Oxidative Stress-Induced STIM2 Cysteine Modifications Suppress Store-Operated Calcium Entry.

Store-operated calcium entry (SOCE) through STIM-gated ORAI channels governs vital cellular functions. In this context, SOCE controls cellular redox signaling and is itself regulated by redox modifications. However, the molecular mechanisms underlying this calcium-redox interplay and the functional outcomes are not fully understood. Here, we examine the role of STIM2 in SOCE redox regulation. Redox proteomics identify cysteine 313 as the main redox sensor of STIM2 in vitro and in vivo. Oxidative stress suppresses SOCE and calcium currents in cells overexpressing STIM2 and ORAI1, an effect that is abolished by mutation of cysteine 313. FLIM and FRET microscopy, together with MD simulations, indicate that oxidative modifications of cysteine 313 alter STIM2 activation dynamics and thereby hinder STIM2-mediated gating of ORAI1. In summary, this study establishes STIM2-controlled redox regulation of SOCE as a mechanism that affects several calcium-regulated physiological processes, as well as stress-induced pathologies.

Identification of molecular determinants that govern distinct STIM2 activation dynamics.

The endoplasmic reticulum (ER) Ca2+ sensors stromal interaction molecule 1 (STIM1) and STIM2, which connect ER Ca2+ depletion with extracellular Ca2+ influx, are crucial for the maintenance of Ca2+ homeostasis in mammalian cells. Despite the recent progress in unraveling the role of STIM2 in Ca2+ signaling, the mechanistic underpinnings of its activation remain underexplored. We use an engineering approach to direct ER-resident STIMs to the plasma membrane (PM) while maintaining their correct membrane topology, as well as Förster resonance energy transfer (FRET) sensors that enabled in cellulo real-time monitoring of STIM activities. This allowed us to determine the calcium affinities of STIM1 and STIM2 both in cellulo and in situ, explaining the current discrepancies in the literature. We also identified the key structural determinants, especially the corresponding G residue in STIM1, which define the distinct activation dynamics of STIM2. The chimeric E470G mutation could switch STIM2 from a slow and weak Orai channel activator into a fast and potent one like STIM1 and vice versa. The systemic dissection of STIM2 activation by protein engineering sets the stage for the elucidation of the regulation and function of STIM2-mediated signaling in mammals.

Structural elements of stromal interaction molecule function.

Stromal interaction molecule (STIM)-1 and -2 are multi-domain, single-pass transmembrane proteins involved in sensing changes in compartmentalized calcium (Ca2+) levels and transducing this cellular signal to Orai1 channel proteins. Our understanding of the molecular mechanisms underlying STIM signaling has been dramatically improved through available X-ray crystal and solution NMR structures. This high-resolution structural data has revealed that intricate intramolecular and intermolecular protein-protein interactions are involved in converting STIMs from the quiescent to activation-competent states. This review article summarizes the current high resolution structural data on specific EF-hand, sterile α motif and coiled-coil interactions which drive STIM function in the activation of Orai1 channels. Further, the work discusses the effects of post-translational modifications on the structure and function of STIMs. Future structural studies on larger STIM:Orai complexes will be critical to fully defining the molecular bases for STIM function and how post-translational modifications influence these mechanisms.

STIM2 regulates both intracellular Ca2+ distribution and Ca2+ movement in skeletal myotubes.

Stromal interaction molecule 1 (STIM1) along with Orai1 mediates extracellular Ca2+ entry into the cytosol through a store-operated Ca2+ entry (SOCE) mechanism in various tissues including skeletal muscle. However, the role(s) of STIM2, a homolog of STIM1, in skeletal muscle has not been well addressed. The present study, first, was focused on searching for STIM2-binding proteins from among proteins mediating skeletal muscle functions. This study used a binding assay, quadrupole time-of-flight mass spectrometry, and co-immunoprecipitation assay with bona-fide STIM2- and SERCA1a-expressing rabbit skeletal muscle. The region for amino acids from 453 to 729 of STIM2 binds to sarcoplasmic/endoplasmic reticulum Ca2+-ATPase 1a (SERCA1a). Next, oxalate-supported 45Ca2+-uptake experiments and various single-myotube Ca2+ imaging experiments using STIM2-knockdown mouse primary skeletal myotubes have suggested that STIM2 attenuates SERCA1a activity during skeletal muscle contraction, which contributes to the intracellular Ca2+ distribution between the cytosol and the SR at rest. In addition, STIM2 regulates Ca2+ movement through RyR1 during skeletal muscle contraction as well as SOCE. Therefore, via regulation of SERCA1a activity, STIM2 regulates both intracellular Ca2+ distribution and Ca2+ movement in skeletal muscle, which makes it both similar to, yet different from, STIM1.

STIM1 and STIM2 cooperatively regulate mouse neutrophil store-operated calcium entry and cytokine production.

Neutrophils are key effector cells of the innate immune system. Calcium-dependent signaling pathways initiated by store-operated calcium entry (SOCE) are known to regulate neutrophil activation; however, the precise mechanism of this process remains unclear. STIM1 and STIM2 are calcium-sensing molecules that link calcium depletion of the endoplasmic reticulum with opening of plasma membrane calcium channels. Although a role for STIM1 in neutrophil SOCE and activation has been established, the function of STIM2 is unknown. Here we use mice with conditional ablation of Stim1 and/or Stim2 to investigate the role of STIM2 in neutrophil activation. We demonstrate that loss of STIM2 results in decreased SOCE, particularly at lower doses of agonists. Reactive oxygen species (ROS) production, degranulation, and phagocytosis are normal in the absence of STIM2, suggesting STIM1 is the dominant calcium sensor required for classical short-term neutrophil responses. However, neutrophil cytokine production required STIM2, but not STIM1, at least in part as a result of redox regulation of cytokine gene expression. In vivo loss of STIM2 results in lower cytokine levels and protection from mortality in a mouse model of systemic inflammatory response syndrome. These data, combined with previous studies focusing on STIM1, define distinct but cooperative functions for STIM1 and STIM2 in modulating neutrophil bactericidal and cytokine responses.

The TRPM7 channel kinase regulates store-operated calcium entry.

KEY POINTS: Pharmacological and molecular inhibition of transient receptor potential melastatin 7 (TRPM7) reduces store-operated calcium entry (SOCE). Overexpression of TRPM7 in TRPM7-/- cells restores SOCE. TRPM7 is not a store-operated calcium channel. TRPM7 kinase rather than channel modulates SOCE. TRPM7 channel activity contributes to the maintenance of store Ca2+ levels at rest. ABSTRACT: The transient receptor potential melastatin 7 (TRPM7) is a protein that combines an ion channel with an intrinsic kinase domain, enabling it to modulate cellular functions either by conducting ions through the pore or by phosphorylating downstream proteins via its kinase domain. In the present study, we report store-operated calcium entry (SOCE) as a novel target of TRPM7 kinase activity. TRPM7-deficient chicken DT40 B lymphocytes exhibit a strongly impaired SOCE compared to wild-type cells as a result of reduced calcium release activated calcium currents, and independently of potassium channel regulation, membrane potential changes or changes in cell-cycle distribution. Pharmacological blockade of TRPM7 with NS8593 or waixenicin A in wild-type B lymphocytes results in a significant decrease in SOCE, confirming that TRPM7 activity is acutely linked to SOCE, without TRPM7 representing a store-operated channel itself. Using kinase-deficient mutants, we find that TRPM7 regulates SOCE through its kinase domain. Furthermore, Ca2+ influx through TRPM7 is essential for the maintenance of endoplasmic reticulum Ca2+ concentration in resting cells, and for the refilling of Ca2+ stores after a Ca2+ signalling event. We conclude that the channel kinase TRPM7 and SOCE are synergistic mechanisms regulating intracellular Ca2+ homeostasis.

STIMs and Orai1 regulate cytokine production in spinal astrocytes.

BACKGROUND: Our previous study demonstrated that a store-operated calcium channel (SOCC) inhibitor (YM-58483) has central analgesic effects. However, the cellular and molecular mechanisms of such effects remain to be determined. It is well-known that glial cells play important roles in central sensitization. SOC entry (SOCE) has been implicated in many cell types including cortical astrocytes. However, the role of the SOCC family in the function of astrocytes has not been determined. Here, we thoroughly investigated the expression and the functional significance of SOCCs in spinal astrocytes. METHODS: Primary cultured astrocytes were prepared from neonatal (P2-P3) CD1 mice. Expressions of mRNAs and proteins were respectively assessed by real-time PCR and Western blot analysis. SOCE was measured using a calcium imaging system. Live-cell STIM1 translocation was detected using a confocal microscope. Cytokine levels were measured by the enzyme-linked immunosorbent assay. RESULTS: We found that the SOCC family is expressed in spinal astrocytes and that depletion of calcium stores from the endoplasmic reticulum by cyclopiazonic acid (CPA) resulted in a large sustained calcium entry, which was blocked by SOCC inhibitors. Using the siRNA knockdown approach, we identified STIM1 and Orai1 as primary components of SOCCs in spinal astrocytes. We also observed thapsigargin (TG)- or CPA-induced puncta formation of STIM1 and Orai1. In addition, activation of SOCCs remarkably promoted TNF-α and IL-6 production in spinal astrocytes, which were greatly attenuated by knockdown of STIM1 or Orai1. Importantly, knockdown of STIM2 and Orai1 dramatically decreased lipopolysaccharide-induced TNF-α and IL-6 production without changing cell viability. CONCLUSIONS: This study presents the first evidence that STIM1, STIM2, and Orai1 mediate SOCE and are involved in cytokine production in spinal astrocytes. Our findings provide the basis for future assessment of SOCCs in pain and other central nervous system disorders associated with abnormal astrocyte activities.

[Store-operated Calcium Entry into B Cells Regulates Autoimmune Inflammation].

Alterations in the cytosolic concentration of calcium ions (Ca(2+)) are important signals for various physiological events. The engagement of B cell receptors (BCR) results in the transient release of Ca(2+) into cytosol from endoplasmic reticulum (ER) stores. In turn, this decrease in ER luminal Ca(2+) concentration triggers the opening of Ca(2+) channels in the plasma membrane, inducing a sustained influx of extracellular Ca(2+) into cells. These processes are referred to as store-operated Ca(2+) entry (SOCE), which is an essential pathway for continuous Ca(2+) signaling. While the ER calcium sensor stromal interaction molecule (STIM) 1 and STIM2 are crucial components for SOCE activation, their physiological roles in B cells are unknown. Here we uncover the physiological function of SOCE in B cells by analyzing mice with B cell-specific deletions of STIM1 and STIM2. Our findings indicate that STIM1 and STIM2 are critical for BCR-induced SOCE, as well as the activation of nuclear factors of activated T cells (NFAT), and the subsequent production of interleukin-10 (IL-10). Although STIM proteins are not essential for B cell development and antibody responses, these molecules are required to suppress experimental autoimmune encephalomyelitis (EAE) via an IL-10-dependent mechanism. Accumulating evidence underscores the importance of IL-10-producing B cells in autoimmunity, although the identity of IL-10-producing B cells with a regulatory function in vivo remains unclear. We addressed this issue and identified plasmablasts as IL-10-producing B cells that can suppress EAE inflammation. Our data established STIM-dependent SOCE as a key signal for the regulatory plasmablasts required to limit autoimmunity.