Drosophila Orai Research Articles

The Impact of Mutation L138F/L210F on the Orai Channel: A Molecular Dynamics Simulation Study.

The calcium release-activated calcium channel, composed of the Orai channel and the STIM protein, plays a crucial role in maintaining the Ca2+ concentration in cells. Previous studies showed that the L138F mutation in the human Orai1 creates a constitutively open channel independent of STIM, causing severe myopathy, but how the L138F mutation activates Orai1 is still unclear. Here, based on the crystal structure of Drosophila melanogaster Orai (dOrai), molecular dynamics simulations for the wild-type (WT) and the L210F (corresponding to L138F in the human Orai1) mutant were conducted to investigate their structural and dynamical properties. The results showed that the L210F dOrai mutant tends to have a more hydrated hydrophobic region (V174 to F171), as well as more dilated basic region (K163 to R155) and selectivity filter (E178). Sodium ions were located deeper in the mutant than in the wild-type. Further analysis revealed two local but essential conformational changes that may be the key to the activation. A rotation of F210, a previously unobserved feature, was found to result in the opening of the K163 gate through hydrophobic interactions. At the same time, a counter-clockwise rotation of F171 occurred more frequently in the mutant, resulting in a wider hydrophobic gate with more hydration. Ultimately, the opening of the two gates may facilitate the opening of the Orai channel independent of STIM.

Cryo-EM structure of the calcium release-activated calcium channel Orai in an open conformation.

The calcium release-activated calcium channel Orai regulates Ca2+ entry into non-excitable cells and is required for proper immune function. While the channel typically opens following Ca2+ release from the endoplasmic reticulum, certain pathologic mutations render the channel constitutively open. Previously, using one such mutation (H206A), we obtained low (6.7 Å) resolution X-ray structural information on Drosophila melanogaster Orai in an open conformation (Hou et al., 2018). Here we present a structure of this open conformation at 3.3 Å resolution using fiducial-assisted cryo-electron microscopy. The improved structure reveals the conformations of amino acids in the open pore, which dilates by outward movements of subunits. A ring of phenylalanine residues repositions to expose previously shielded glycine residues to the pore without significant rotational movement of the associated helices. Together with other hydrophobic amino acids, the phenylalanines act as the channel's gate. Structured M1-M2 turrets, not evident previously, form the channel's extracellular entrance.

Molecular understanding of calcium permeation through the open Orai channel.

The Orai channel is characterized by voltage independence, low conductance, and high Ca2+ selectivity and plays an important role in Ca2+ influx through the plasma membrane (PM). How the channel is activated and promotes Ca2+ permeation is not well understood. Here, we report the crystal structure and cryo-electron microscopy (cryo-EM) reconstruction of a Drosophila melanogaster Orai (dOrai) mutant (P288L) channel that is constitutively active according to electrophysiology. The open state of the Orai channel showed a hexameric assembly in which 6 transmembrane 1 (TM1) helices in the center form the ion-conducting pore, and 6 TM4 helices in the periphery form extended long helices. Orai channel activation requires conformational transduction from TM4 to TM1 and eventually causes the basic section of TM1 to twist outward. The wider pore on the cytosolic side aggregates anions to increase the potential gradient across the membrane and thus facilitate Ca2+ permeation. The open-state structure of the Orai channel offers insights into channel assembly, channel activation, and Ca2+ permeation.

Structures Reveal Opening of the Store-Operated Calcium Channel ORAI

The store-operated calcium (Ca2+) channel Orai governs Ca2+ influx through the plasma membrane of many non-excitable cells in metazoans. The channel opens in response to the depletion of Ca2+ stored in the endoplasmic reticulum (ER). Loss- and gain-of-function mutants of Orai cause disease. Our previous work revealed the structure of Orai with a closed pore. Here, using a gain-of-function mutation that constitutively activates the channel, we present an X-ray structure of Drosophila melanogaster Orai in an open conformation. Well-defined electron density maps reveal that the pore is dramatically dilated on its cytosolic side in comparison to the slender closed pore. Cations and anions bind in different regions of the open pore, informing mechanisms for ion permeation and Ca2+ selectivity. Opening of the pore requires the release of cytosolic latches. Together with additional X-ray structures of an unlatched-but-closed conformation, we propose a sequence for store-operated activation.

Structures reveal opening of the store-operated calcium channel Orai.

The store-operated calcium (Ca2+) channel Orai governs Ca2+ influx through the plasma membrane of many non-excitable cells in metazoans. The channel opens in response to the depletion of Ca2+ stored in the endoplasmic reticulum (ER). Loss- and gain-of-function mutants of Orai cause disease. Our previous work revealed the structure of Orai with a closed pore. Here, using a gain-of-function mutation that constitutively activates the channel, we present an X-ray structure of Drosophila melanogaster Orai in an open conformation. Well-defined electron density maps reveal that the pore is dramatically dilated on its cytosolic side in comparison to the slender closed pore. Cations and anions bind in different regions of the open pore, informing mechanisms for ion permeation and Ca2+ selectivity. Opening of the pore requires the release of cytosolic latches. Together with additional X-ray structures of an unlatched-but-closed conformation, we propose a sequence for store-operated activation.

Pore properties of Orai1 calcium channel dimers and their activation by the STIM1 ER calcium sensor

Store-operated Ca2+ entry signals are mediated by plasma membrane Orai channels activated through intermembrane coupling with Ca2+-sensing STIM proteins in the endoplasmic reticulum (ER). The nature of this elaborate Orai-gating mechanism has remained enigmatic. Based on the Drosophila Orai structure, mammalian Orai1 channels are hexamers comprising three dimeric subunit pairs. We utilized concatenated Orai1 dimers to probe the function of key domains within the channel pore and gating regions. The Orai1-E106Q selectivity-filter mutant, widely considered a dominant pore blocker, was surprisingly nondominant within concatenated heterodimers with Orai1-WT. The Orai1-E106Q/WT heterodimer formed STIM1-activated nonselective cation channels with significantly enlarged apparent pore diameter. Other Glu-106 substitutions entirely blocked the function of heterodimers with Orai1-WT. The hydrophobic pore-lining mutation V102C, which constitutively opens channels, was suppressed by Orai1-WT in the heterodimer. In contrast, the naturally occurring R91W pore-lining mutation associated with human immunodeficiency was completely dominant-negative over Orai-WT in heterodimers. Heterodimers containing the inhibitory K85E mutation extending outward from the pore helix gave an interesting partial effect on both channel activation and STIM1 binding, indicating an important allosteric link between the cytosolic Orai1 domains. The Orai1 C-terminal STIM1-binding domain mutation L273D powerfully blocked STIM1-induced channel activation. The Orai1-L273D/WT heterodimer had drastically impaired STIM1-induced channel gating but, unexpectedly, retained full STIM1 binding. This reveals the critical role of Leu-273 in transducing the STIM1-binding signal into the allosteric conformational change that initiates channel gating. Overall, our results provide important new insights into the role of key functional domains that mediate STIM1-induced gating of the Orai1 channel.

The STIM-Orai Pathway: STIM-Orai Structures: Isolated and in Complex.

Considerable progress has been made elucidating the molecular mechanisms of calcium (Ca2+) sensing by stromal interaction molecules (STIMs) and the basis for Orai channel activity. This chapter focuses on the available high-resolution structural details of STIM and Orai proteins with respect to the regulation of store-operated Ca2+ entry (SOCE). Solution structures of the Ca2+-sensing domains of STIM1 and STIM2 are reviewed in detail, crystal structures of cytosolic coiled-coil STIM fragments are discussed, and an overview of the closed Drosophila melanogaster Orai hexameric structure is provided. Additionally, we highlight structures of human Orai1 N-terminal and C-terminal domains in complex with calmodulin and human STIM1, respectively. Ultimately, the accessible structural data are discussed in terms of potential mechanisms of action and cohesiveness with functional observations.

Functional Analysis of Orai1 Concatemers Supports a Hexameric Stoichiometry for the CRAC Channel

Store-operated Ca2+ entry occurs through the binding of the endoplasmic reticulum (ER) Ca2+ sensor STIM1 to Orai1, the pore-forming subunit of the Ca2+ release-activated Ca2+ (CRAC) channel. Although the essential steps leading to channel opening have been described, fundamental questions remain, including the functional stoichiometry of the CRAC channel. The crystal structure of Drosophila Orai indicates a hexameric stoichiometry, while studies of linked Orai1 concatemers and single-molecule photobleaching suggest that channels assemble as tetramers. We assessed CRAC channel stoichiometry by expressing hexameric concatemers of human Orai1 and comparing in detail their ionic currents to those of native CRAC channels and channels generated from monomeric Orai1 constructs. Cell surface biotinylation results indicated that Orai1 channels in the plasma membrane were assembled from intact hexameric polypeptides and not from truncated protein products. In addition, the L273D mutation depressed channel activity equally regardless of which Orai1 subunit in the concatemer carried the mutation. Thus, functional channels were generated from intact Orai1 hexamers in which all subunits contributed equally. These hexameric Orai1 channels displayed the biophysical fingerprint of native CRAC channels, including the distinguishing characteristics of gating (store-dependent activation, Ca2+-dependent inactivation, open probability), permeation (ion selectivity, affinity for Ca2+ block, La3+ sensitivity, unitary current magnitude), and pharmacology (enhancement and inhibition by 2-aminoethoxydiphenyl borate). Because permeation characteristics depend strongly on pore geometry, it is unlikely that hexameric and tetrameric pores would display identical Ca2+ affinity, ion selectivity, and unitary current magnitude. Thus, based on the highly similar pore properties of the hexameric Orai1 concatemer and native CRAC channels, we conclude that the CRAC channel functions as a hexamer of Orai1 subunits.

Orai1 pore residues control CRAC channel inactivation independently of calmodulin.

Ca(2+) entry through CRAC channels causes fast Ca(2+)-dependent inactivation (CDI). Previous mutagenesis studies have implicated Orai1 residues W76 and Y80 in CDI through their role in binding calmodulin (CaM), in agreement with the crystal structure of Ca(2+)-CaM bound to an Orai1 N-terminal peptide. However, a subsequent Drosophila melanogaster Orai crystal structure raises concerns about this model, as the side chains of W76 and Y80 are predicted to face the pore lumen and create a steric clash between bound CaM and other Orai1 pore helices. We further tested the functional role of CaM using several dominant-negative CaM mutants, none of which affected CDI. Given this evidence against a role for pretethered CaM, we altered side-chain volume and charge at the Y80 and W76 positions to better understand their roles in CDI. Small side chain volume had different effects at the two positions: it accelerated CDI at position Y80 but reduced the extent of CDI at position W76. Positive charges at Y80 and W76 permitted partial CDI with accelerated kinetics, whereas introducing negative charge at any of five consecutive pore-lining residues (W76, Y80, R83, K87, or R91) completely eliminated CDI. Noise analysis of Orai1 Y80E and Y80K currents indicated that reductions in CDI for these mutations could not be accounted for by changes in unitary current or open probability. The sensitivity of CDI to negative charge introduced into the pore suggested a possible role for anion binding in the pore. However, although Cl(-) modulated the kinetics and extent of CDI, we found no evidence that CDI requires any single diffusible cytosolic anion. Together, our results argue against a CDI mechanism involving CaM binding to W76 and Y80, and instead support a model in which Orai1 residues Y80 and W76 enable conformational changes within the pore, leading to CRAC channel inactivation.

Light generation of intracellular Ca(2+) signals by a genetically encoded protein BACCS.

Ca2+ signals are highly regulated in a spatiotemporal manner in numerous cellular physiological events. Here we report a genetically engineered blue light-activated Ca2+ channel switch (BACCS), as an optogenetic tool for generating Ca2+ signals. BACCS opens Ca2+-selective ORAI ion channels in response to light. A BACCS variant, dmBACCS2, combined with Drosophila Orai, elevates the Ca2+ concentration more rapidly, such that Ca2+ elevation in mammalian cells is observed within 1 s on light exposure. Using BACCSs, we successfully control cellular events including NFAT-mediated gene expression. In the mouse olfactory system, BACCS mediates light-dependent electrophysiological responses. Furthermore, we generate BACCS mutants, which exhibit fast and slow recovery of intracellular Ca2+. Thus, BACCSs are a useful optogenetic tool for generating temporally various intracellular Ca2+ signals with a large dynamic range, and will be applicable to both in vitro and in vivo studies.

Conformational Changes in the Orai1 C-Terminus Evoked by STIM1 Binding.

Store-operated CRAC channels regulate a wide range of cellular functions including gene expression, chemotaxis, and proliferation. CRAC channels consist of two components: the Orai proteins (Orai1-3), which form the ion-selective pore, and STIM proteins (STIM1-2), which form the endoplasmic reticulum (ER) Ca2+ sensors. Activation of CRAC channels is initiated by the migration of STIM1 to the ER-plasma membrane (PM) junctions, where it directly interacts with Orai1 to open the Ca2+-selective pores of the CRAC channels. The recent elucidation of the Drosophila Orai structure revealed a hexameric channel wherein the C-terminal helices of adjacent Orai subunits associate in an anti-parallel orientation. This association is maintained by hydrophobic interactions between the Drosophila equivalents of human Orai1 residues L273 and L276. Here, we used mutagenesis and chemical cross-linking to assess the nature and extent of conformational changes in the self-associated Orai1 C-termini during STIM1 binding. We find that linking the anti-parallel coiled-coils of the adjacent Orai1 C-termini through disulfide cross-links diminishes STIM1-Orai1 interaction, as assessed by FRET. Conversely, prior binding of STIM1 to the Orai1 C-terminus impairs cross-linking of the Orai1 C-termini. Mutational analysis indicated that a bend of the Orai1 helix located upstream of the self-associated coils (formed by the amino acid sequence SHK) establishes an appropriate orientation of the Orai1 C-termini that is required for STIM1 binding. Together, our results support a model wherein the self-associated Orai1 C-termini rearrange modestly to accommodate STIM1 binding.

Functional Reconstitution and Structural Flexibility of the CRAC Channel Orai

Calcium (Ca2+) release-activated Ca2+ (CRAC) channels mediate Ca2+ influx across the plasma membrane in response to Ca2+ store depletion in the endoplasmic reticulum (ER). CRAC channel function requires two components: the plasma membrane Ca2+ channel Orai and its regulator located in the ER membrane, the Ca2+ store sensor STIM. Using liposomes reconstituted with a purified fusion protein of human Orai1 and cytosolic fragments of human STIM1 (hO1-SS), we show that Orai1 and STIM1 are sufficient to form active CRAC channels in vitro. Reconstituted hO1-SS recapitulates CRAC channel properties as shown by detection of sodium (Na+) flux in the absence of Ca2+ and by direct detection of Ca2+ flux. 2-APB, a known CRAC channel inhibitor, blocks both fluxes. Our findings confirm that human STIM1 gates the pore of Orai1 and demonstrates that the two proteins are sufficient to form functional channels in the absence of other cellular factors. Previously, we published the crystal structure of drosophila Orai in a closed state. Here we present low-resolution X-ray diffraction data of human Orai1, which indicate an overall structure that is indistinguishable from drosophila Orai. In addition, a new 4.25 A resolution X-ray structure of drosophila Orai reveals an extended conformation of the fourth transmembrane helix (M4) at the periphery of the channel that extends into the cytosol and is strikingly different from the arrangement of these helices in the previous structure. The comparison of structures reveals conformational flexibility that starts from the M4 helices and continues into the cytosolic M4 extension helices. In all of the structures, the pore adopts the same conformation and remains closed. The conformational flexibility observed for the M4 and M4 extension helices may have a role in the binding of STIM and in signal transduction from the ER.

Stoichiometry of CRAC Channel Assembly and Gating

CRAC channels are opened by binding of the ER calcium sensor STIM1 to the C-terminus of the channel subunit Orai1. Previous functional experiments suggested a tetrameric channel stoichiometry, but the crystal structure of Drosophila Orai is a trimer of dimers, with each C-terminus forming a coiled-coil with its neighbor. This raises two fundamental questions: what is the stoichiometry of the CRAC channel, and does STIM1 bind to individual or pairs of C-termini to open it? To address these questions, we constructed hexameric concatemers of Orai1. Orai1 hexamers produced currents with properties that were indistinguishable from native ICRAC, including Ca2+ selectivity, Ca2+-dependent inactivation, and modulation by 2-APB. The inhibitory effects of single L273D mutations confirmed that all 6 subunits participated equally in forming the functional channel. STIM1-Orai1 binding was studied using E-FRET between STIM1-YFP and CFP-Orai1. While the Orai1(L273D) C-terminus alone did not bind STIM1, it enhanced binding when paired with a neighboring WT C-terminus. To compare how monomer vs dimer binding are coupled to channel opening, we constructed hexamers with a single truncated or L273D C-terminus. The truncated hexamer showed significantly less activity than the L273D mutant, arguing against a pure monomeric gating mode. The relationship between STIM1 occupancy and channel activation was examined using Orai1 hexamers containing 1-3 STIM-binding mutations (producing 1-3 Orai1 heterodimers per channel). For both strong (L273D) and weak (L286S) inhibitory mutations, channel activity was well described by a model that assumes independent and equal energetic contributions from each heterodimer. In summary, we present the first functional evidence that hexameric Orai1 channels have the same properties as native CRAC channels. Our data suggest that STIM1 binds pairs of Orai C-termini and opens CRAC channels as a trimer of dimers, with each dimer contributing a constant amount of gating energy.

Atomistic Molecular Dynamics Simulations of Drosophila Orai in a Hydrated Lipid Bilayer

Orai channels are prominent (calcium) Ca2+ signal mediators in many cell types. In T lymphocytes, Orai1 and STIM1 form the Ca2+ release-activated Ca2+ (CRAC) channel. Following endoplasmic reticulum (ER) Ca2+ store depletion, ER membrane-resident Ca2+ sensor STIM1 physically interacts with plasma membrane-bound, pore-forming Orai1 subunits. The STIM1-Orai1 interaction results in Ca2+ influx and ultimately changes in gene expression that underlie the immune response. In 2012, the Drosophila Orai (dOrai) crystal structure was solved to 3.34 A resolution. To develop an atomistic view of Orai channel function, given the high sequence identity (73%) between dOrai and human Orai1, we constructed a complete dOrai structure by building in flexible loops missing in the crystal structure and adjusting the protonation state of several pore residues. The model was placed into a lipid bilayer with excess hydration and simulated for over 200 ns. We find shifted pKa values for several titratable residues in the pore interior, and that changing protonation states of these key residues is essential to maintain pore stability. To explore the mechanism of channel block, we also simulated the model with either Ca2+ or (gadolinium) Gd3+ in the selectivity filter. Taken together, our results help to address the stability of the closed structure in a lipid bilayer while also yielding novel atomistic insights into channel block and pH sensitivity.This work was supported by NIH grants F30CA171717 to M.L.W., P01GM86685 to D.J.T., and NS-14609 to M.D.C.1. Hou, X, et al. Science. (2012); 338; 1308-13.

Structural Modeling of Hexameric and Tetrameric Ion Conduction Pathways of Orai1 Channel

Ca2+ release-activated Ca2+ (CRAC) channels mediate Ca2+ entry in response to store depletion in a variety of cell types. Endogenous CRAC channels in mammals are formed by a homomeric assembly of Orai1 proteins. Earlier functional studies involving chemical cross-linking of different numbers of wild-type Orai1 subunits, electrophysiological recordings and single molecule fluorescence analysis techniques as well as high-resolution electron microscopic examination of purified Orai proteins strongly indicated that functional Orai channels were most likely tetramers. In contrast, study of crystals containing holo-dOrai channel complexes from Drosophila melanogaster, revealed that a single channel complex contains six dOrai subunits. The hexameric CRAC channel stoichiometry was further supported by cross-linking and size-exclusion chromatography studies of the Drosophila Orai. Recent functional study revealed that expression of concatenated hexameric Orai1 channel produces a non-selective cation channel with biophysical properties essentially different from those of endogenous CRAC channels or channels formed by expressed concatenated tetrameric Orai1 proteins. Thus, the studies reported are far from conclusive but they do indicate the need for further investigation of Orai1 channel stoichiometry. Accordingly, we generated structural models of Orai1 ion conduction pathway formed by tetrameric or hexameric assembly of Orai1 pore-lining TM1 segments using Rosetta fold and dock protocol. Based on available experimental data we constrained proximity of several key residues lining Orai1 ion conduction pathway during simulations. Among the lowest energy and most frequently sampled conformations of Orai TM1 hexamers generated by Rosetta, we identified structural models that were in close agreement with Orai structure in the transmembrane region proposed based on crystallographic data analysis. Our preliminary models of Orai TM1 tetramers suggest alternative structural topology forming ion conduction pathway, which may account for the differences in ionic selectivity of hexameric and tetrameric Orai1 channels.

Orai1 Pore Mutations and Calcium-Dependent Inactivation

Ca2+ entry through CRAC channels causes rapid Ca2+-dependent inactivation (CDI) via interaction with a site located nanometers from the pore mouth. We previously found that mutations of W76 and Y80 in human Orai1 greatly alter the extent and kinetics of CDI (Mullins et al. 2009). Based on a recent crystal structure of a Drosophila Orai hexamer (Hou et al. 2012), the side chains of W76, Y80 and a ring of basic residues hypothesized to form a gate (R83, K87, R91) are all predicted to face the human Orai1 pore lumen. We mutated these residues to explore their contributions to CDI. STIM1 and variants of Orai1 were expressed in HEK cells, and the kinetics and extent of CDI were measured during 200-ms hyperpolarizations in 20 mM Ca2+. Ca2+-selectivity and magnitude of mutant currents were comparable to those of WT Orai1, indicating that observed changes in CDI were not likely due to changes in the local [Ca2+]i near the pore.At position 80, kinetics of CDI were affected by the size and charge of side chains: F was similar to Y (WT), while A, C, and S were significantly faster. Adding a positive charge (R or K) increased the speed further. These data suggest that Y80 limits the rate of inactivation by steric and/or hydrophobic interactions. The extent of inactivation was also influenced by substitutions: at position 80, Y(WT)≈F≈A≈C≈S > K≈R≈Q, while D, E, and W did not inactivate. Like W76E, Y80D, and Y80E, the R83E, K87E, and R91E mutations eliminated or markedly reduced CDI. Overall, CDI is disrupted by introduction of negatively charged side chains in the pore, but positive side chains permit or facilitate CDI. These effects suggest that CDI involves the interaction of an anionic species with basic pore-lining side chains in Orai1.

Interplay of Orai1-Loop3 with Extracellular Ca2+ Binding Sites in Loop1 Controls Crac Channel Activity

Ca2+ release-activated Ca2+ (CRAC) channels are a major pathway for Ca2+ entry in a vast majority of cell types. The identification of the ER Ca2+ sensor STIM1 and the plasma membrane channel subunit Orai1 lead to a substantial progress in our understanding of molecular structures underlying the unique permeation properties of CRAC channels. Nevertheless, Ca2+ coordination at the pore entrance and the role of the extracellular loop1 and loop3 of hOrai1 proteins still remain rather unclear. Based on a recent crystal structure of the drosophila Orai channel we generated an all-atom model of human Orai1 including the missing extracellular loops. Using a combined approach of patch-clamp, cysteine-crosslinking and molecular dynamics simulations we investigated the pore entrance of hOrai1 channels. Our experiments showed that the extracellular loop3 of hOrai1 exhibited a high degree of crosslinking along the whole loop compatible with a remarkable flexibility. Crosslinking studies between loop1 and loop3 demonstrated close proximity, suggesting a movement of loop3 affecting the pore entrance. Molecular dynamics simulations indicated that Ca2+ ions bind to the extracellular aspartates located in the first loop. Further, an interaction of the third loop with these negatively charged amino acids seems to compete with the Ca2+ ions, thereby reducing CRAC channel activity. Sequence alignment of Orai1 isoforms identified that the essential residues for hOrai1 loop1-loop3 coupling are conserved in higher mammals. With this work we suggest a pivotal/regulatory role of the third loop in modulating hOrai1 function. Via movement and subsequent coupling of loop3 to aspartates in the first loop it may regulate the Ca2+ sink at the pore entrance and thus permeation through CRAC channels. This work was supported by the Austrian Science Foundation (FWF): P22747, P26067 (R.S.), P25172 (C.R.), V286 (I.F.).

Stim1 Binds to Pairs of Orai1 Subunits to Open the Crac Channel

CRAC channels are activated by binding of the ER Ca2+-sensor STIM1 to the cytoplasmic C-terminus of the channel subunit Orai1. The crystal structure of Drosophila Orai describes a trimer of Orai dimers in which each C-terminus forms an antiparallel coiled-coil with its neighbor. This unexpected arrangement raises the question of whether pairs of C-termini cooperate to bind STIM1 and thereby function as a unit, or whether single C-termini act independently to bind STIM1 and activate Orai1.We assayed binding by E-FRET between CFP-labeled Orai1 tandem dimers and YFP-labeled STIM1 CRAC activation domain (aa#342-448) expressed in HEK cells. FRET indicated strong binding to normal (WT-WT) dimers and no binding to L273D-L273D dimers, as expected (the C-terminal L273D mutation prevents STIM1 binding). Dimers with one C-terminus deleted (WT-CT) had weak FRET, indicating that STIM1 binds weakly to the 3 monomeric C-termini in the assembled channel. However, WT-L273D heterodimers produced significantly higher FRET, showing that the “non-binding” L273D C-terminus contributes to STIM1 binding when paired with a WT C-terminus. These results suggest that STIM1 binds to pairs of Orai1 C-termini in the native channel. To quantify the effects of L273D on Orai1 activation, we generated hexameric concatemers with L273D mutations in subunits 1, 1+3, or 1+3+5, producing channels with one, two, or three heterodimers, respectively. When co-expressed with saturating levels of STIM1, WT concatemers generated normal CRAC-like currents. L273D reduced the current magnitude in a highly nonlinear manner; each heterodimer reduced activity by ∼2/3, while the three-heterodimer channel produced no detectable current. Thus, although STIM1 can bind WT-L273D heterodimers, the binding energy provided does not open the channel. Future experiments will test whether L273 acts simply to bind STIM1 or whether it also contributes to coupling binding to channel opening.

Mechanism of Activation of Calcium Channel Orai1 by its Regulatory Partner Stim1

Store-operated calcium entry in T lymphocytes depends on the sensing by STIM1 of cellular calcium store depletion, leading to an interaction of STIM1 with ORAI1 and culminating in calcium flux through the ORAI1 channel. Some important features of the STIM1-ORAI1 interaction have been inferred from studies of mutated channels. A recently published structure of the Drosophila Orai channel, which has high sequence homology with human ORAI1, revealed the resting state pore architecture of the channel and confirmed evidence from biochemical and electrophysiological studies that transmembrane helices 1 line the pore. However, the critical steps of STIM1 binding and the resulting transition of the channel to its active calcium-conducting state are yet to be delineated. Here we demonstrate a STIM1-mediated conformational change in the purified wildtype ORAI1 channel that correlates with calcium flux through the channel reconstituted into liposomes. Introduction of the known disabling mutation R91W blocks the conformational change as well as calcium flux through the reconstituted channel. Mutations in the N-terminal cytoplasmic region of ORAI1 that disrupt STIM1 interaction with this part of ORAI1 are sufficient to abrogate the gating signal, although these mutations do not affect STIM1 interaction with the C-terminal region of ORAI1. Our study offers key insights into the STIM1-dependent gating of the ORAI1 channel.

How Many Orai's Does It Take to Make a CRAC Channel?

CRAC (Calcium Release-Activated Calcium) channels represent the primary pathway for so-called “store-operated calcium entry” – the cellular entry of calcium induced by depletion of intracellular calcium stores. These channels play a key role in diverse cellular activities, most noticeably in the differentiation and activation of Tcells, and in the response of mast cells to inflammatory signals. CRAC channels are formed by members of the recently discovered Orai protein family, with previous studies indicating that the functional channel is formed by a tetramer of Orai subunits. However, a recent report has shown that crystals obtained from the purified Drosophila Orai protein display a hexameric channel structure. Here, by comparing the biophysical properties of concatenated hexameric and tetrameric human Orai1 channels expressed in HEK293 cells, we show that the tetrameric channel displays the highly calcium-selective conductance properties consistent with endogenous CRAC channels, whilst the hexameric construct forms an essentially non-selective cation channel.