Orai1 pore residues control CRAC channel inactivation independently of calmodulin

Ca²⁺ entry through CRAC channels causes fast Ca²⁺-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 Ca²⁺–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.     

Mapping interactions between the CRAC activation domain and CC1 regulating the activity of the ER Ca2+ sensor STIM1

Stromal interaction molecule 1 (STIM1) is a widely expressed protein that functions as the endoplasmic reticulum (ER) Ca²⁺ sensor and activator of Orai1 channels. In resting cells with replete Ca²⁺ stores, an inhibitory clamp formed by the coiled-coil 1 (CC1) domain interacting with the CRAC-activation domain (CAD) of STIM1 helps keep STIM1 in a quiescent state. Following depletion of ER Ca²⁺ stores, the brake is released, allowing CAD to extend away from the ER membrane and enabling it to activate Orai1 channels. However, the molecular determinants of CC1–CAD interactions that enforce the inhibitory clamp are incompletely understood. Here, we performed Ala mutagenesis in conjunction with live-cell FRET analysis to examine residues in CC1 and CAD that regulate the inhibitory clamp. Our results indicate that in addition to previously identified hotspots in CC1⍺1 and CC3, several hydrophobic residues in CC2 and the apex region of CAD are critical for CC1–CAD interactions. Mutations in these residues loosen the CC1-CAD inhibitory clamp to release CAD from CC1 in cells with replete Ca²⁺ stores. By contrast, altering the hydrophobic residues L265 and L273 strengthens the clamp to prevent STIM1 activation. Inclusion of the inactivation domain of STIM1 helps stabilize CC1–CAD interaction in several mutants to prevent spontaneous STIM1 activation. In addition, R426C, a human disease–linked mutation in CC3, affects the clamp but also impairs Orai1 binding to inhibit CRAC channel activation. These results identify the CC2, apex, and inactivation domain regions of STIM1 as important determinants of STIM1 activation.     

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.     

Complex role of STIM1 in the activation of store-independent Orai1/3 channels

Orai proteins contribute to Ca²⁺ entry into cells through both store-dependent, Ca²⁺ release–activated Ca²⁺ (CRAC) channels (Orai1) and store-independent, arachidonic acid (AA)-regulated Ca²⁺ (ARC) and leukotriene C₄ (LTC₄)-regulated Ca²⁺ (LRC) channels (Orai1/3 heteromultimers). Although activated by fundamentally different mechanisms, CRAC channels, like ARC and LRC channels, require stromal interacting molecule 1 (STIM1). The role of endoplasmic reticulum–resident STIM1 (ER-STIM1) in CRAC channel activation is widely accepted. Although ER-STIM1 is necessary and sufficient for LRC channel activation in vascular smooth muscle cells (VSMCs), the minor pool of STIM1 located at the plasma membrane (PM-STIM1) is necessary for ARC channel activation in HEK293 cells. To determine whether ARC and LRC conductances are mediated by the same or different populations of STIM1, Orai1, and Orai3 proteins, we used whole-cell and perforated patch-clamp recording to compare AA- and LTC₄-activated currents in VSMCs and HEK293 cells. We found that both cell types show indistinguishable nonadditive LTC₄- and AA-activated currents that require both Orai1 and Orai3, suggesting that both conductances are mediated by the same channel. Experiments using a nonmetabolizable form of AA or an inhibitor of 5-lipooxygenase suggested that ARC and LRC currents in both cell types could be activated by either LTC₄ or AA, with LTC₄ being more potent. Although PM-STIM1 was required for current activation by LTC₄ and AA under whole-cell patch-clamp recordings in both cell types, ER-STIM1 was sufficient with perforated patch recordings. These results demonstrate that ARC and LRC currents are mediated by the same cellular populations of STIM1, Orai1, and Orai3, and suggest a complex role for both ER-STIM1 and PM-STIM1 in regulating these store-independent Orai1/3 channels.     

The Extended Transmembrane Orai1 N-terminal (ETON) Region Combines Binding Interface and Gate for Orai1 Activation by STIM1

STIM1 and Orai1 represent the two molecular key components of the Ca²⁺ release-activated Ca²⁺ channels. Their activation involves STIM1 C terminus coupling to both the N terminus and the C terminus of Orai. Here we focused on the extended transmembrane Orai1 N-terminal (ETON, aa73–90) region, conserved among the Orai family forming an elongated helix of TM1 as recently shown by x-ray crystallography. To identify “hot spot” residues in the ETON binding interface for STIM1 interaction, numerous Orai1 constructs with N-terminal truncations or point mutations within the ETON region were generated. N-terminal truncations of the first four residues of the ETON region or beyond completely abolished STIM1-dependent Orai1 function. Loss of Orai1 function resulted from neither an impairment of plasma membrane targeting nor pore damage, but from a disruption of STIM1 interaction. In a complementary approach, we monitored STIM1-Orai interaction via Orai1 V102A by determining restored Ca²⁺ selectivity as a consequence of STIM1 coupling. Orai1 N-terminal truncations that led to a loss of function consistently failed to restore Ca²⁺ selectivity of Orai1 V102A in the presence of STIM1, demonstrating impairment of STIM1 binding. Hence, the major portion of the ETON region (aa76–90) is essential for STIM1 binding and Orai1 activation. Mutagenesis within the ETON region revealed several hydrophobic and basic hot spot residues that appear to control STIM1 coupling to Orai1 in a concerted manner. Moreover, we identified two basic residues, which protrude into the elongated pore to redound to Orai1 gating. We suggest that several hot spot residues in the ETON region contribute in aggregate to the binding of STIM1, which in turn is coupled to a conformational reorientation of the gate.     

Graded activation of CRAC channel by binding of different numbers of STIM1 to Orai1 subunits

The Ca²⁺ release-activated Ca²⁺ (CRAC) channel pore is formed by Orai1 and gated by STIM1 after intracellular Ca²⁺ store depletion. To resolve how many STIM1 molecules are required to open a CRAC channel, we fused different numbers of Orai1 subunits with functional two-tandem cytoplasmic domains of STIM1 (residues 336-485, designated as S domain). Whole-cell patch clamp recordings of these chimeric molecules revealed that CRAC current reached maximum at a stoichiometry of four Orai1 and eight S domains. Further experiments indicate that two-tandem S domains specifically interact with the C-terminus of one Orai1 subunit, and CRAC current can be gradually increased as more Orai1 subunits can interact with S domains or STIM1 proteins. Our data suggest that maximal opening of one CRAC channel requires eight STIM1 molecules, and support a model that the CRAC channel activation is not in an “all-or-none” fashion but undergoes a graded process via binding of different numbers of STIM1.     

Structural and stoichiometric determinants of Ca release-activated Ca (CRAC) channel Ca-dependent inactivation

Depletion of intracellular Ca^2 + stores in mammalian cells results in Ca^2 + entry across the plasma membrane mediated primarily by Ca^2 + release-activated Ca^2 + (CRAC) channels. Ca^2 + influx through these channels is required for the maintenance of homeostasis and Ca^2 + signaling in most cell types. One of the main features of native CRAC channels is fast Ca^2 +-dependent inactivation (FCDI), where Ca^2 + entering through the channel binds to a site near its intracellular mouth and causes a conformational change, closing the channel and limiting further Ca^2 + entry. Early studies suggested that FCDI of CRAC channels was mediated by calmodulin. However, since the discovery of STIM1 and Orai1 proteins as the basic molecular components of the CRAC channel, it has become apparent that FCDI is a more complex phenomenon. Data obtained using heterologous overexpression of STIM1 and Orai1 suggest that, in addition to calmodulin, several cytoplasmic domains of STIM1 and Orai1 and the selectivity filter within the channel pore are required for FCDI. The stoichiometry of STIM1 binding to Orai1 also has emerged as an important determinant of FCDI. Consequently, STIM1 protein expression levels have the potential to be an endogenous regulator of CRAC channel Ca^2 + influx. This review discusses the current understanding of the molecular mechanisms governing the FCDI of CRAC channels, including an evaluation of further experiments that may delineate whether STIM1 and/or Orai1 protein expression is endogenously regulated to modulate CRAC channel function, or may be dysregulated in some pathophysiological states.     

State-dependent block of Orai3 TM1 and TM3 cysteine mutants: Insights into 2-APB activation

After endoplasmic reticulum (ER) Ca²⁺ store depletion, Orai channels in the plasma membrane (PM) are activated directly by ER-resident stromal interacting molecule (STIM) proteins to form the Ca²⁺-selective Ca²⁺ release-activated Ca²⁺ (CRAC) channel. Of the three human Orai channel homologues, only Orai3 can be activated by high concentrations (>50 µM) of 2-aminoethyl diphenylborinate (2-APB). 2-APB activation of Orai3 occurs without STIM1–Orai3 interaction or store depletion, and results in a cationic, nonselective current characterized by biphasic inward and outward rectification. Here we use cysteine scanning mutagenesis, thiol-reactive reagents, and patch-clamp analysis to define the residues that assist in formation of the 2-APB–activated Orai3 pore. Mutating transmembrane (TM) 1 residues Q83, V77, and L70 to cysteine results in potentiated block by cadmium ions (Cd²⁺). TM1 mutants E81C, G73A, G73C, and R66C form channels that are not sensitive to 2-APB activation. We also find that Orai3 mutant V77C is sensitive to block by 2-aminoethyl methanethiosulfonate (MTSEA), but not 2-(trimethylammonium)ethyl methanethiosulfonate (MTSET). Block induced by reaction with MTSEA is state dependent, as it occurs only when Orai3-V77C channels are opened by either 2-APB or by cotransfection with STIM1 and concurrent passive store depletion. We also analyzed TM3 residue E165. Mutation E165A in Orai3 results in diminished 2-APB–activated currents. However, it has little effect on store-operated current density. Furthermore, mutation E165C results in Cd²⁺-induced block that is state dependent: Cd²⁺ only blocks 2-APB–activated, not store-operated, mutant channels. Our data suggest that the dilated pore of 2-APB–activated Orai3 is lined by TM1 residues, but also allows for TM3 E165 to approach the central axis of the channel that forms the conducting pathway, or pore.     

The Intracellular Loop of Orai1 Plays a Central Role in Fast Inactivation of Ca2+ Release-activated Ca2+ Channels

Store-operated Ca²⁺ entry (SOCE) due to activation of Ca²⁺ release-activated Ca²⁺ (CRAC) channels leads to sustained elevation of cytoplasmic Ca²⁺ and activation of lymphocytes. CRAC channels consisting of four pore-forming Orai1 subunits are activated by STIM1, an endoplasmic reticulum Ca²⁺ sensor that senses intracellular store depletion and migrates to plasma membrane proximal regions to mediate SOCE. One of the fundamental properties of CRAC channels is their Ca²⁺-dependent fast inactivation. To identify the domains of Orai1 involved in fast inactivation, we have mutated residues in the Orai1 intracellular loop linking transmembrane segment II to III. Mutation of four residues, V¹⁵¹SNV¹⁵⁴, at the center of the loop (MutA) abrogated fast inactivation, leading to increased SOCE as well as higher CRAC currents. Point mutation analysis identified five key amino acids, N¹⁵³VHNL¹⁵⁷, that increased SOCE in Orai1 null murine embryonic fibroblasts. Expression or direct application of a peptide comprising the entire intracellular loop or the sequence N¹⁵³VHNL¹⁵⁷ blocked CRAC currents from both wild type (WT) and MutA Orai1. A peptide incorporating the MutA mutations had no blocking effect. Concatenated Orai1 constructs with four MutA monomers exhibited high CRAC currents lacking fast inactivation. Reintroduction of a single WT monomer (MutA-MutA-MutA-WT) was sufficient to fully restore fast inactivation, suggesting that only a single intracellular loop can block the channel. These data suggest that the intracellular loop of Orai1 acts as an inactivation particle, which is stabilized in the ion permeation pathway by the N¹⁵³VHNL¹⁵⁷ residues. These results along with recent reports support a model in which the N terminus and the selectivity filter of Orai1 as well as STIM1 act in concert to regulate the movement of the intracellular loop and evoke fast inactivation.     

Orai1 mutations alter ion permeation and Ca2+-dependent fast inactivation of CRAC channels: evidence for coupling of permeation and gating

Ca²⁺ entry through store-operated Ca²⁺ release-activated Ca²⁺ (CRAC) channels is an essential trigger for lymphocyte activation and proliferation. The recent identification of Orai1 as a key CRAC channel pore subunit paves the way for understanding the molecular basis of Ca²⁺ selectivity, ion permeation, and regulation of CRAC channels. Previous Orai1 mutagenesis studies have indicated that a set of conserved acidic amino acids in trans membrane domains I and III and in the I–II loop (E106, E190, D110, D112, D114) are essential for the CRAC channel's high Ca²⁺ selectivity. To further dissect the contribution of Orai1 domains important for ion permeation and channel gating, we examined the role of these conserved acidic residues on pore geometry, properties of Ca²⁺ block, and channel regulation by Ca²⁺. We find that alteration of the acidic residues lowers Ca²⁺ selectivity and results in striking increases in Cs⁺ permeation. This is likely the result of enlargement of the unusually narrow pore of the CRAC channel, thus relieving steric hindrance for Cs⁺ permeation. Ca²⁺ binding to the selectivity filter appears to be primarily affected by changes in the apparent on-rate, consistent with a rate-limiting barrier for Ca²⁺ binding. Unexpectedly, the mutations diminish Ca²⁺-mediated fast inactivation, a key mode of CRAC channel regulation. The decrease in fast inactivation in the mutant channels correlates with the decrease in Ca²⁺ selectivity, increase in Cs⁺ permeability, and enlargement of the pore. We propose that the structural elements involved in ion permeation overlap with those involved in the gating of CRAC channels.     

The STIM1 CTID domain determines access of SARAF to SOAR to regulate Orai1 channel function

Ca²⁺ influx by store-operated Ca²⁺ channels (SOCs) mediates all Ca²⁺-dependent cell functions, but excess Ca²⁺ influx is highly toxic. The molecular components of SOC are the pore-forming Orai1 channel and the endoplasmic reticulum Ca²⁺ sensor STIM1. Slow Ca²⁺-dependent inactivation (SCDI) of Orai1 guards against cell damage, but its molecular mechanism is unknown. Here, we used homology modeling to identify a conserved STIM1(448–530) C-terminal inhibitory domain (CTID), whose deletion resulted in spontaneous clustering of STIM1 and full activation of Orai1 in the absence of store depletion. CTID regulated SCDI by determining access to and interaction of the STIM1 inhibitor SARAF with STIM1 Orai1 activation region (SOAR), the STIM1 domain that activates Orai1. CTID had two lobes, STIM1(448–490) and STIM1(490–530), with distinct roles in mediating access of SARAF to SOAR. The STIM1(448–490) lobe restricted, whereas the STIM1(490–530) lobe directed, SARAF to SOAR. The two lobes cooperated to determine the features of SCDI. These findings highlight the central role of STIM1 in SCDI and provide a molecular mechanism for SCDI of Orai1.     

Fine-tuning of store-operated calcium entry by fast and slow Ca2+-dependent inactivation: Involvement of SARAF

Store-operated Ca²⁺ entry (SOCE) is a functionally relevant mechanism for Ca²⁺ influx present in electrically excitable and non-excitable cells. Regulation of Ca²⁺ entry through store-operated channels is essential to maintain an appropriate intracellular Ca²⁺ homeostasis and prevent cell damage. Calcium-release activated channels exhibit Ca²⁺-dependent inactivation mediated by two temporally separated mechanisms: fast Ca²⁺-dependent inactivation takes effect in the order of milliseconds and involves the interaction of Ca²⁺ with residues in the channel pore while slow Ca²⁺-dependent inactivation (SCDI) develops over tens of seconds, requires a global rise in [Ca²⁺]_cyt and is a mechanism regulated by mitochondria. Recent studies have provided evidence that the protein SARAF (SOCE-associated regulatory factor) is involved in the mechanism underlying SCDI of Orai1. SARAF is an endoplasmic reticulum (ER) membrane protein that associates with STIM1 and translocate to plasma membrane-ER junctions in a STIM1-dependent manner upon store depletion to modulate SOCE. SCDI mediated by SARAF depends on the location of the STIM1-Orai1 complex within a PI(4,5)P₂-rich microdomain. SARAF also interacts with Orai1 and TRPC1 in cells endogenously expressing STIM1 and cells with a low STIM1 expression and modulates channel function. This review focuses on the modulation by SARAF of SOCE and other forms of Ca²⁺ influx mediated by Orai1 and TRPC1 in order to provide spatio-temporally regulated Ca²⁺ signals.     

Atomic force microscopy (AFM) imaging suggests that stromal interaction molecule 1 (STIM1) binds to Orai1 with sixfold symmetry

Depletion of Ca²⁺ from the endoplasmic reticulum (ER) lumen triggers the opening of Ca²⁺ release-activated Ca²⁺ (CRAC) channels at the plasma membrane. CRAC channels are activated by stromal interaction molecule 1 (STIM1), an ER resident protein that senses Ca²⁺ store depletion and interacts with Orai1, the pore-forming subunit of the channel. The subunit stoichiometry of the CRAC channel is controversial. Here we provide evidence, using atomic force microscopy (AFM) imaging, that Orai1 assembles as a hexamer, and that STIM1 binds to Orai1 with sixfold symmetry. STIM1 associates with Orai1 in the form of monomers, dimers, and multimeric string-like structures that form links between the Orai1 hexamers. Our results provide new insights into the nature of the interactions between STIM1 and Orai1.     

Protein Kinase C-induced Phosphorylation of Orai1 Regulates the Intracellular Ca2+ Level via the Store-operated Ca2+ Channel

Ca²⁺ signals through store-operated Ca²⁺ (SOC) channels, activated by the depletion of Ca²⁺ from the endoplasmic reticulum, regulate various physiological events. Orai1 is the pore-forming subunit of the Ca²⁺ release-activated Ca²⁺ (CRAC) channel, the best characterized SOC channel. Orai1 is activated by stromal interaction molecule (STIM) 1, a Ca²⁺ sensor located in the endoplasmic reticulum. Orai1 and STIM1 are crucial for SOC channel activation, but the molecular mechanisms regulating Orai1 function are not fully understood. In this study, we demonstrate that protein kinase C (PKC) suppresses store-operated Ca²⁺ entry (SOCE) by phosphorylation of Orai1. PKC inhibitors and knockdown of PKCβ both resulted in increased Ca²⁺ influx. Orai1 is strongly phosphorylated by PKC in vitro and in vivo at N-terminal Ser-27 and Ser-30 residues. Consistent with these results, substitution of endogenous Orai1 with an Orai1 S27A/S30A mutant resulted in increased SOCE and CRAC channel currents. We propose that PKC suppresses SOCE and CRAC channel function by phosphorylation of Orai1 at N-terminal serine residues Ser-27 and Ser-30.     

Regulation of CRAC channels by Ca2+-dependent inactivation

CRAC channels are a major route for Ca²⁺ influx in eukaryotic cells. The channels show prominent Ca²⁺-dependent inactivation through two spatially and temporally distinct mechanisms: fast inactivation, which develops over milliseconds and is triggered by Ca²⁺ near the mouth of the channel and slow inactivation, which arises over tens of seconds and requires a rise in global cytosolic Ca²⁺. Slow inactivation is controlled physiologically by Ca²⁺ uptake into mitochondria through the MCU. Site-directed mutagenesis studies on STIM1 and Orai1 have led to new molecular insight into how fast inactivation occurs. This review describes properties and molecular mechanisms that contribute to these important Ca²⁺-dependent inhibitory pathways.     

Stromal Interaction Molecule 1 (STIM1) and Orai1 Mediate Histamine-evoked Calcium Entry and Nuclear Factor of Activated T-cells (NFAT) Signaling in Human Umbilical Vein Endothelial Cells

Histamine is an important immunomodulator involved in allergic reactions and inflammatory responses. In endothelial cells, histamine induces Ca²⁺ mobilization by releasing Ca²⁺ from the endoplasmic reticulum and eliciting Ca²⁺ entry across the plasma membrane. Herein, we show that histamine-evoked Ca²⁺ entry in human umbilical vein endothelial cells (HUVECs) is sensitive to blockers of Ca²⁺ release-activated Ca²⁺ (CRAC) channels. RNA interference against STIM1 or Orai1, the activating subunit and the pore-forming subunit of CRAC channels, respectively, abolishes this histamine-evoked Ca²⁺ entry. Furthermore, overexpression of dominant-negative CRAC channel subunits inhibits while co-expression of both STIM1 and Orai1 enhances histamine-induced Ca²⁺ influx. Interestingly, gene silencing of STIM1 or Orai1 also interrupts the activation of calcineurin/nuclear factor of activated T-cells (NFAT) pathway and the production of interleukin 8 triggered by histamine in HUVECs. Collectively, these results suggest a central role of STIM1 and Orai1 in mediating Ca²⁺ mobilization linked to inflammatory signaling of endothelial cells upon histamine stimulation.