Molecular determinants of fast Ca2+-dependent inactivation and gating of the Orai channels

Ca²⁺ influx by store-operated Ca²⁺ influx channels (SOCs) mediates many cellular functions regulated by Ca²⁺, and excessive SOC-mediated Ca²⁺ influx is cytotoxic and associated with disease. One form of SOC is the CRAC current that is mediated by Orai channels activated by STIM1. A fundamental property of the native CRAC and of the Orais is fast Ca²⁺-dependent inactivation, which limits Ca²⁺ influx to guard against cellular damage. The molecular mechanism of this essential regulatory mechanism is unknown. We report here the fast Ca²⁺-dependent inactivation is mediated by three conserved glutamates in the C termini (CT) of Orai2 and Orai3, which show prominent fast Ca²⁺-dependent inactivation compared with Orai1. Transfer of the CT between the Orais transfers both the extent of channel opening and the mode of fast Ca²⁺-dependent inactivation. Fast Ca²⁺-dependent inactivation of the Orais also requires a domain of STIM1; fragments of STIM1 that efficiently open Orai channels do not evoke fast inactivation unless they include an anionic sequence that is C-terminal to the STIM1-Orai activating region (SOAR). Our studies suggest that Orai CT are necessary and sufficient to control pore opening and uncover the molecular mechanism of fast Ca²⁺-dependent inactivation that has implications for Ca²⁺ influx by SOC in physiological and pathological states.     

Functional stoichiometry of the unitary calcium-release-activated calcium channel

Two proteins, STIM1 in the endoplasmic reticulum and Orai1 in the plasma membrane, are required for the activation of Ca²⁺ release-activated Ca²⁺ (CRAC) channels at the cell surface. How these proteins interact to assemble functional CRAC channels has remained uncertain. Here, we determine how many Orai1 and STIM1 molecules are required to form a functional CRAC channel. We engineered several genetically expressed fluorescent Orai1 tandem multimers and a fluorescent, constitutively active STIM1 mutant. The tandem multimers assembled into CRAC channels, as seen by rectifying inward currents and by cytoplasmic calcium elevations. CRAC channels were visualized as fluorescent puncta in total internal reflection microscopy. With single-molecule imaging techniques, it was possible to observe photo-bleaching of individual fluorophores and to count the steps of bleaching as a measure of the stoichiometry of each CRAC channel complex. We conclude that the subunit stoichiometry in an active CRAC channel is four Orai1 molecules and two STIM1 molecules. Fluorescence resonance energy transfer experiments also showed that four Orai1 subunits form the assembled channel. From the fluorescence intensity of single fluorophores, we could estimate that our transfected HEK293 cells had almost 400,000 CRAC channels and that, when intracellular Ca²⁺ stores were depleted, the channels clustered in aggregates containing ≈1,300 channels, amplifying the local Ca²⁺ entry.     

STIM1 and calmodulin interact with Orai1 to induce Ca2+-dependent inactivation of CRAC channels

Ca²⁺-dependent inactivation (CDI) is a key regulator and hallmark of the Ca²⁺ release-activated Ca²⁺ (CRAC) channel, a prototypic store-operated Ca²⁺ channel. Although the roles of the endoplasmic reticulum Ca²⁺ sensor STIM1 and the channel subunit Orai1 in CRAC channel activation are becoming well understood, the molecular basis of CDI remains unclear. Recently, we defined a minimal CRAC activation domain (CAD; residues 342–448) that binds directly to Orai1 to activate the channel. Surprisingly, CAD-induced CRAC currents lack fast inactivation, revealing a critical role for STIM1 in this gating process. Through truncations of full-length STIM1, we identified a short domain (residues 470–491) C-terminal to CAD that is required for CDI. This domain contains a cluster of 7 acidic amino acids between residues 475 and 483. Neutralization of aspartate or glutamate pairs in this region either reduced or enhanced CDI, whereas the combined neutralization of six acidic residues eliminated inactivation entirely. Based on bioinformatics predictions of a calmodulin (CaM) binding site on Orai1, we also investigated a role for CaM in CDI. We identified a membrane-proximal N-terminal domain of Orai1 (residues 68–91) that binds CaM in a Ca²⁺-dependent manner and mutations that eliminate CaM binding abrogate CDI. These studies identify novel structural elements of STIM1 and Orai1 that are required for CDI and support a model in which CaM acts in concert with STIM1 and the N terminus of Orai1 to evoke rapid CRAC channel inactivation.     

Orai1 internalization and STIM1 clustering inhibition modulate SOCE inactivation during meiosis

Store-operated Ca²⁺ entry (SOCE) is a ubiquitous Ca²⁺ influx pathway activated in response to depletion of intracellular Ca²⁺ stores. SOCE is a primary modulator of intracellular Ca²⁺ dynamics, which specify cellular responses. Interestingly, SOCE inactivates during M phase but the mechanisms involved remain unclear. SOCE is mediated by clustering of the ER Ca²⁺ sensor STIM1 in response to Ca²⁺ store depletion, leading to gating of the plasma membrane SOCE channel Orai1. Here we show that SOCE inactivation in meiosis is the result of internalization of Orai1 into an intracellular vesicular compartment and to the inability of STIM1 to cluster in response to store depletion. At rest, Orai1 continuously recycles between the cell membrane and an endosomal compartment. We further show that STIM1–STIM1 interactions are inhibited during meiosis, which appears to mediate the inability of STIM1 to form puncta following store depletion. In contrast, STIM1–Orai1 interactions remain functional during meiosis. Combined, the removal of Orai1 from the cell membrane and STIM1 clustering inhibition effectively uncouple store depletion from SOCE activation in meiosis. Although STIM1 is phosphorylated during meiosis, phosphomimetic and alanine substitution mutations do not modulate STIM1 clustering, arguing that phosphorylation does not mediate STIM1 clustering inhibition during meiosis.     

STIM protein coupling in the activation of Orai channels

STIM proteins are sensors of endoplasmic reticulum (ER) luminal Ca²⁺ changes and rapidly translocate into near plasma membrane (PM) junctions to activate Ca²⁺ entry through the Orai family of highly Ca²⁺-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 Ca²⁺ release-activated Ca²⁺ (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.     

Plasticity in Ca2+ selectivity of Orai1/Orai3 heteromeric channel

A general cellular response following depletion of intracellular calcium stores involves activation of store-operated channels (SOCs). While Orai1 forms the native Ca²⁺ release-activated Ca²⁺ (CRAC) channel in mast and T cells, the molecular architecture of less Ca²⁺ selective SOCs is insufficiently defined. Here we present evidence that diminished Ca²⁺ selectivity and robust Cs⁺ permeation together with a reduced fast inactivation are characteristics of heteromeric Orai1 and Orai3 channels in contrast to their homomeric forms. The first extracellular loop of these Orai isoforms differs by two aspartates replacing glutamates that affect the selectivity. Co-expression of an Orai3 mutant that mimicked the first loop of Orai1 with either Orai1 or Orai3 recovered or decreased Ca²⁺ selectivity, respectively. Heteromeric Orai1/3 protein assembly provides a concept for less Ca²⁺-selective SOCs.     

Nanoscale patterning of STIM1 and Orai1 during store-operated Ca2+ entry

Stromal interacting molecule (STIM) and Orai proteins constitute the core machinery of store-operated calcium entry. We used transmission and freeze–fracture electron microscopy to visualize STIM1 and Orai1 at endoplasmic reticulum (ER)–plasma membrane (PM) junctions in HEK 293 cells. Compared with control cells, thin sections of STIM1-transfected cells possessed far more ER elements, which took the form of complex stackable cisternae and labyrinthine structures adjoining the PM at junctional couplings (JCs). JC formation required STIM1 expression but not store depletion, induced here by thapsigargin (TG). Extended molecules, indicative of STIM1, decorated the cytoplasmic surface of ER, bridged a 12-nm ER-PM gap, and showed clear rearrangement into small clusters following TG treatment. Freeze–fracture replicas of the PM of Orai1-transfected cells showed extensive domains packed with characteristic “particles”; TG treatment led to aggregation of these particles into sharply delimited “puncta” positioned upon raised membrane subdomains. The size and spacing of Orai1 channels were consistent with the Orai crystal structure, and stoichiometry was unchanged by store depletion, coexpression with STIM1, or an Orai1 mutation (L273D) affecting STIM1 association. Although the arrangement of Orai1 channels in puncta was substantially unstructured, a portion of channels were spaced at ∼15 nm. Monte Carlo analysis supported a nonrandom distribution for a portion of channels spaced at ∼15 nm. These images offer dramatic, direct views of STIM1 aggregation and Orai1 clustering in store-depleted cells and provide evidence for the interaction of a single Orai1 channel with small clusters of STIM1 molecules.Specialized junctions linking the endoplasmic reticulum (ER) to the plasma membrane (PM) were first described by Porter and Palade (1) in skeletal and cardiac muscle. In skeletal muscle, excitation–contraction coupling is mediated by direct physical contact between voltage-gated Ca²⁺ channels (dihydropyridine receptors) in invaginated transverse tubules of the PM and Ca²⁺-release channels (ryanodine receptors) in ER membrane (2). A second type of ER-PM junction mediates inside-out signaling by linking depletion of Ca²⁺ in the ER lumen to Ca²⁺ influx across the PM in a process termed store-operated Ca²⁺ entry (SOCE). In addition to being a mechanism of ionic homeostasis, SOCE supports long-lasting Ca²⁺ signals in many cell types. ER stromal interacting molecule (STIM) and PM Orai proteins were identified by RNAi screening as required for SOCE (3–7). Overexpression of both proteins is required to amplify Ca²⁺ influx through Orai channels (7–10). In Drosophila, STIM and Orai are the sole members of a gene family, which in mammals, includes two STIM and three Orai proteins. STIM1 and Orai1 are predominant in the immune system; human mutations in either gene can cause lethal severe combined immune deficiencies (SCID) (11). ER STIM proteins trigger SOCE by sensing ER Ca²⁺ store depletion, translocating as oligomers to the PM, and binding to PM Orai proteins to promote clustering and channel opening (3, 12–16). These events have been extensively documented by microscopy of cells expressing fluorescently tagged proteins. Numerous studies have defined domains and amino acid residues of STIM1 and Orai1 that are vital for channel function (17, 18).ER-PM junctions underlying SOCE have been visualized by electron microscopy (EM), using either HRP-tagged STIM1 (13, 19) or immunogold labeling of STIM1 (20). However, little is known about the nanometer-scale subcellular organization of STIM and Orai proteins, although they define a basic unit of Ca²⁺ signaling. Here, through a close examination of transmission and freeze–fracture electron micrographs of transfected cells expressing STIM1 and Orai1, we further define the microanatomy of the ER-PM, as well as of ER-ER junctions in store-depleted and untreated cells. These images provide direct candidate signatures for STIM1 molecules bridging the ER-PM and ER-ER gaps and for individual Orai1 channels in puncta. Taken together, our observations provide visual confirmation of STIM1 and Orai1 function, constrain models of STIM1 and Orai1 assembly and interaction, and suggest new aspects of molecular interactions between STIM1 and Orai1.     

Orai1 and Stim1 regulate normal and hypertrophic growth in cardiomyocytes

Cardiac hypertrophy is an independent risk for heart failure (HF) and sudden death. Deciphering signalling pathways dependent on extracellular calcium (Ca²⁺) influx that control normal and pathological cardiac growth may enable identification of novel therapeutic targets. The objective of the present study is to determine the role of the Ca²⁺ release-activated Ca²⁺ (CRAC) channel Orai1 and stromal interaction molecule 1 (Stim1) in postnatal cardiomyocyte store operated Ca²⁺ entry (SOCE) and impact on normal and hypertrophic postnatal cardiomyocyte growth. Employing a combination of siRNA-mediated gene silencing, cultured neonatal rat ventricular cardiomyocytes together with indirect immunofluorescence, epifluorescent Ca²⁺ imaging and site-specific protein phosphorylation and real-time mRNA expression analysis, we show for the first time that both Orai1 and Stim1 are present in cardiomyocytes and required for SOCE due to intracellular Ca²⁺ store depletion by thapsigargin. Stim1-KD but not Orai1-KD significantly decreased diastolic Ca²⁺ levels and caffeine-releasable Ca²⁺ from the sarcoplasmic reticulum (SR). Conversely, Orai1-KD but not Stim1-KD significantly diminished basal NRCM cell size, anp and bnp mRNA levels and activity of the calcineurin (CnA) signalling pathway although diminishing both Orai1 and Stim1 proteins similarly attenuated calmodulin kinase II (CamKII) and ERK1/2 activity under basal conditions. Both Orai1- and Stim1-KD completely abrogated phenylephrine (PE) mediated hypertrophic NRCM growth and enhanced natriuretic factor expression by inhibiting G_q-protein conveyed activation of the CamKII and ERK1/2 signalling pathway. Interestingly, only Orai1-KD but not Stim1-KD prevented Gq-mediated CaN-dependent prohypertrophic signalling. This study shows for the first time that both Orai1 and Stim1 have a key role in cardiomyocyte SOCE regulating both normal and hypertrophic postnatal cardiac growth in vitro.     

Structural and mechanistic insights into the activation of Stromal interaction molecule 1 (STIM1)

Calcium influx through the Ca(2+) release-activated Ca(2+) (CRAC) channel is an essential process in many types of cells. Upon store depletion, the calcium sensor in the endoplasmic reticulum, STIM1, activates Orai1, a CRAC channel in the plasma membrane. We have determined the structures of SOAR from Homo sapiens (hSOAR), which is part of STIM1 and is capable of constitutively activating Orai1, and the entire coiled coil region of STIM1 from Caenorhabditis elegans (ceSTIM1-CCR) in an inactive state. Our studies reveal that the formation of a SOAR dimer is necessary to activate the Orai1 channel. Mutations that disrupt SOAR dimerization or remove the cluster of positive residues abolish STIM1 activation of Orai1. We identified a possible inhibitory helix within the structure of ceSTIM1-CCR that tightly interacts with SOAR. Functional studies suggest that the inhibitory helix may keep the C-terminus of STIM1 in an inactive state. Our data allowed us to propose a model for STIM1 activation.     

Protein histidine phosphatase 1 negatively regulates CD4 T cells by inhibiting the K+ channel KCa3.1

The calcium activated K⁺ channel KCa3.1 plays an important role in T lymphocyte Ca²⁺ signaling by helping to maintain a negative membrane potential, which provides an electrochemical gradient to drive Ca²⁺ influx. We previously showed that nucleoside diphosphate kinase beta (NDPK-B), a mammalian histidine kinase, is required for KCa3.1 channel activation in human CD4 T lymphocytes. We now show that the mammalian protein histidine phosphatase (PHPT-1) directly binds and inhibits KCa3.1 by dephosphorylating histidine 358 on KCa3.1. Overexpression of wild-type, but not a phosphatase dead, PHPT-1 inhibited KCa3.1 channel activity. Decreased expression of PHPT-1 by siRNA in human CD4 T cells resulted in an increase in KCa3.1 channel activity and increased Ca²⁺ influx and proliferation after T cell receptor (TCR) activation, indicating that endogenous PHPT-1 functions to negatively regulate CD4 T cells. Our findings provide a previously unrecognized example of a mammalian histidine phosphatase negatively regulating TCR signaling and are one of the few examples of histidine phosphorylation/dephosphorylation influencing a biological process in mammals.     

The store-operated Ca2+ entry complex comprises a small cluster of STIM1 associated with one Orai1 channel

Increases in cytosolic Ca²⁺ concentration regulate diverse cellular activities and are usually evoked by opening of Ca²⁺ channels in intracellular Ca²⁺ stores and the plasma membrane (PM). For the many signals that evoke formation of inositol 1,4,5-trisphosphate (IP₃), IP₃ receptors coordinate the contributions of these two Ca²⁺ sources by mediating Ca²⁺ release from the endoplasmic reticulum (ER). Loss of Ca²⁺ from the ER then activates store-operated Ca²⁺ entry (SOCE) by causing dimers of STIM1 to cluster and unfurl cytosolic domains that interact with the PM Ca²⁺ channel, Orai1, causing its pore to open. The relative concentrations of STIM1 and Orai1 are important, but most analyses of their interactions use overexpressed proteins that perturb the stoichiometry. We tagged endogenous STIM1 with EGFP using CRISPR/Cas9. SOCE evoked by loss of ER Ca²⁺ was unaffected by the tag. Step-photobleaching analysis of cells with empty Ca²⁺ stores revealed an average of 14.5 STIM1 molecules within each sub-PM punctum. The fluorescence intensity distributions of immunostained Orai1 puncta were minimally affected by store depletion, and similar for Orai1 colocalized with STIM1 puncta or remote from them. We conclude that each native SOCE complex is likely to include only a few STIM1 dimers associated with a single Orai1 channel. Our results, demonstrating that STIM1 does not assemble clusters of interacting Orai channels, suggest mechanisms for digital regulation of SOCE by local depletion of the ER.

In generating the cytosolic Ca²⁺ signals that regulate cellular activities, cells call upon two sources of Ca²⁺: the extracellular space, accessed through Ca²⁺ channels in the plasma membrane (PM), and Ca²⁺ sequestered within intracellular stores, primarily within the endoplasmic reticulum (ER). In animal cells, the many receptors that stimulate formation of inositol 1,4,5-trisphosphate (IP₃) provide coordinated access to both Ca²⁺ sources (1). IP₃ stimulates the opening of IP₃ receptors (IP₃R), which are large Ca²⁺-permeable channels expressed mostly within ER membranes. IP₃ thereby triggers Ca²⁺ release from the ER (2, 3). The link to extracellular Ca²⁺ is provided by store-operated Ca²⁺ entry (SOCE), which is activated by loss of Ca²⁺ from the ER. The reduction in ER free-Ca²⁺ concentration causes Ca²⁺ to dissociate from the luminal Ca²⁺-binding sites of stromal interaction molecule 1 (STIM1), a dimeric protein embedded in ER membranes. This loss of Ca²⁺ causes STIM1 to unfurl cytosolic domains that interact with the PM Ca²⁺ channel, Orai1, causing its pore to open and Ca²⁺ to flow into the cell through the SOCE pathway (Fig. 1A) (4, 5). Available evidence suggests that STIM1 must bind to the C-terminal tail of each of the six subunits of an Orai1 channel for optimal activity, with lesser occupancies reducing activity and modifying channel properties (6–10). The interactions between STIM1 and Orai1 occur at membrane contact sites (MCS), where the two membranes are organized to provide a gap of about 10–30 nm, across which the two proteins directly interact (11–13). Orai channels are unusual in having no structural semblance to other ion channels and in having their opening controlled by direct interactions between proteins in different membranes (Fig. 1A). Competing models suggest that dimeric STIM1 binds either to a pair of C-terminal tails within a single channel (6 STIM1 molecules per hexameric Orai1 channel) (Fig. 1 B, a), or that each dimer interacts with only a single C-terminal tail leaving the remaining STIM1 subunit free to cross-link with a different Orai1 channel (12 STIM1 molecules around a single Orai1 channel) (Fig. 1 B, b) (see references in ref. 14). The latter arrangement has been proposed to allow assembly of close-packed Orai1 clusters (Fig. 1 B, c) and to explain the variable stoichiometry of Orai1 to STIM1 at MCS (14).Open in a separate windowFig. 1.SOCE is unaffected by tagging of endogenous STIM1. (A) SOCE is activated when loss of Ca²⁺ from the ER, usually mediated by IP₃Rs, causes Ca²⁺ to dissociate from the EF hands of dimeric STIM1. This causes STIM1 to unfurl its cytosolic domain, unmasking the C-terminal polybasic tail (PBT) and CRAC (Ca²⁺-release-activated channel)-activation domain (CAD) Association of the PBT with PM phosphoinositides causes STIM1 to accumulate at MCS, where the CAD captures the C-terminal tail of Orai1. Binding of STIM1 to each of the six subunits of Orai1 opens the Ca²⁺ channel, allowing SOCE to occur (9). (B) Orai1 is a hexamer, comprising three pairs of dimers (33). Dimeric STIM1 may activate Orai1 by binding as three dimers (B, a), or as six dimers (B, b) with the residual STIM1 subunit free to interact with another Orai1 channel (B, c) (14). (C) Structure of the edited STIM1-EGFP. (D) TIRF images of STIM1-EGFP HeLa cells treated with STIM1 or nonsilencing (NS) shRNA before emptying of Ca²⁺ stores. (Scale bar, 10 µm.) (E) Summary results (individual values, mean ± SD, n = 3 independent experiments, each with ∼30 cells analyzed) show whole-cell fluorescence intensities from TIRF images of STIM1-EGFP HeLa cells treated with the indicated shRNA. Results from WT cells are also shown (n = 4). ****P < 0.0001, ANOVA with Bonferroni test, relative to WT cells. (F) In-gel fluorescence of lysates from WT or STIM1-EGFP HeLa cells (protein loadings in μg). The STIM1-EGFP band (arrow) and molecular mass markers (kDa) are shown. Similar results were obtained in four independent analyses. (G) WB for STIM1 and β-actin for WT and STIM1-EGFP HeLa cells. Protein loadings (μg) and molecular mass markers (kDa) are shown. Arrows show positions of native and EGFP-tagged STIM1. (H) Summary results (individual values, mean ± SD, n = 9) show expression of STIM1-EGFP relative to all STIM1 in STIM1-EGFP HeLa cells (red), and total STIM1 expression in WT and edited cells (black). (I) Effects of histamine in Ca²⁺-free HBS on the peak increase in [Ca²⁺]_c (Δ[Ca²⁺]_c) in populations of WT and STIM1-EGFP HeLa cells. Mean ± SEM from four experiments, each with six determinations. (J) Effects of CPA in Ca²⁺-free HBS on the peak increase in [Ca²⁺]_c (Δ[Ca²⁺]_c) in populations of WT and STIM1-EGFP HeLa cells. Mean ± SEM from four experiments, each with six determinations. (K) Populations of cells were treated (5 min) with CPA in Ca²⁺-free HBS to evoke graded depletion of ER Ca²⁺ stores before addition of extracellular Ca²⁺ (final free [Ca²⁺] ∼10 mM). Results (mean ± SEM, n = 6, each with six determinations) show the amplitude of the SOCE in WT and STIM1-EGFP HeLa cells. See also SI Appendix, Figs. S1 and S2.Opening of most ion channels is regulated by changes in membrane potential or by binding of soluble stimuli, where the relationship between stimulus intensity and response is readily amenable to experimental analysis. The unusual behavior of SOCE, where direct interactions between proteins embedded in different membranes control channel opening (Fig. 1A), makes it more difficult to define stimulus–response relationships and highlights the need to understand the amounts of STIM1 and Orai1 within the MCS where the interactions occur. When STIM1 or Orai1 are overexpressed their behaviors are perturbed, yet most analyses of their interactions have involved overexpression of the proteins. These difficulties motivated the present study, which was designed to determine the number of native STIM1 molecules associated with each SOCE signaling complex.     

STIM1 enhances SR Ca2+ content through binding phospholamban in rat ventricular myocytes

In ventricular myocytes, the physiological function of stromal interaction molecule 1 (STIM1), an endo/sarcoplasmic reticulum (ER/SR) Ca²⁺ sensor, is unclear with respect to its cellular localization, its Ca²⁺-dependent mobilization, and its action on Ca²⁺ signaling. Confocal microscopy was used to measure Ca²⁺ signaling and to track the cellular movement of STIM1 with mCherry and immunofluorescence in freshly isolated adult rat ventricular myocytes and those in short-term primary culture. We found that endogenous STIM1 was expressed at low but measureable levels along the Z-disk, in a pattern of puncta and linear segments consistent with the STIM1 localizing to the junctional SR (jSR). Depleting SR Ca²⁺ using thapsigargin (2–10 µM) changed neither the STIM1 distribution pattern nor its mobilization rate, evaluated by diffusion coefficient measurements using fluorescence recovery after photobleaching. Two-dimensional blue native polyacrylamide gel electrophoresis and coimmunoprecipitation showed that STIM1 in the heart exists mainly as a large protein complex, possibly a multimer, which is not altered by SR Ca²⁺ depletion. Additionally, we found no store-operated Ca²⁺ entry in control or STIM1 overexpressing ventricular myocytes. Nevertheless, STIM1 overexpressing cells show increased SR Ca²⁺ content and increased SR Ca²⁺ leak. These changes in Ca²⁺ signaling in the SR appear to be due to STIM1 binding to phospholamban and thereby indirectly activating SERCA2a (Sarco/endoplasmic reticulum Ca²⁺ ATPase). We conclude that STIM1 binding to phospholamban contributes to the regulation of SERCA2a activity in the steady state and rate of SR Ca²⁺ leak and that these actions are independent of store-operated Ca²⁺ entry, a process that is absent in normal heart cells.Store-operated Ca²⁺ entry (SOCE) is a cellular mechanism to ensure that sufficient levels of Ca²⁺ are present in the intracellular Ca²⁺ stores to enable robust signaling (1). SOCE depends on the presence and interaction of two proteins, STIM1 (stromal interaction molecule 1) and Orai1 (a low conductance plasma/sarcolemmal Ca²⁺ channel), or their equivalents (2–5). STIM1 is an endo/sarcoplasmic reticulum (ER/SR) Ca²⁺-sensitive protein that interacts with Orai1 to activate the channel function of Orai1, a Ca²⁺ selective channel, and thus permit Ca²⁺ entry. SOCE is clearly present in nonexcitable cells such as T lymphocytes and some excitable cells including skeletal muscle cells (4, 6–13). STIM1 is a membrane-spanning ER/SR protein with a single transmembrane domain and a luminal Ca²⁺ ([Ca²⁺]_ER/SR)-sensing domain. When luminal Ca²⁺ is low (i.e., [Ca²⁺]_ER/SR drops to less than 300 µM), then STIM1 self-aggregates and associates with Orai1 to activate it, producing a SOCE current (I_SOCE) (2, 14–16) and Ca²⁺ entry (with a reversal potential E_SOCE ∼ +50 mV or more) (17, 18). Then, as [Ca²⁺]_ER/SR increases in response to the Ca²⁺ influx, the process reverses.In adult skeletal muscle cells, Ca²⁺ influx is normally low, and it has been suggested that SOCE is needed for maintaining an appropriate level of [Ca²⁺]_ER/SR and correct Ca²⁺ signaling (6, 7, 9, 19). In skeletal muscle, it has been hypothesized that STIM1 is prelocalized in the SR terminal cisternae (6, 20) and hence can more rapidly respond to changes in [Ca²⁺]_ER/SR. The putative importance of SOCE in skeletal muscle was further supported by the observation that the skeletal muscle dysfunction is significant in STIM1-null mice where 91% (30/33) of the animals died in the perinatal period from a skeletal myopathy (6). Furthermore, in humans, STIM1 mutations were identified as a genetic cause of tubular aggregate myopathy (21).Despite the clarity of the SOCE paradigm, the canonical SOCE activation process described above does not apply to all conditions in which STIM1 and Orai1 interact. For example, in T lymphocytes, STIM1 clustering is necessary and sufficient to activate SOCE, regardless of whether [Ca²⁺]_ER/SR is low (4). When present, the STIM1 EF hand mutation causes STIM1 oligomerization and constitutive Ca²⁺ influx across the plasma membrane into cells with full Ca²⁺ stores (4). Although this is consistent with the use of STIM1 clusters and puncta to measure the activation of Orai1 (15, 16, 22, 23), it does not necessarily reflect the state of [Ca²⁺]_ER/SR. Furthermore, several small-molecule bioactive reagents, such as 2-APB and FCCP, neither of which causes [Ca²⁺]_ER/SR depletion, induce STIM1 clustering (24). Thus, STIM1 may have actions that are more complicated than simple [Ca²⁺]_ER/SR sensing and Orai1 signaling.Cardiomyocytes have been reported to have SOCE (8, 13, 25, 26) but are very different from many of the cells noted above that exhibit significant [Ca²⁺]_ER/SR depletion-sensitive Ca²⁺ entry through the Ca²⁺-selective Orai1. Cardiac ventricular myocytes are different from the other cells in that they have large, regular, and dynamic changes in [Ca²⁺]_i and robust influx and extrusion pathways across the sarcolemmal membrane. For example, it is not unusual for investigators to measure a 10–20 nA calcium current (I_Ca,L) in single cardiac ventricular myocytes that is readily extruded by the sarcolemmal Na⁺/Ca²⁺ exchanger. Because of these large fluxes, adult ventricular myocytes have no “need” for SOCE and the same logic applies to neonatal cardiomyocytes. Nevertheless, reports of SOCE in neonatal cardiac myocytes are clear (10, 12, 13). Against this background, we have attempted to determine if STIM1 is present in adult cardiomyocytes and, if so, where the protein is located, how it is mobilized, and how it may interact with other Ca²⁺ signal proteins. In the work presented here, we show that STIM1 is present but that its function in heart is distinct from the canonical SOCE behavior and does not contribute to Ca²⁺ influx through I_SOCE. Instead we show that STIM1 binds phospholamban (PLN), an endogenous SERCA2a inhibitor in the heart (27), and by doing so reduces the PLN-dependent inhibition of SERCA2a and thereby indirectly activates SERCA2a.     

Functional communication between IP3R and STIM2 at subthreshold stimuli is a critical checkpoint for initiation of SOCE

Stromal interaction molecules, STIM1 and STIM2, sense decreases in the endoplasmic reticulum (ER) [Ca²⁺] ([Ca²⁺]_ER) and cluster in ER–plasma membrane (ER–PM) junctions where they recruit and activate Orai1. While STIM1 responds when [Ca²⁺]_ER is relatively low, STIM2 displays constitutive clustering in the junctions and is suggested to regulate basal Ca²⁺ entry. The cellular cues that determine STIM2 clustering under basal conditions is not known. By using gene editing to fluorescently tag endogenous STIM2, we report that endogenous STIM2 is constitutively localized in mobile and immobile clusters. The latter associate with ER–PM junctions and recruit Orai1 under basal conditions. Agonist stimulation increases immobile STIM2 clusters, which coordinate recruitment of Orai1 and STIM1 to the junctions. Extended synaptotagmin (E-Syt)2/3 are required for forming the ER–PM junctions, but are not sufficient for STIM2 clustering. Importantly, inositol 1,4,5-triphosphate receptor (IP₃R) function and local [Ca²⁺]_ER are the main drivers of immobile STIM2 clusters. Enhancing, or decreasing, IP₃R function at ambient [IP₃] causes corresponding increase, or attenuation, of immobile STIM2 clusters. We show that immobile STIM2 clusters denote decreases in local [Ca²⁺]_ER mediated by IP₃R that is sensed by the STIM2 N terminus. Finally, under basal conditions, ambient PIP₂-PLC activity of the cell determines IP₃R function, immobilization of STIM2, and basal Ca²⁺ entry while agonist stimulation augments these processes. Together, our findings reveal that immobilization of STIM2 clusters within ER–PM junctions, a first response to ER-Ca²⁺ store depletion, is facilitated by the juxtaposition of IP₃R and marks a checkpoint for initiation of Ca²⁺ entry.

Store-operated calcium entry (SOCE), which provides critical cytosolic Ca²⁺ signals for regulation of cell functions, is activated in response to depletion of Ca²⁺ stores within the endoplasmic reticulum (ER) (1, 2). Decreases in [Ca²⁺]_ER are sensed by resident ER proteins Stromal Interaction Molecules 1 and 2 (STIM1 and STIM2) via their luminal N-terminal Ca²⁺-binding domains. This triggers their clustering in ER–plasma membrane (PM) junctions (3–5) where they recruit and activate the PM channel Orai1 (6–10). STIM1, the primary regulator of Orai1, has a relatively high Ca²⁺ affinity and responds to substantial decreases in [Ca²⁺]_ER. In contrast, STIM2, a relatively weak activator of Orai1, has a lower Ca²⁺ affinity and can thus respond to minimal decreases in [Ca²⁺]_ER (4, 9–12). When overexpressed, STIM2 displays constitutive clustering within ER–PM junctions where it recruits and activates Orai1 channels to cause Ca²⁺ entry in unstimulated cells (4, 12, 13). We previously demonstrated that preclustering of STIM2 promotes recruitment of Orai1/STIM1 and facilitates STIM1 activation under conditions when [Ca²⁺]_ER is not sufficiently depleted to activate STIM1 (9, 10). These data suggest that preclustered STIM2 is in an activated state in unstimulated cells. There is, however, little information regarding the clustering of endogenous STIM2 and the molecular mechanisms, or cellular cues, that regulate its preclustering at ER–PM junctions in the cell. A particular concern is that exogenous overexpression of STIM2 alters the stoichiometry of endogenous STIM/Orai complexes, which might artificially force them into the junctions to cause preclustering.We have used CRISPR/Cas9 to knockin mVenus into the N terminus of the native Stim2 gene and generated HEK293 cell lines expressing fluorescently tagged endogenous STIM2 (mV-STIM2). Herein we report that endogenous STIM2 is preclustered in the ER–PM junctional region of cells under basal conditions. While the majority of STIM2 clusters are mobile, there is a small population of relatively immobile STIM2 clusters. Importantly, immobilization of native STIM2 clusters is triggered by decreases in local [Ca²⁺]_ER that are mediated by functional IP₃ receptors (IP₃R) and sensed by STIM2 N terminus. In the absence of added agonist, constitutive PIP₂-PLC activity, together with cAMP/protein kinase A (PKA) signaling, determines IP₃R function. Consistent with this response of STIM2 at ambient stimuli, there is an increase in immobile STIM2 clusters following simulation of cells with a Ca²⁺-mobilizing agonist. Further, the immobile STIM2 clusters demarcate sites where Orai1 clusters in basal conditions and both Orai1 and STIM1 cluster following agonist stimulation. Together, our findings suggest that a critical functional link between IP₃R and STIM2 underlies preclustering of STIM2 and is a checkpoint for initiation of SOCE in response to decreases in [Ca²⁺]_ER.     

Activating mutations in STIM1 and ORAI1 cause overlapping syndromes of tubular myopathy and congenital miosis

Signaling through the store-operated Ca²⁺ release-activated Ca²⁺ (CRAC) channel regulates critical cellular functions, including gene expression, cell growth and differentiation, and Ca²⁺ homeostasis. Loss-of-function mutations in the CRAC channel pore-forming protein ORAI1 or the Ca²⁺ sensing protein stromal interaction molecule 1 (STIM1) result in severe immune dysfunction and nonprogressive myopathy. Here, we identify gain-of-function mutations in the cytoplasmic domain of STIM1 (p.R304W) associated with thrombocytopenia, bleeding diathesis, miosis, and tubular myopathy in patients with Stormorken syndrome, and in ORAI1 (p.P245L), associated with a Stormorken-like syndrome of congenital miosis and tubular aggregate myopathy but without hematological abnormalities. Heterologous expression of STIM1 p.R304W results in constitutive activation of the CRAC channel in vitro, and spontaneous bleeding accompanied by reduced numbers of thrombocytes in zebrafish embryos, recapitulating key aspects of Stormorken syndrome. p.P245L in ORAI1 does not make a constitutively active CRAC channel, but suppresses the slow Ca²⁺-dependent inactivation of the CRAC channel, thus also functioning as a gain-of-function mutation. These data expand our understanding of the phenotypic spectrum of dysregulated CRAC channel signaling, advance our knowledge of the molecular function of the CRAC channel, and suggest new therapies aiming at attenuating store-operated Ca²⁺ entry in the treatment of patients with Stormorken syndrome and related pathologic conditions.Ca²⁺ influx in response to the depletion of intracellular Ca²⁺ stores, or store-operated Ca²⁺ entry, constitutes one of the major routes of Ca²⁺ entry in all animal cells (1). Under physiological conditions, Ca²⁺ influx is activated in response to numerous G protein-coupled receptors and receptor tyrosine kinases signaling via inositol-1,4,5-trisphosphate as a second messenger (2). Store-operated Ca²⁺ entry is mediated primarily by the Ca²⁺ release-activated Ca²⁺ (CRAC) channel (3), which consists of the pore-forming subunits ORAI1–3 (or CRAC modulators 1–3) and Ca²⁺ sensors, STIM1 and STIM2 (4–7). STIM proteins reside in the membrane of endoplasmic reticulum (ER), whereas ORAI proteins reside in the plasma membrane. STIM1 is a single transmembrane-spanning protein (8–12) that, in resting cells, exists as a dimer that binds Ca²⁺ through two EF hand-containing domains located in the ER lumen (13). Depletion of Ca²⁺ in the ER induces a series of molecular events in the conformation and localization of STIM1, initiated by the formation of higher-order oligomers, protein unfolding, and accumulation at discrete sites in the cell where the ER membrane is in close proximity to the plasma membrane (11, 13–16). In these sites, STIM1 binds to the cytosolic C and N termini of ORAI1 (17, 18), resulting in channel activation and generation of a highly Ca²⁺-selective CRAC current, or I_CRAC (3, 19, 20). I_CRAC is responsible not only for restoring cytosolic and ER Ca²⁺ concentration, thus maintaining the cell in a Ca²⁺ signaling-competent stage (1), but also for many cellular functions such as regulation of gene expression, exocytosis, proliferation, and apoptosis (1).Consistent with a fundamental role of the CRAC channel in cell signaling, loss-of-function mutations in STIM1 or ORAI1 lead to immune deficiency and nonprogressive myopathy (21–23). However, evidence that gain-of-function mutations in STIM1 and ORAI1 can affect human health is only recently starting to emerge. It was shown that mutations in the domain of STIM1 that binds Ca²⁺ (EF hand domain) in resting conditions are associated with nonsyndromic myopathy with tubular aggregates (24). Functional studies demonstrated that these mutations cause hyperactivation of the CRAC channel (24). However, it remains unknown whether myopathy with tubular aggregates is caused by the increased activity of the CRAC channel, increased activity of another Ca²⁺ channel using STIM1 as a sensor (25), or a function of STIM1 that is unrelated to Ca²⁺ signaling, as STIM1 can function independently of ORAI1 (26-28).Stormorken syndrome [Mendelian Inheritance in Man (MIM) 185070] is a rare autosomal-dominant condition with a constellation of symptoms, including congenital miosis, bleeding diathesis, thrombocytopenia, functional (or anatomical) asplenia, and proximal muscle weakness (29). Other manifestations include ichthyosis, headaches, and dyslexia (30). Patients typically display increased creatine kinase (CK) levels and histologic evidence of myopathy with tubular aggregates (30, 31). Here, we show that Stormorken syndrome is caused by an activating mutation in STIM1. We also identify a mutation in the STIM1-interacting molecule, ORAI1, in a Stormorken-like syndrome that presented with miosis and tubular myopathy. Functional analyses reveal that both mutations enhance the activity of the CRAC channel, but by different molecular mechanisms. These data expand the phenotypic spectrum of activating mutations in the CRAC channels from myopathy with tubular aggregates to miosis, bleeding diathesis, thrombocytopenia, asplenia, ichthyosis, headaches, and dyslexia.     

Junctophilin-4, a component of the endoplasmic reticulum–plasma membrane junctions,regulates Ca2+ dynamics in T cells

Orai1 and stromal interaction molecule 1 (STIM1) mediate store-operated Ca²⁺ entry (SOCE) in immune cells. STIM1, an endoplasmic reticulum (ER) Ca²⁺ sensor, detects store depletion and interacts with plasma membrane (PM)-resident Orai1 channels at the ER–PM junctions. However, the molecular composition of these junctions in T cells remains poorly understood. Here, we show that junctophilin-4 (JP4), a member of junctional proteins in excitable cells, is expressed in T cells and localized at the ER–PM junctions to regulate Ca²⁺ signaling. Silencing or genetic manipulation of JP4 decreased ER Ca²⁺ content and SOCE in T cells, impaired activation of the nuclear factor of activated T cells (NFAT) and extracellular signaling-related kinase (ERK) signaling pathways, and diminished expression of activation markers and cytokines. Mechanistically, JP4 directly interacted with STIM1 via its cytoplasmic domain and facilitated its recruitment into the junctions. Accordingly, expression of this cytoplasmic fragment of JP4 inhibited SOCE. Furthermore, JP4 also formed a complex with junctate, a Ca²⁺-sensing ER-resident protein, previously shown to mediate STIM1 recruitment into the junctions. We propose that the junctate–JP4 complex located at the junctions cooperatively interacts with STIM1 to maintain ER Ca²⁺ homeostasis and mediate SOCE in T cells.The endoplasmic reticulum (ER)–plasma membrane (PM) junctions are ubiquitous structures essential for intermembrane communications (1–3). These junctions play an important role in lipid transfer and regulation of Ca²⁺ dynamics, including ER Ca²⁺ homeostasis and Ca²⁺ entry after receptor stimulation (1, 4). Four major categories of components of the ER–PM junctions have been identified so far: (i) dyad/triad junctional proteins in the heart and skeletal muscle (e.g., junctophilins and junctin), (ii) ER-resident vesicle-associated membrane protein-associated proteins (VAPs) that form the lipid transfer machinery by interacting with phospholipid-binding proteins, (iii) extended synaptogamin-like proteins (E-Syts) that tether membranes, and (iv) the Orai1–stromal interaction molecule 1 (STIM1) complex that forms the primary Ca²⁺ channel in T cells, the Ca²⁺ release-activated Ca²⁺ (CRAC) channels. Among these proteins, the dyad/triad junctional proteins and the Orai1–STIM1 complex are known to play a crucial role in Ca²⁺ dynamics, including excitation–contraction coupling in muscle and store-operated Ca²⁺ entry (SOCE) in immune cells, respectively (2, 5).Stimulation of T-cell receptors (TCRs) triggers activation of SOCE primarily mediated by the PM-resident Orai1 channels and ER-resident STIM1 protein that senses ER Ca²⁺ concentration (6–11). Upon store depletion, STIM1 translocates and interacts with Orai1 at the preformed ER–PM junctions (12, 13). STIM1 uses two major mechanisms to translocate into the ER–PM junctions: by interactions with phosphatidylinositol-4,5-bisphosphate (PIP₂) in the PM via its C-terminal polybasic residues and by interaction with Orai1 or the ER-resident junctate proteins (14, 15). Recently, septin filaments were shown to play a role in PIP₂ enrichment at the ER–PM junctions before STIM1 recruitment (16). Subsequently, membrane-tethering VAP and E-Syt proteins were shown to be important for PIP₂ replenishment after store depletion (17). The importance of protein interaction in STIM1 recruitment was demonstrated by a STIM1ΔK mutant truncated in its C-terminal polybasic domain. Interaction with Orai1 or junctate facilitated recruitment of this PIP₂ binding-deficient mutant into the junctions (15, 18, 19). It was thought that the roles of dyad/triad junctional proteins are limited to muscle cells. However, identification of junctate as a STIM1-interacting partner implied that some components (or homologs) of ER–PM junctions in excitable cells may be shared in immune cells.The junctophilin family consists of four genes (JP1, JP2, JP3, and JP4) that are expressed in a tissue-specific manner and are known to form ER–PM junctions in excitable cells (20, 21). Junctophilins contain eight repeats of the membrane occupation and recognition nexus (MORN) motifs that bind to phospholipids in the N terminus and a C-terminal ER membrane-spanning transmembrane segment (20, 22). In this study, we observed expression of JP4 in both human and mouse T cells, which was further enhanced by TCR stimulation. Depletion or deficiency of JP4 reduced ER Ca²⁺ content, SOCE, and activation of the nuclear factor of activated T cells (NFAT) and ERK mitogen-activated protein kinase (MAPK) pathways. Mechanistically, JP4 depletion reduced accumulation of STIM1 at the junctions without affecting the number and length of the ER–PM junctions. We observed a direct interaction between the cytoplasmic regions of JP4 and STIM1, and, correspondingly, overexpression of the STIM1-interacting JP4 fragment had a dominant negative effect on SOCE. Finally, we identified a protein complex consisting of JP4 and junctate at the ER–PM junctions, which may have a synergistic effect in recruiting STIM1 to the junctions. Therefore, our studies identify a PIP₂-independent, but protein interaction-mediated, mechanism by which the junctate–JP4 complex recruits STIM1 into the ER–PM junctions to maintain ER Ca²⁺ homeostasis and activate SOCE in T cells.     

Stoichiometric requirements for trapping and gating of Ca2+ release-activated Ca2+ (CRAC) channels by stromal interaction molecule 1 (STIM1)

Store-operated Ca(2+) entry depends critically on physical interactions of the endoplasmic reticulum (ER) Ca(2+) sensor stromal interaction molecule 1 (STIM1) and the Ca(2+) release-activated Ca(2+) (CRAC) channel protein Orai1. Recent studies support a diffusion-trap mechanism in which ER Ca(2+) depletion causes STIM1 to accumulate at ER-plasma membrane (PM) junctions, where it binds to Orai1, trapping and activating mobile CRAC channels in the overlying PM. To determine the stoichiometric requirements for CRAC channel trapping and activation, we expressed mCherry-STIM1 and Orai1-GFP at varying ratios in HEK cells and quantified CRAC current (I(CRAC)) activation and the STIM1:Orai1 ratio at ER-PM junctions after store depletion. By competing for a limited amount of STIM1, high levels of Orai1 reduced the junctional STIM1:Orai1 ratio to a lower limit of 0.3-0.6, indicating that binding of one to two STIM1s is sufficient to immobilize the tetrameric CRAC channel at ER-PM junctions. In cells expressing a constant amount of STIM1, CRAC current was a highly nonlinear bell-shaped function of Orai1 expression and the minimum stoichiometry for channel trapping failed to evoke significant activation. Peak current occurred at a ratio of ～2 STIM1:Orai1, suggesting that maximal CRAC channel activity requires binding of eight STIM1s to each channel. Further increases in Orai1 caused channel activity and fast Ca(2+)-dependent inactivation to decline in parallel. The data are well described by a model in which STIM1 binds to Orai1 with negative cooperativity and channels open with positive cooperativity as a result of stabilization of the open state by STIM1.     

Junctate is a Ca2+-sensing structural component of Orai1 and stromal interaction molecule 1 (STIM1)

Orai1 and stromal interaction molecule (STIM)1 are critical components of Ca(2+) release-activated Ca(2+) (CRAC) channels. Orai1 is a pore subunit of CRAC channels, and STIM1 acts as an endoplasmic reticulum (ER) Ca(2+) sensor that detects store depletion. Upon store depletion after T-cell receptor stimulation, STIM1 translocates and coclusters with Orai1 at sites of close apposition of the plasma membrane (PM) and the ER membrane. However, the molecular components of these ER-PM junctions remain poorly understood. Using affinity protein purification, we uncovered junctate as an interacting partner of Orai1-STIM1 complex. Furthermore, we identified a Ca(2+)-binding EF-hand motif in the ER-luminal region of junctate. Mutation of this EF-hand domain of junctate impaired its Ca(2+) binding and resulted in partial activation of CRAC channels and clustering of STIM1 independently of store depletion. In addition to the known mechanisms of STIM1 clustering (i.e., phosphoinositide and Orai1 binding), our study identifies an alternate mechanism to recruit STIM1 into the ER-PM junctions via binding to junctate. We propose that junctate, a Ca(2+)-sensing ER protein, is a structural component of the ER-PM junctions where Orai1 and STIM1 cluster and interact in T cells.