STIM1–Orai1 interactions and Orai1 conformational changes revealed by live‐cell FRET microscopy

Navarro-Borelly, Laura; Somasundaram, Agila; Yamashita, Megumi; Ren, Dongjun; Miller, Richard J.; Prakriya, Murali

doi:10.1113/jphysiol.2008.162503

Cited by 200 publications

(308 citation statements)

References 40 publications

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“…We also observe no changes of 2-APB on endogenous STIM1 puncta formation. In contrast, 2-APB was reported to prevent STIM1 puncta (22,25), although a recent study revealed this effect occurs only with extended 2-APB exposure (35). Importantly, in the same study, FRET analysis between coexpressed full-length STIM1 and Orai1 revealed that 50 M 2-APB could induce an increased association between the proteins after store depletion, but it did not visibly alter STIM1 or Orai1 in puncta.…”

Section: Resultsmentioning

confidence: 79%

STIM protein coupling in the activation of Orai channels

Wang

Deng

Zhou

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

125

141

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STIM proteins are sensors of endoplasmic reticulum (ER) luminalCa 2؉ changes and rapidly translocate into near plasma membrane (PM) junctions to activate Ca 2؉ entry through the Orai family of highly Ca 2؉ -selective ''store-operated'' channels (SOCs). Dissecting the STIM-Orai coupling process is restricted by the abstruse nature of the ER-PM junctional domain. To overcome this problem, we studied coupling by using STIM chimera and cytoplasmic C-terminal domains of STIM1 and STIM2 (S1ct and S2ct) and identifying a fundamental action of the powerful SOC modifier, 2-aminoethoxydiphenyl borate (2-APB), the mechanism of which has eluded recent scrutiny. We reveal that 2-APB induces profound, rapid, and direct interactions between S1ct or S2ct and Orai1, effecting full Ca 2؉ release-activated Ca 2؉ (CRAC) current activation. The short 235-505 S1ct coiled-coil region was sufficient for functional Orai1 coupling. YFP-tagged S1ct or S2ct fragments cleared from the cytosol seconds after 2-APB addition, binding avidly to Orai1-CFP with a rapid increase in FRET and transiently increasing CRAC current 200-fold above basal levels. Functional S1ct-Orai1 coupling occurred in STIM1/STIM2 ؊/؊ DT40 chicken B cells, indicating ct fragments operate independently of native STIM proteins. The 2-APB-induced S1ct-Orai1 and S2-ct-Orai1 complexes undergo rapid reorganization into discrete colocalized PM clusters, which remain stable for >100 s, well beyond CRAC activation and subsequent deactivation. In addition to defining 2-APB's action, the locked STIMct-Orai complex provides a potentially useful probe to structurally examine coupling.calcium signaling ͉ DT40 cells ͉ CRAC channel A dynamic interplay between 2 membrane proteins, STIM and Orai, underlies an intricate coupling between Ca 2ϩ release from endoplasmic reticulum (ER) stores and Ca 2ϩ entry across the plasma membrane (PM) (1-3). The single spanning transmembrane proteins STIM1 and STIM2 function as sensors of ER luminal Ca 2ϩ changes (4-7). Depletion of luminal Ca 2ϩ within ER stores triggers STIM proteins to aggregate and undergo rapid translocation into closely juxtaposed ER-PM junctions to activate Ca 2ϩ entry through one or more of the 3-member family of PM tetra-spanning channel proteins, Orai1, Orai2, and Orai3 (8-10). The Orai proteins function as highly Ca 2ϩ -selective ''storeoperated'' channels (SOCs) (11,12). The activation of SOCs is crucial in mediating longer-term cytosolic Ca 2ϩ signals, replenishing intracellular stores, and homeostatic control of both cytosolic and ER luminal Ca 2ϩ levels (3,(11)(12)(13)(14). Orai1 coexpressed with STIM1 reconstitutes high levels of the inwardly-rectifying Ca 2ϩ release-activated Ca 2ϩ (CRAC) current (10, 15-17), the hallmark of SOCs (11,12).Despite close interactions (5, 18), the coupling mechanism between STIM and Orai proteins to activate Ca 2ϩ entry remains to be elucidated. Our approach was to examine the STIM-Orai interactions by using the reactive borate 2-aminoethoxydiphenyl borate (2-APB), a powerful modifier of SOC ...

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Section: Resultsmentioning

confidence: 79%

STIM protein coupling in the activation of Orai channels

Wang

Deng

Zhou

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

125

141

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“…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)(36)(37)(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)(36)(37)(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).…”

Section: Discussionmentioning

confidence: 99%

Pore architecture of the ORAI1 store-operated calcium channel

Zhou

Ramachandran

Oh‐hora

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

135

176

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ORAI1 is the pore-forming subunit of the calcium release-activated calcium (CRAC) channel, a store-operated channel that is central to Ca 2þ 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 Ca 2þ . 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 Ca 2þ 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 T he electrophysiologically defined calcium release-activated calcium (CRAC) channel is responsible for physiological Ca 2þ influx in T cells and mast cells and for store-operated Ca 2þ 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 Ca 2þ 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)(13)(14). The hallmarks of native CRAC channels and of recombinant ORAI1 channels are that they open in response to a reduction of Ca 2þ concentration in the endoplasmic reticulum (ER) lumen, that they are highly selective for Ca 2þ 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 Ca 2þ stores has been traced to the ER-resident Ca 2þ sensor protein STIM1 (15)(16)(17)(18)(19)(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 Ca 2þ and Na þ under physiological conditions and reduce the decided preference of the channel for Na þ over Cs þ 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 ...

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“…ORAI1 has been assumed to act in concert with STIM1 (10,19,20), activating inward Ca 2ϩ currents after store depletion. We and others have recently provided evidence that store depletion leads to a dynamic coupling of STIM1 to ORAI1 (21)(22)(23), probably involving the putative coiled-coil domain in the C terminus of ORAI1 (22). Furthermore, the C terminus of STIM1 has been established as the key fragment for CRAC as well as ORAI1 activation because its expression alone, without the necessity to deplete ER store, is sufficient for constitutive current activation (18,22,24).…”

Section: Store-operated Camentioning

confidence: 99%

A Cytosolic Homomerization and a Modulatory Domain within STIM1 C Terminus Determine Coupling to ORAI1 Channels

Muik

Fahrner

Derler

et al. 2009

Journal of Biological Chemistry

299

340

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In immune cells, generation of sustained Ca2؉ levels is mediated by the Ca 2؉ release-activated Ca 2؉ (CRAC) current. Molecular key players in this process comprise the stromal interaction molecule 1 (STIM1) that functions as a Ca 2؉ sensor in the endoplasmic reticulum and ORAI1 located in the plasma membrane. Depletion of endoplasmic reticulum Ca 2؉ stores leads to STIM1 multimerization into discrete puncta, which co-cluster with ORAI1 to couple to and activate ORAI1 channels. The cytosolic C terminus of STIM1 is sufficient to activate ORAI1 currents independent of store depletion. Here we identified an ORAI1-activating small fragment (OASF, amino acids 233-450/474) within STIM1 C terminus comprising the two coiled-coil domains and additional 50 -74 amino acids that exhibited enhanced interaction with ORAI1, resulting in 3-fold increased Ca 2؉ currents. This OASF, similar to the complete STIM1 C terminus, displayed the ability to homomerize by a novel assembly domain that occurred subsequent to the coiled-coil domains. A smaller fragment (amino acids 233-420) generated by a further deletion of 30 amino acids substantially reduced the ability to homomerize concomitant to a loss of coupling to as well as activation of ORAI1. Extending OASF by 35 amino acids (233-485) did not alter homomerization but substantially decreased efficiency in coupling to and activation of ORAI1. Expressing OASF in rat basophilic leukemia (RBL) mast cells demonstrated its enhanced plasma membrane targeting associated with 2.5-fold larger CRAC currents in comparison with the complete STIM1 C terminus. In aggregate, we have identified two cytosolic key regions within STIM1 C terminus that control ORAI1/CRAC activation: a homomerization domain indispensable for coupling to ORAI1 and a modulatory domain that controls the extent of coupling to ORAI1.Store-operated Ca 2ϩ entry is key to cellular regulation of short term responses such as contraction and secretion as well as long term processes like proliferation and cell growth (1). The prototypic and best characterized store-operated channel is the Ca 2ϩ release-activated Ca 2ϩ (CRAC) 5 channel (2-6). However, its molecular components have remained elusive until 3 years ago; the stromal interacting molecule 1 (STIM1) (7, 8) and later on ORAI1 (9 -11) have been identified as the two limiting components for CRAC activation. STIM1 is an ER-located Ca 2ϩ sensor (7,8,12), and store depletion triggers its aggregation into puncta close to the plasma membrane, resulting in stimulation of CRAC currents (13,14). Its N terminus is located in the ER lumen and contains an EF-hand Ca 2ϩ binding motif that senses the ER Ca 2ϩ level and a sterile ␣ motif that is suggested to mediate homomeric STIM1 aggregation (15, 16). In the cytosolic STIM1 C terminus, two coiled-coil regions overlapping with the ezrin-radixin-moesin (ERM)-like domain and a lysine-rich region have been proposed as essential for CRAC activation (15,17,18). ORAI1 has been assumed to act in concert with STIM1 (10,19,20), activating inward Ca...

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STIM1–Orai1 interactions and Orai1 conformational changes revealed by live‐cell FRET microscopy

Cited by 200 publications

References 40 publications

STIM protein coupling in the activation of Orai channels

STIM protein coupling in the activation of Orai channels

Pore architecture of the ORAI1 store-operated calcium channel

A Cytosolic Homomerization and a Modulatory Domain within STIM1 C Terminus Determine Coupling to ORAI1 Channels

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