Essential role of N-terminal SAM regions in STIM1 multimerization and function.

The single-pass transmembrane protein Stromal Interaction Molecule 1 (STIM1), located in the endoplasmic reticulum (ER) membrane, possesses two main functions: It senses the ER-Ca2+ concentration and directly binds to the store-operated Ca2+ channel Orai1 for its activation when Ca2+ recedes. At high resting ER-Ca2+ concentration, the ER-luminal STIM1 domain is kept monomeric but undergoes di/multimerization once stores are depleted. Luminal STIM1 multimerization is essential to unleash the STIM C-terminal binding site for Orai1 channels. However, structural basis of the luminal association sites has so far been elusive. Here, we employed molecular dynamics (MD) simulations and identified two essential di/multimerization segments, the α7 and the adjacent region near the α9-helix in the sterile alpha motif (SAM) domain. Based on MD results, we targeted the two STIM1 SAM domains by engineering point mutations. These mutations interfered with higher-order multimerization of ER-luminal fragments in biochemical assays and puncta formation in live-cell experiments upon Ca2+ store depletion. The STIM1 multimerization impeded mutants significantly reduced Ca2+ entry via Orai1, decreasing the Ca2+ oscillation frequency as well as store-operated Ca2+ entry. Combination of the ER-luminal STIM1 multimerization mutations with gain of function mutations and coexpression of Orai1 partially ameliorated functional defects. Our data point to a hydrophobicity-driven binding within the ER-luminal STIM1 multimer that needs to switch between resting monomeric and activated multimeric state. Altogether, these data reveal that interactions between SAM domains of STIM1 monomers are critical for multimerization and activation of the protein.

Impact of Targeting Moiety Type and Protein Corona Formation on the Uptake of Fn14-Targeted Nanoparticles by Cancer Cells.

The TWEAK receptor, Fn14, is a promising candidate for active targeting of cancer nanotherapeutics to many solid tumor types, including metastatic breast and primary brain cancers. Targeting of therapeutic nanoparticles (NPs) has been accomplished using a range of targeting moieties including monoclonal antibodies and related fragments, peptides, and small molecules. Here, we investigated a full-length Fn14-specific monoclonal antibody, ITEM4, or an ITEM4-Fab fragment as a targeting moiety to guide the development of a clinical formulation. We formulated NPs with varying densities of the targeting moieties while maintaining the decreased nonspecific adhesivity with receptor targeting (DART) characteristics. To model the conditions that NPs experience following intravenous infusion, we investigated the impact of serum exposure in relation to the targeting moiety type and surface density. To further evaluate performance at the cancer cell level, we performed experiments to assess differences in cellular uptake and trafficking in several cancer cell lines using confocal microscopy, imaging flow cytometry, and total internal reflection fluorescence microscopy. We observed that Fn14-targeted NPs exhibit enhanced cellular uptake in Fn14-high compared to Fn14-low cancer cells and that in both cell lines uptake levels were greater than observed with control, nontargeted NPs. We found that serum exposure increased Fn14-targeted NP specificity while simultaneously reducing the total NP uptake. Importantly, serum exposure caused a larger reduction in cancer cell uptake over time when the targeting moiety was an antibody fragment (Fab region of the monoclonal antibody) compared with the full-length monoclonal antibody targeting moiety. Lastly, we uncovered that full monoclonal antibody-targeted NPs enter cancer cells via clathrin-mediated endocytosis and traffic through the endolysosomal pathway. Taken together, these results support a pathway for developing a clinical formulation using a full-length Fn14 monoclonal antibody as the targeting moiety for a DART cancer nanotherapeutic agent.

STIM Proteins and Regulation of SOCE in ER-PM Junctions.

ER-PM junctions are membrane contact sites formed by the endoplasmic reticulum (ER) and plasma membrane (PM) in close apposition together. The formation and stability of these junctions are dependent on constitutive and dynamic enrichment of proteins, which either contribute to junctional stability or modulate the lipid levels of both ER and plasma membranes. The ER-PM junctions have come under much scrutiny recently as they serve as hubs for assembling the Ca2+ signaling complexes. This review summarizes: (1) key findings that underlie the abilities of STIM proteins to accumulate in ER-PM junctions; (2) the modulation of Orai/STIM complexes by other components found within the same junction; and (3) how Orai1 channel activation is coordinated and coupled with downstream signaling pathways.

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.

PIP2 and septin control STIM1/Orai1 assembly by regulating cytoskeletal remodeling via a CDC42-WASP/WAVE-ARP2/3 protein complex.

Store-operated calcium entry (SOCE) is triggered by assembly of Orai1 with STIM proteins in ER-PM junctions. Plasma membrane PIP2 as well as PIP2-binding protein, SEPT4, significantly impact Orai1-STIM1 interaction. While septins and PIP2 can organize the actin cytoskeleton, it is unclear whether the status of actin within the junctions contributes to SOCE. We report herein that actin remodeling modulates STIM1 clustering. Our findings show that a PIP2- and SEPT4-dependent mechanism involving CDC42, WASP/WAVE, and ARP2 regulates actin remodeling into a ring-like structure around STIM1 puncta. CDC42 localization in the ER-plasma membrane region is enhanced following ER-Ca2+ store depletion. PIP2 depletion or knockdown of SEPT4 attenuate the recruitment of CDC42 to the ER-PM region. Importantly, knockdown of SEPT4, or CDC42+ARP2, disrupts the organization of actin as well as STIM1 clustering. Consequently, Orai1 recruitment to STIM1 puncta, SOCE, and NFAT translocation to the nucleus are all attenuated. Ca2+ influx induced by STIM1-C terminus is not affected by CDC42 knockdown. In aggregate, our findings reveal that PIP2 and SEPT4 affect Orai1/STIM1 clustering by coordinating actin remodeling within ER-PM junctions. This dynamic reorganization of actin has an important role in regulation of SOCE and downstream Ca2+-dependent effector functions.

RACK1 plays a critical role in mast cell secretion and Ca2+ mobilization by modulating F-actin dynamics.

Although RACK1 is known to act as a signaling hub in immune cells, its presence and role in mast cells (MCs) is undetermined. MC activation via antigen stimulation results in mediator release and is preceded by cytoskeleton reorganization and Ca2+ mobilization. In this study, we found that RACK1 was distributed throughout the MC cytoplasm both in vivo and in vitro. After RACK1 knockdown (KD), MCs were rounded, and the cortical F-actin was fragmented. Following antigen stimulation, in RACK1 KD MCs, there was a reduction in cortical F-actin, an increase in monomeric G-actin and a failure to organize F-actin. RACK1 KD also increased and accelerated degranulation. CD63+ secretory granules were localized in F-actin-free cortical regions in non-stimulated RACK1 KD MCs. Additionally, RACK1 KD increased antigen-stimulated Ca2+ mobilization, but attenuated antigen-stimulated depletion of ER Ca2+ stores and thapsigargin-induced Ca2+ entry. Following MC activation there was also an increase in interaction of RACK1 with Orai1 Ca2+-channels, ß-actin and the actin-binding proteins vinculin and MyoVa. These results show that RACK1 is a critical regulator of actin dynamics, affecting mediator secretion and Ca2+ signaling in MCs. This article has an associated First Person interview with the first author of the paper.

No social distancing between ORAI channels.

ORAI1 is established as an essential component of Ca2+ release-activated Ca2+ (CRAC) channel which mediates store-operated Ca2+ entry (SOCE). However, the contributions of ORAI2 and ORAI3 to SOCE are not understood. We highlight a recent study which shows that ORAI proteins form heteromeric channels which tune SOCE over a range of stimulus intensities.

STIM2 targets Orai1/STIM1 to the AKAP79 signaling complex and confers coupling of Ca2+ entry with NFAT1 activation.

The Orai1 channel is regulated by stromal interaction molecules STIM1 and STIM2 within endoplasmic reticulum (ER)-plasma membrane (PM) contact sites. Ca2+ signals generated by Orai1 activate Ca2+-dependent gene expression. When compared with STIM1, STIM2 is a weak activator of Orai1, but it has been suggested to have a unique role in nuclear factor of activated T cells 1 (NFAT1) activation triggered by Orai1-mediated Ca2+ entry. In this study, we examined the contribution of STIM2 in NFAT1 activation. We report that STIM2 recruitment of Orai1/STIM1 to ER-PM junctions in response to depletion of ER-Ca2+ promotes assembly of the channel with AKAP79 to form a signaling complex that couples Orai1 channel function to the activation of NFAT1. Knockdown of STIM2 expression had relatively little effect on Orai1/STIM1 clustering or local and global [Ca2+]i increases but significantly attenuated NFAT1 activation and assembly of Orai1 with AKAP79. STIM1ΔK, which lacks the PIP2-binding polybasic domain, was recruited to ER-PM junctions following ER-Ca2+ depletion by binding to Orai1 and caused local and global [Ca2+]i increases comparable to those induced by STIM1 activation of Orai1. However, in contrast to STIM1, STIM1ΔK induced less NFAT1 activation and attenuated the association of Orai1 with STIM2 and AKAP79. Orai1-AKAP79 interaction and NFAT1 activation were recovered by coexpressing STIM2 with STIM1ΔK. Replacing the PIP2-binding domain of STIM1 with that of STIM2 eliminated the requirement of STIM2 for NFAT1 activation. Together, these data demonstrate an important role for STIM2 in coupling Orai1-mediated Ca2+ influx to NFAT1 activation.

PunctaSpecks: A tool for automated detection, tracking, and analysis of multiple types of fluorescently labeled biomolecules.

Recent advances in imaging technology and fluorescent probes have made it possible to gain information about the dynamics of subcellular processes at unprecedented spatiotemporal scales. Unfortunately, a lack of automated tools to efficiently process the resulting imaging data encoding fine details of the biological processes remains a major bottleneck in utilizing the full potential of these powerful experimental techniques. Here we present a computational tool, called PunctaSpecks, that can characterize fluorescence signals arising from a wide range of biological molecules under normal and pathological conditions. Among other things, the program can calculate the number, areas, life-times, and amplitudes of fluorescence signals arising from multiple sources, track diffusing fluorescence sources like moving mitochondria, and determine the overlap probability of two processes or organelles imaged using indicator dyes of different colors. We have tested PunctaSpecks on synthetic time-lapse movies containing mobile fluorescence objects of various sizes, mimicking the activity of biomolecules. The robustness of the software is tested by varying the level of noise along with random but known pattern of appearing, disappearing, and movement of these objects. Next, we use PunctaSpecks to characterize protein-protein interaction involved in store-operated Ca2+ entry through the formation and activation of plasma membrane-bound ORAI1 channel and endoplasmic reticulum membrane-bound stromal interaction molecule (STIM), the evolution of inositol 1,4,5-trisphosphate (IP3)-induced Ca2+ signals from sub-micrometer size local events into global waves in human cortical neurons, and the activity of Alzheimer's disease-associated ß amyloid pores in the plasma membrane. The tool can also be used to study other dynamical processes imaged through fluorescence molecules. The open source algorithm allows for extending the program to analyze more than two types of biomolecules visualized using markers of different colors.

TRIC-A shapes oscillatory Ca2+ signals by interaction with STIM1/Orai1 complexes.

Trimeric intracellular cation (TRIC) channels have been proposed to modulate Ca2+ release from the endoplasmic reticulum (ER) and determine oscillatory Ca2+ signals. Here, we report that TRIC-A-mediated amplitude and frequency modulation of ryanodine receptor 2 (RyR2)-mediated Ca2+ oscillations and inositol 1,4,5-triphosphate receptor (IP3R)-induced cytosolic signals is based on attenuating store-operated Ca2+ entry (SOCE). Further, TRIC-A-dependent delay in ER Ca2+ store refilling contributes to shaping the pattern of Ca2+ oscillations. Upon ER Ca2+ depletion, TRIC-A clusters with stromal interaction molecule 1 (STIM1) and Ca2+-release-activated Ca2+ channel 1 (Orai1) within ER-plasma membrane (PM) junctions and impairs assembly of the STIM1/Orai1 complex, causing a decrease in Orai1-mediated Ca2+ current and SOCE. Together, our findings demonstrate that TRIC-A is a negative regulator of STIM1/Orai1 function. Thus, aberrant SOCE could contribute to muscle disorders associated with loss of TRIC-A.

The Endoplasmic Reticulum-Plasma Membrane Junction: A Hub for Agonist Regulation of Ca2+ Entry.

Stimulation of cell-surface receptors induces cytosolic Ca2+ ([Ca2+]i) increases that are detected and transduced by effector proteins for regulation of cell function. Intracellular Ca2+ release, via endoplasmic reticulum (ER) proteins inositol 1,4,5-trisphosphate receptors (IP3R) and ryanodine receptors (RyR), and Ca2+ influx, via store-operated Ca2+ entry (SOCE), contribute to the increase in [Ca2+]i The amplitude, frequency, and spatial characteristics of the [Ca2+]i increases are controlled by the compartmentalization of proteins into signaling complexes such as receptor-signaling complexes and SOCE complexes. Both complexes include protein and lipid components, located in the plasma membrane (PM) and ER. Receptor signaling initiates in the PM via phospholipase C (PLC)-mediated hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2), and culminates with the activation of IP3R in the ER. Conversely, SOCE is initiated in the ER by Ca2+-sensing stromal interaction molecule (STIM) proteins, which then interact with PM channels Orai1 and TRPC1 to activate Ca2+ entry. This review will address how ER-PM junctions serve a central role in agonist regulation of SOCE.

Tuning store-operated calcium entry to modulate Ca2+-dependent physiological processes.

The intracellular calcium signaling processes are tightly regulated to ensure the generation of calcium signals with the specific spatiotemporal characteristics required for regulating various cell functions. Compartmentalization of the molecular components involved in the generation of these signals at discrete intracellular sites ensures the signaling specificity and transduction fidelity of the signal for regulating downstream effector processes. Store-operated calcium entry (SOCE) is ubiquitously present in cells and is critical for essential cell functions in a variety of tissues. SOCE is mediated via plasma membrane Ca2+ channels that are activated when luminal [Ca2+] of the endoplasmic reticulum ([Ca2+]ER) is decreased. The ER-resident stromal interaction molecules, STIM1 and STIM2, respond to decreases in [Ca2+]ER by undergoing conformational changes that cause them to aggregate at the cell periphery in ER-plasma membrane (ER-PM) junctions. At these sites, STIM proteins recruit Orai1 channels and trigger their activation. Importantly, the two STIM proteins concertedly modulate Orai1 function as well as the sensitivity of SOCE to ER-Ca2+ store depletion. Another family of plasma membrane Ca2+ channels, known as the Transient Receptor Potential Canonical (TRPC) channels (TRPC1-7) also contribute to sustained [Ca2+]i elevation. Although Ca2+ signals generated by these channels overlap with those of Orai1, they regulate distinct functions in the cells. Importantly, STIM1 is also required for plasma membrane localization and activation of some TRPCs. In this review, we will discuss various molecular components and factors that govern the activation, regulation and modulation of the Ca2+ signal generated by Ca2+ entry pathways in response to depletion of ER-Ca2+ stores. This article is part of a Special Issue entitled: ECS Meeting edited by Claus Heizmann, Joachim Krebs and Jacques Haiech.

TRP Channel Involvement in Salivary Glands-Some Good, Some Bad.

Salivary glands secrete saliva, a mixture of proteins and fluids, which plays an extremely important role in the maintenance of oral health. Loss of salivary secretion causes a dry mouth condition, xerostomia, which has numerous deleterious consequences including opportunistic infections within the oral cavity, difficulties in eating and swallowing food, and problems with speech. Secretion of fluid by salivary glands is stimulated by activation of specific receptors on acinar cell plasma membrane and is mediated by an increase in cytosolic [Ca2+] ([Ca2+]i). The increase in [Ca2+]i regulates a number of ion channels and transporters that are required for establishing an osmotic gradient that drives water flow via aquaporin water channels in the apical membrane. The Store-Operated Ca2+ Entry (SOCE) mechanism, which is regulated in response to depletion of ER-Ca2+, determines the sustained [Ca2+]i increase required for prolonged fluid secretion. Core components of SOCE in salivary gland acinar cells are Orai1 and STIM1. In addition, TRPC1 is a major and non-redundant contributor to SOCE and fluid secretion in salivary gland acinar and ductal cells. Other TRP channels that contribute to salivary flow are TRPC3 and TRPV4, while presence of others, including TRPM8, TRPA1, TRPV1, and TRPV3, have been identified in the gland. Loss of salivary gland function leads to dry mouth conditions, or xerostomia, which is clinically seen in patients who have undergone radiation treatment for head-and-neck cancers, and those with the autoimmune exocrinopathy, Sjögren's syndrome (pSS). TRPM2 is a unique TRP channel that acts as a sensor for intracellular ROS. We will discuss recent studies reported by us that demonstrate a key role for TRPM2 in radiation-induced salivary gland dysfunction. Further, there is increasing evidence that TRPM2 might be involved in inflammatory processes. These interesting findings point to the possible involvement of TRPM2 in Sjögren's Syndrome, although further studies will be required to identify the exact role of TRPM2 in this disease.

STIM2 Induces Activated Conformation of STIM1 to Control Orai1 Function in ER-PM Junctions.

Ca2+ entry mediated by the calcium channel, Orai1, provides critical Ca2+ signals that regulate cell function. The ER-Ca2+ sensor protein, STIM1, recruits and strongly activates Orai1 within ER-PM junctions. STIM2 is a poor activator of Orai1, and its physiological role is not well understood. Herein, we report a crucial function for STIM2 in inducing the activated conformation of STIM1. By using conformational sensors of STIM2 and STIM1, together with protein interaction and functional studies, we show that STIM2 is constitutively localized within ER-PM junctions in ER-Ca2+ store replete cells. Importantly, STIM2 traps STIM1 and triggers remodeling of STIM1 C terminus, causing STIM1/Orai1 coupling and enhancement of Orai1 function in cells with relatively high ER-[Ca2+]. The increase in Ca2+ entry controls Ca2+-dependent transcription factor, NFAT, activation at low [agonist]. Our findings reveal that STIM2 modulates STIM1/Orai1 function to tune the fidelity of receptor-evoked Ca2+ signaling and the physiological response of cells.

STIM-TRP Pathways and Microdomain Organization: Contribution of TRPC1 in Store-Operated Ca2+ Entry: Impact on Ca2+ Signaling and Cell Function.

Store-operated calcium entry (SOCE) is a ubiquitous Ca2+ entry pathway that is activated in response to depletion of ER-Ca2+ stores and critically controls the regulation of physiological functions in a wide variety of cell types. The transient receptor potential canonical (TRPC) channels (TRPCs 1-7), which are activated by stimuli leading to PIP2 hydrolysis, were first identified as molecular components of SOCE channels. While TRPC1 was associated with SOCE and regulation of function in several cell types, none of the TRPC members displayed I CRAC, the store-operated current identified in lymphocytes and mast cells. Intensive search finally led to the identification of Orai1 and STIM1 as the primary components of the CRAC channel. Orai1 was established as the pore-forming channel protein and STIM1 as the ER-Ca2+ sensor protein involved in activation of Orai1. STIM1 also activates TRPC1 via a distinct domain in its C-terminus. However, TRPC1 function depends on Orai1-mediated Ca2+ entry, which triggers recruitment of TRPC1 into the plasma membrane where it is activated by STIM1. TRPC1 and Orai1 form distinct store-operated Ca2+ channels that regulate specific cellular functions. It is now clearly established that regulation of TRPC1 trafficking can change plasma membrane levels of the channel, the phenotype of the store-operated Ca2+ current, as well as pattern of SOCE-mediated [Ca2+]i signals. Thus, TRPC1 is activated downstream of Orai1 and modifies the initial [Ca2+]i signal generated by Orai1. This review will highlight current concepts of the activation and regulation of TRPC1 channels and its impact on cell function.

Radiation inhibits salivary gland function by promoting STIM1 cleavage by caspase-3 and loss of SOCE through a TRPM2-dependent pathway.

Store-operated Ca2+ entry (SOCE) is critical for salivary gland fluid secretion. We report that radiation treatment caused persistent salivary gland dysfunction by activating a TRPM2-dependent mitochondrial pathway, leading to caspase-3-mediated cleavage of stromal interaction molecule 1 (STIM1) and loss of SOCE. After irradiation, acinar cells from the submandibular glands of TRPM2+/+ , but not those from TRPM2-/- mice, displayed an increase in the concentrations of mitochondrial Ca2+ and reactive oxygen species, a decrease in mitochondrial membrane potential, and activation of caspase-3, which was associated with a sustained decrease in STIM1 abundance and attenuation of SOCE. In a salivary gland cell line, silencing the mitochondrial Ca2+ uniporter or caspase-3 or treatment with inhibitors of TRPM2 or caspase-3 prevented irradiation-induced loss of STIM1 and SOCE. Expression of exogenous STIM1 in the salivary glands of irradiated mice increased SOCE and fluid secretion. We suggest that targeting the mechanisms underlying the loss of STIM1 would be a potentially useful approach for preserving salivary gland function after radiation therapy.

TRPC1, Orai1, and STIM1 in SOCE: Friends in tight spaces.

Store-operated calcium entry (SOCE) is a ubiquitous Ca2+ entry pathway that is activated in response to depletion of ER-Ca2+ stores and critically controls the regulation of physiological functions in miscellaneous cell types. The transient receptor potential canonical 1 (TRPC1) is the first member of the TRPC channel subfamily to be identified as a molecular component of SOCE. While TRPC1 has been shown to contribute to SOCE and regulate various functions in many cells, none of the reported TRPC1-mediated currents resembled ICRAC, the highly Ca2+-selective store-dependent current first identified in lymphocytes and mast cells. Almost a decade after the cloning of TRPC1 two proteins were identified as the primary components of the CRAC channel. The first, STIM1, is an ER-Ca2+ sensor protein involved in activating SOCE. The second, Orai1 is the pore-forming component of the CRAC channel. Co-expression of STIM1 and Orai1 generated robust ICRAC. Importantly, STIM1 was shown to also activate TRPC1 via its C-terminal polybasic domain, which is distinct from its Orai1-activating domain, SOAR. In addition, TRPC1 function critically depends on Orai1-mediated Ca2+ entry which triggers recruitment of TRPC1 into the plasma membrane where it is then activated by STIM1. TRPC1 and Orai1 form discrete STIM1-gated channels that generate distinct Ca2+ signals and regulate specific cellular functions. Surface expression of TRPC1 can be modulated by trafficking of the channel to and from the plasma membrane, resulting in changes to the phenotype of TRPC1-mediated current and [Ca2+]i signals. Thus, TRPC1 is activated downstream of Orai1 and modifies the initial [Ca2+]i signal generated by Orai1 following store depletion. This review will summarize the important findings that underlie the current concepts for activation and regulation of TRPC1, as well as its impact on cell function.

Assembly of ER-PM Junctions: A Critical Determinant in the Regulation of SOCE and TRPC1.

Store-operated calcium entry (SOCE), a unique plasma membrane Ca2+ entry mechanism, is activated when ER-[Ca2+] is decreased. SOCE is mediated via the primary channel, Orai1, as well as others such as TRPC1. STIM1 and STIM2 are ER-Ca2+ sensor proteins that regulate Orai1 and TRPC1. SOCE requires assembly of STIM proteins with the plasma membrane channels which occurs within distinct regions in the cell that have been termed as endoplasmic reticulum (ER)-plasma membrane (PM) junctions. The PM and ER are in close proximity to each other within this region, which allows STIM1 in the ER to interact with and activate either Orai1 or TRPC1 in the plasma membrane. Activation and regulation of SOCE involves dynamic assembly of various components that are involved in mediating Ca2+ entry as well as those that determine the formation and stabilization of the junctions. These components include proteins in the cytosol, ER and PM, as well as lipids in the PM. Recent studies have also suggested that SOCE and its components are compartmentalized within ER-PM junctions and that this process might require remodeling of the plasma membrane lipids and reorganization of structural and scaffolding proteins. Such compartmentalization leads to the generation of spatially- and temporally-controlled Ca2+signals that are critical for regulating many downstream cellular functions.

Targeting the Ca(2+) Sensor STIM1 by Exosomal Transfer of Ebv-miR-BART13-3p is Associated with Sjögren's Syndrome.

Primary Sjögren's syndrome (pSS) is a systemic autoimmune disease that is associated with inflammation and dysfunction of salivary and lacrimal glands. The molecular mechanism(s) underlying this exocrinopathy is not known, although the syndrome has been associated with viruses, such as the Epstein Barr Virus (EBV). We report herein that an EBV-specific microRNA (ebv-miR-BART13-3p) is significantly elevated in salivary glands (SGs) of pSS patients and we show that it targets stromal interacting molecule 1 (STIM1), a primary regulator of the store-operated Ca(2+) entry (SOCE) pathway that is essential for SG function, leading to loss of SOCE and Ca(2+)-dependent activation of NFAT. Although EBV typically infects B cells and not salivary epithelial cells, ebv-miR-BART13-3p is present in both cell types in pSS SGs. Importantly, we further demonstrate that ebv-miR-BART13-3p can be transferred from B cells to salivary epithelial cells through exosomes and it recapitulates its functional effects on calcium signaling in a model system.