqueous iodine are mapped onto the predicted α-helix.

Drosophila Orai (28, 29). There is contrasting electrophysiological evidence that ORAI1-ORAI3 heteromeric channels are pentamers (30). Our data are consistent with a tetrameric ORAI1 complex, but, because of the relatively low efficiency of crosslinking to tetramers, we cannot exclude a pentameric ORAI1 complex. Regardless of the oligomeric state, however, the major conclusion is that TM1 is centrally located in the ORAI1 channel complex. Experimentally, the Ca2þ selectivity of ORAI channels depends critically on the single glutamate residue in TM1, residue E106 in the case of human ORAI1 (5–7, 31). Monomers of ORAI1(E106C) cross-link efficiently, indicating that in wild-type ORAI1 the E106 side chains are situated close together. The protein chemistry complements electrophysiological evidence that ORAI1(E106D) channels have lower affinity for Ca2þ than wild-type channels, as measured by the ability of Ca2þ to block monovalent ion currents (6, 32), and strengthens the conclusion that E106 carboxyl groups form a binding site or binding sites for Ca2þ . This conclusion is further supported by the findings that 4898 ∣


ORAI1(E106A) channels do not conduct Ca2þ (6, 33) and that a single E106Q substitution in a concatenated ORAI1 tetramer prevents Ca2þ current through the channel (27). In contrast, a second residue that has been implicated in Ca2þ selectivity, E190 in TM3, does not contribute to a Ca2þ binding site in the channel pore. The isosteric replacement E190Q in TM3 results in a decrease in the selectivity of the ORAI1 channel for Ca2þ (6, 7) and a substantial increase in its estimated pore diameter, from 3.8 Å to 7.0 Å (32). However, monomers of ORAI1(E190C) do not cross-link efficiently in our experiments, nor do ORAI1 monomers with cysteine substituted at neighboring TM3 sites, indicating that pairs of E190 side chains are distant from each other in the ORAI1 channel. The distinction between the roles of E190 and E106 in ion permeation is also underlined by the finding that Ca2þ influx is not altered by the E190A replacement (6). We conclude that E190 side chains do not constitute a Ca2þ binding site in the channel and that the E190Q substitution decreases ion selectivity and widens the channel pore through an allosteric change in the selectivity filter. Zhou et al.

Fig. 3. Cross-linking of TM1 residues in a more extended CuP-catalyzed reaction. (A) Membranes containing ORAI1 with engineered cysteine residues at positions indicated were incubated on ice for 30 min with 10 mM CuP. Samples were separated by SDS-PAGE under nonreducing conditions, and ORAI1 was detected by staining with anti-FLAG monoclonal antibody. ORAI1 proteins with cysteine at positions showing efficient cross-linking in Fig. 2 were not included in this experiment. (B) Quantitation of cross-linking efficiency in the extended CuP-catalyzed reaction. Error bars represent the range of duplicate measurements.

The rigidity or close packing of the segment containing residues 99–104 may serve to hold E106 carboxyl groups in position to coordinate Ca2þ . The voltage dependence conferred by the V102I substitution (33) demonstrates that the channel is exquisitely sensitive to the packing of side chains in this segment of TM1. The central location of TM1 in the ORAI1 channel ensures that Ca2þ or other ions leaving the E106 site and passing inward through the channel, whatever their detailed path, will confront a part of the channel lined at least in part by nonpolar side chains projecting from the helical turns of TM1 immediately internal to E106. This architecture and the local rigidity between residues 99 and 104 are likely to contribute to the low single-channel currents of the ORAI1 channel and to the narrow pore diameter determined with small organic cations. There is considerable evidence for a role of TM1 in ORAI channel gating. TM1 is nearly invariant in ORAI1 orthologues across vertebrate and insect species (Fig. S5), and we show here that this helix is centrally located and that its inner (N-terminal) portion is relatively more flexible than the outer (C-terminal) part. Moreover, A88C in the TM1-TM1′ interface is the most Zhou et al.

readily cross-linked position in TM1. Functional STIM1 fragments interact with the peptide ORAI1(68–91) in a yeast splitubiquitin assay and with the isolated peptide ORAI1(65–87) in vitro (8, 34), an interaction that maps to a conserved part of ORAI overlapping the N-terminal cytoplasmic region and the initial residues of TM1. Electrophysiological data point in the same direction. Substitutions for residues S89, S90, and R91 near the N terminus of TM1 can decrease or increase store-operated currents in cellular assays. Attention has focused mainly on ORAI1 channels with the SCID mutation R91W (2) or with the experimental replacements R91V, R91L, or R91F (35), which fail to carry current upon store depletion even though the proteins are at the cell surface and interact with STIM1 (35–38). R91 itself is not essential, as evidenced by the normal currents carried by R91E and R91G channels (35), and therefore the defect in the R91W channel has been interpreted as interference by the bulky tryptophan side chain with channel gating or with ion flux through the pore (35–38). Other substitutions of interest are R91T, which reduces the amplitude of whole-cell currents compared to wild-type ORAI1; and S89G/S90G, which increases currents (35). In the two latter cases, the inward rectifying current–voltage (I–V) curve in standard recording conditions implies that the substitutions do not alter the selectivity filter (35). The existing data are compatible with the possibility that movement of TM1 gates the channel, either through a displacement of the lower portion of TM1 or through a reorientation of TM1 that brings E106, key nonpolar side chains in the selectivity filter, or both, into a geometry that allows ion flux through the channel. In a complementary study, McNally et al. have probed the ion permeation pathway of the ORAI1 channel electrophysiologically, by examining the sensitivity of inward current to block PNAS ∣ March 16, 2010 ∣

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Fig. 4. E190C and surrounding TM3 residues fail to cross-link. (A) Analysis of ORAI1(E106C) and ORAI1(E190C) proteins incubated with CuP or with aqueous iodine under identical conditions. (B) Analysis of ORAI1 proteins with cysteine engineered into TM3 at the positions indicated. Treatment with 1 mM CuP or with aqueous iodine was carried out as in Fig. 2.

arrangement of the helices inferred from Cd2þ block might overstate the similarity between the conducting ORAI1 channel examined electrophysiologically and the nonconducting channel characterized by cross-linking, because L95C and R91C are in a flexible region of TM1, and the position of the helices in the Cd2þ -blocked channel might not reflect precisely their configuration in the open channel. Nevertheless, the closely similar findings of the two studies suggest that a comparatively small structural change suffices to open the channel. Methods Plasmids. cDNA encoding cysteineless human ORAI1 was synthesized (DNA2.0), with the region encoding N-terminal residues 1–64 replaced by a FLAG epitope (DYKDDDDK), with valine codons substituted for the native cysteine codons (C126V/C143V/C195V), and with an alanine codon at protein position 223 to eliminate the N-glycosylation site (N223A). For expression in HEK293 cells, the cysteineless ORAI1 cDNA was amplified by PCR and inserted between the EcoRI and KpnI sites of the vector pCMVTnT (Promega). Engineered cysteine replacements within the predicted transmembrane segments TM1 and TM3 of ORAI1 were generated by site-directed mutagenesis. The presence of the intended coding sequence in all mutated cDNAs was confirmed by DNA sequencing. Retroviral expression plasmids containing wild-type human ORAI1 cDNA (accession number NM_032790) or the synthetic cysteineless ORAI1 cDNA were constructed by inserting the corresponding coding sequences between the XhoI and EcoRI sites of the bicistronic vector pMSCV-CITE-eGFP-PGK-Puro (Clontech), which allows for the coexpression of the inserted gene, GFP, and a puromycin resistance gene. GFP expression was used to estimate transduction efficiency and to select transduced cells during single-cell real-time ½Ca2þ i measurement.

Fig. 5. ORAI1 assembles as a tetramer or larger oligomer. (A) Membranes from HEK293 cells expressing the ORAI1 mutant A88C/V102C were subjected to cross-linking for 10 min on ice with the specified concentrations of CuP. (B) Membranes from HEK293 cells expressing the paired-cysteine mutants A88C/V102C, A88C/L95C, and L95C/V102C were subjected to cross-linking with 1 mM CuP for the specified periods on ice. (C) Diagram of cross-linking patterns that would produce covalently linked dimers, trimers, and tetramers. Only selected examples are shown.

by methanethiosulfonate reagents or Cd2þ , in ORAI1 channels with cysteine residues engineered into TM1 and TM3 (39). That study arrived at the same principal conclusions: that E106 is part of a Ca2þ binding site, that E190 is not, and that one face of the TM1 helix lines the pore. The two studies begin to define the pore architecture. First, the ORAI1 protein backbone exhibits flexibility at the mouth of the pore. The electrophysiological experiments map a flexible segment from residues 107–110 (39), and cross-linking indicates that the segment from residues 105–107 is flexible, at least in experiments conducted in the nominal absence of Ca2þ . Second, E106 is the key Ca2þ -binding site within the pore. Third, a narrowing of the pore is likely to shape ion selectivity. The blockade of currents through the V102C channel by Cd2þ , but not by the larger methanethiosulfonate reagents, is evidence that the pore narrows at or just below the level of E106 (39). This constriction coincides roughly with the tightly constrained segment of TM1 between residues 99 and 104 identified in cross-linking experiments. Fourth, the inner portions of TM1 helices of separate ORAI1 monomers are in close proximity and line the pore at least to the level of residue 91 as judged by Cd2þ block (39) and remain in proximity to the level of residue 88 as assessed by physical cross-linking. The 4900 ∣


Membrane Preparation. HEK293 cells cultured in six-well plates were transfected with 1 μg plasmid plus 3 μL of Lipofectamine 2000 (Invitrogen) following the manufacturer’s instructions. Forty-eight hours posttransfection, cells (∼107 cells) were detached from the culture plates with a nonenzymatic lifting solution consisting of HBSS with 1 mM EDTA and washed twice with PBS. Cells were collected by centrifugation for 5 min at 1,000 × g, and cell pellets were resuspended in a homogenization buffer [25 mM Tris-HCl pH 7.4, 50 mM NaCl, Protease Inhibitor Cocktail (Roche)] on ice and lysed by passing 20–30 times through a 27G 1∕200 needle (BD Biosciences). Cell lysates were centrifuged at 1,000 × g in a benchtop centrifuge for 15 min at 4 °C to remove nuclei and debris. The postnuclear supernatants were collected and centrifuged at 167,000 × g in an Airfuge (Beckman Coulter) for 30–60 min at 4 ° C. The supernatants were removed, and the pelleted membranes were resuspended in ice-cold resuspension buffer (25 mM Tris-HCl pH 7.4, 150 mM NaCl, 0.3 mM DTT, Protease Inhibitor Cocktail), snap frozen, and stored at −80 °C. Disulfide Cross-Linking. For CuP cross-linking, 1 mM CuSO4 ∕1.3 mM o-phenanthroline final concentration (Sigma), or a higher concentration where indicated, was added to 40 μL resuspended membranes by 5-fold dilution from a solution freshly prepared in 50 mM Tris-HCl pH 7.4, 150 mM NaCl, 0.3 mM DTT, and incubation was carried out on ice for 10 min or for the time indicated. Reactions with aqueous iodine were similarly carried out by the addition of Lugol solution (Sigma) to the final total iodine concentration indicated. Concentrations of aqueous iodine, which consists of I− , I2 , I3 − , and other species in proportions that vary with dilution (40), are given as moles atomic iodine per liter. The actual concentrations of oxidizing species are lower. Reactions were stopped by the addition of an equal volume of quenching solution consisting of 50 mM Tris-HCl, 20 mM N-ethylmaleimide, 20 mM EDTA, pH 7.4. Samples were mixed with 5× nonreducing SDS loading buffer, heated at 55 °C for 15 min, and subjected to SDS-PAGE using NuPAGE 4–12% Bis-Tris gradient gels (Invitrogen). The separated proteins were transferred to nitrocellulose membrane and immunoblotted with anti-FLAG monoclonal antibody M2 (Sigma). Each cross-linking experiment was carried out independently at least twice. Quantitation of the fraction of ORAI1 cross-linked was performed using the program ImageJ (National Institute of Mental Health). Single-Cell ½Ca2þ i Measurement. Primary CD4þ cells were purified from spleen and lymph nodes of Orai1−∕− mice (13) using Dynal magnetic beads (Invitrogen) following the manufacturer’s instructions. Purified T cells were stimulated and further expanded in an IL-2-containing medium. Retrovirus-transduced Orai1−∕− CD4þ cells were incubated overnight in IL-2-free RPMI medium 1640 and loaded with 2 μM Fura-2 acetoxymethyl ester

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Bioinformatic Analyses. The membrane topology of ORAI1 was predicted using the programs TMpred (http://www.ch.embnet.org/software/TMPRED_ 1. Parekh AB, Putney JW Jr (2005) Store-operated calcium channels. Physiol Rev 85:757–810. 2. Feske S, et al. (2006) A mutation in Orai1causes immune deficiency by abrogating CRAC channel function. Nature 441:179–185. 3. Vig M, et al. (2006) CRACM1 is a plasma membrane protein essential for storeoperated Ca2þ entry. Science 312:1220–1223. 4. Zhang SL, et al. (2006) Genome-wide RNAi screen of Ca2þ influx identifies genes that regulate Ca2þ release-activated Ca2þ channel activity. Proc Natl Acad Sci USA 103:9357–9362. 5. Yeromin AV, et al. (2006) Molecular identification of the CRAC channel by altered ion selectivity in a mutant of Orai. Nature 443:226–229. 6. Prakriya M, et al. (2006) Orai1 is an essential pore subunit of the CRAC channel. Nature 443:230–233. 7. Vig M, et al. (2006) CRACM1 multimers form the ion-selective pore of the CRAC channel. Curr Biol 16:2073–2079. 8. Zhou Y, et al. (2010) STIM1 gates the store-operated calcium channel ORAI1 in vitro. Nat Struct Mol Biol 17:112–116. 9. Hogan PG, Rao A (2007) Dissecting ICRAC , a store-operated calcium current. Trends Biochem Sci 32:235–245. 10. Putney JW (2009) Capacitative calcium entry: From concept to molecules. Immunol Rev 231:10–22. 11. Hogan PG, Lewis RS, Rao A (2010) Molecular basis of calcium signaling in lymphocytes: STIM and ORAI. Annu Rev Immunol 28:491–533. 12. McCarl CA, et al. (2009) ORAI1 deficiency and lack of store-operated Ca2þ entry cause immunodeficiency, myopathy, and ectodermal dysplasia. J Allergy Clin Immunol 124:1311–1318. 13. Gwack Y, et al. (2008) Hair loss and defective T- and B-cell function in mice lacking ORAI1. Mol Cell Biol 28:5209–5222. 14. Vig M, et al. (2008) Defective mast cell effector functions in mice lacking the CRACM1 pore subunit of store-operated calcium release-activated calcium channels. Nat Immunol 9:89–96. 15. Roos J, et al. (2005) STIM1, an essential and conserved component of store-operated Ca2þ channel function. J Cell Biol 169:435–445. 16. Liou J, et al. (2005) STIM is a Ca2þ sensor essential for Ca2þ -store-depletion-triggered Ca2þ influx. Curr Biol 15:1235–1241. 17. Zhang SL, et al. (2005) STIM1 is a Ca2þ sensor that activates CRAC channels and migrates from the Ca2þ store to the plasma membrane. Nature 437:902–905. 18. Wu MM, et al. (2006) Ca2þ store depletion causes STIM1 to accumulate in ER regions closely associated with the plasma membrane. J Cell Biol 174:803–813. 19. Luik RM, et al. (2006) The elementary unit of store-operated Ca2þ entry: Local activation of CRAC channels by STIM1 at ER-plasma membrane junctions. J Cell Biol 174:815–825. 20. Stathopulos PB, et al. (2008) Structural and mechanistic insights into STIM1-mediated initiation of store-operated calcium entry. Cell 135:110–122. 21. Falke JJ, Koshland DE Jr (1987) Global flexibility in a sensory receptor: A site-directed cross-linking approach. Science 237:1596–1600. 22. Pakula AA, Simon MI (1992) Determination of transmembrane protein structure by disulfide cross-linking: The Escherichia coli Tar receptor. Proc Natl Acad Sci USA 89:4144–4148.

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form.html) (41), HMMTOP2 (http://www.enzim.hu/hmmtop/index.html) (42), and TMHMM (http://www.cbs.dtu.dk/services/TMHMM/) (43) and has been experimentally confirmed (6). Multiple sequence alignment of ORAI proteins was carried out using ClustalW2 (http://www.ebi.ac.uk/clustalw/). ACKNOWLEDGMENTS. This work was funded by NIH grants AI84167 and AI40127 (to A.R. and P.G.H.). Y.Z. was supported by an Irvington Fellowship from the Cancer Research Institute and a postdoctoral fellowship from the Leukemia and Lymphoma Society.

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(Invitrogen) in the dark for 45 min at ambient temperature. T cells were further attached to coverslips precoated with 0.01% poly-L-lysine, mounted in an RC-20 closed-bath flow chamber (Warner Instrument), and bathed in Ringer’s solution (155 mM NaCl, 4.5 mM KCl, 2 mM CaCl2 , 1 mM MgCl2 , 10 mM D-glucose, 5 mM Hepes pH 7.4). ½Ca2þ i measurements were performed and analyzed as previously described (2). In each experiment, for each experimental condition, 50–100 GFP-positive CD4þ T cells were analyzed.