Physiological Functions of CRAC Channels.

Store-operated Ca2+ entry (SOCE) is a ubiquitous Ca2+ signaling pathway that is evolutionarily conserved across eukaryotes. SOCE is triggered physiologically when the endoplasmic reticulum (ER) Ca2+ stores are emptied through activation of inositol 1,4,5-trisphosphate receptors. SOCE is mediated by the Ca2+ release-activated Ca2+ (CRAC) channels, which are highly Ca2+ selective. Upon store depletion, the ER Ca2+-sensing STIM proteins aggregate and gain extended conformations spanning the ER-plasma membrane junctional space to bind and activate Orai, the pore-forming proteins of hexameric CRAC channels. In recent years, studies on STIM and Orai tissue-specific knockout mice and gain- and loss-of-function mutations in humans have shed light on the physiological functions of SOCE in various tissues. Here, we describe recent findings on the composition of native CRAC channels and their physiological functions in immune, muscle, secretory, and neuronal systems to draw lessons from transgenic mice and human diseases caused by altered CRAC channel activity.

Janus 3D printed dynamic scaffolds for nanovibration-driven bone regeneration.

The application of physical stimuli to cell cultures has shown potential to modulate multiple cellular functions including migration, differentiation and survival. However, the relevance of these in vitro models to future potential extrapolation in vivo depends on whether stimuli can be applied "externally", without invasive procedures. Here, we report on the fabrication and exploitation of dynamic additive-manufactured Janus scaffolds that are activated on-command via external application of ultrasounds, resulting in a mechanical nanovibration that is transmitted to the surrounding cells. Janus scaffolds were spontaneously formed via phase-segregation of biodegradable polycaprolactone (PCL) and polylactide (PLA) blends during the manufacturing process and behave as ultrasound transducers (acoustic to mechanical) where the PLA and PCL phases represent the active and backing materials, respectively. Remote stimulation of Janus scaffolds led to enhanced cell proliferation, matrix deposition and osteogenic differentiation of seeded human bone marrow derived stromal cells (hBMSCs) via formation and activation of voltage-gated calcium ion channels.

Store-Operated Calcium Entry in Mouse Cardiomyocytes.

The fluorescent dye fura-2 AM was employed to record activation of Ca2+ entry in response to a decrease in Ca2+ concentration in the endoplasmic reticulum. Using whole-cell voltage clamp technique, we revealed Ca2+ currents with an amplitude of 0.46±0.13 pA/pF that passed through selective channels with current-voltage characteristics similar to those of classical store-operated CRAC channels. These currents were sensitive to 2-APB (50 µM), an inhibitor of store-operated channels. The data suggest that store-operated calcium entry is a characteristic feature of mature ventricular cardiomyocytes. Pathological alterations in store-operated Ca2+ entry can be implicated in the development of heart diseases.

Tissue resident and follicular Treg cell differentiation is regulated by CRAC channels.

T regulatory (Treg) cells maintain immunological tolerance and organ homeostasis. Activated Treg cells differentiate into effector Treg subsets that acquire tissue-specific functions. Ca2+ influx via Ca2+ release-activated Ca2+ (CRAC) channels formed by STIM and ORAI proteins is required for the thymic development of Treg cells, but its function in mature Treg cells remains unclear. Here we show that deletion of Stim1 and Stim2 genes in mature Treg cells abolishes Ca2+ signaling and prevents their differentiation into follicular Treg and tissue-resident Treg cells. Transcriptional profiling of STIM1/STIM2-deficient Treg cells reveals that Ca2+ signaling regulates transcription factors and signaling pathways that control the identity and effector differentiation of Treg cells. In the absence of STIM1/STIM2 in Treg cells, mice develop a broad spectrum of autoantibodies and fatal multiorgan inflammation. Our findings establish a critical role of CRAC channels in controlling lineage identity and effector functions of Treg cells.

Patch-Clamp Recording of the CRAC Channel Current in STIM-Orai Overexpressing Cells.

Induced by the depletion of ER calcium store, the calcium influx through calcium release-activated calcium (CRAC) channels is an ubiquitous way of Ca2+ influx for most cell types. This process is mediated by STIM protein, ER calcium sensor, and PM localized Orai calcium channels. Biophysical characterization of this STIM-Orai-mediated current, or ICRAC, with whole-cell patch-clamp technique is essential for revealing the molecular mechanisms underlying the process of STIM-Orai activation or modulation. Here we describe one commonly used procedure of monitoring CRAC activity with whole-cell patch-clamp technique.

Fluorescence-Based Ratiometric Measurement of CRAC Channel Activity in STIM-Orai-Overexpressing HEK-293 Cells.

Calcium influx through store-operated Ca2+ entry (SOCE), mediated by STIM-operated Orai channels, is crucial for many cellular functions. To dissect the molecular mechanisms underlying the process of STIM-Orai activation and identify regulators that modify this process, ratiometric imaging of SOCE responses in HEK cells overexpressing STIM and Orai is a routinely used method. Here we describe one commonly used procedure of monitoring SOCE activity with a ratiometric membrane-permeable dye fura-2-AM. Other ratiometric indicators suitable for SOCE measurements are also discussed.

Indirect Measurement of CRAC Channel Activity Using NFAT Nuclear Translocation by Flow Cytometry in Jurkat Cells.

Flow cytometry is a powerful technology to assess the presence of NFAT in the nuclei after CRAC channel activation. Here we described a simplified procedure for the analysis of CRAC channel activity using NFAT nuclear translocation by flow cytometry, based on the isolation of Jurkat E6-1 cell nuclei.

Engineered Cross-Linking to Study the Pore Architecture of the CRAC Channel.

ORAI1 constitutes the pore-forming subunit of the calcium release-activated calcium (CRAC) channel, a prototypical store-operated channel that is essential for the activation of cells of the immune system. Here we describe a convenient yet powerful cross-linking approach to examine the pore architecture of CRAC channels using ORAI1 proteins engineered to contain one or two cysteine residues. The generalizable cross-linking in situ approach can also be readily extended to study other integral membrane proteins expressed in various types of cells.

Spatial Ca2+ profiling: decrypting the universal cytosolic Ca2+ oscillation.

Stimulation of cell-surface receptors that couple to phospholipase C to generate the second messenger inositol trisphosphate often evokes repetitive oscillations in cytosolic Ca2+ . Signalling information is encoded in both the amplitude and frequency of the Ca2+ spikes. Recent studies have revealed that the spatial profile of the oscillation also imparts signalling power; Ca2+ microdomains near store-operated CRAC channels in the plasma membrane and inositol trisphosphate-gated channels in the endoplasmic reticulum both signal to distinct downstream targets. Spatial profiling therefore increases the transduction power of the universal oscillatory cytosolic Ca2+ signal.

CRAC channels are required for [Ca(2+)]i oscillations and c-fos gene expression after muscarinic acetylcholine receptor activation in leukemic T cells.

AIMS: T lymphocytes express muscarinic acetylcholine receptors (mAChRs) involved in regulating their proliferation, differentiation and cytokine release. Activation of M1, M3 or M5 mAChRs increases the intracellular Ca(2+) concentration ([Ca(2+)]i) through inositol-1,4,5-phosphate (IP3)-mediated Ca(2+) release from endoplasmic reticulum Ca(2+) stores. In addition, T lymphocytes express Ca(2+)-release activated Ca(2+) (CRAC) channels to induce Ca(2+) influx and to regulate diverse immune functions. Our aim in the present study was to assess the role of CRAC channels during mAChR activation in the Ca(2+)-dependent transduction that contributes to the regulation of T cell function. MAIN METHODS: Changes in [Ca(2+)]i following mAChR activation on human leukemic T cells, CCRF-CEM (CEM), were monitored using fura-2, based on the ratio of 510nm fluorescences elicited by excitation at 340nm and 380nm (R340/380). KEY FINDINGS: We demonstrate that CEM cells express mainly M3 and M5 mAChRs, but little the M1 subtype, and that oxotremorine-M (Oxo-M), an mAChR agonist, induces an initial transient increase in [Ca(2+)]i followed by repetitive [Ca(2+)]i oscillations. Removing extracellular Ca(2+) or pharmacological blockade of CRAC channels abolished the [Ca(2+)]i oscillations without affecting the initial [Ca(2+)]i transient induced by Oxo-M. Moreover, CRAC channel blockade also suppressed Oxo-M-induced c-fos and interleukin-2 expression. SIGNIFICANCE: These results suggest that upon M3 or M5 mAChR activation, IP3-mediated Ca(2+) release induces extracellular Ca(2+) influx through CRAC channels, which generates repetitive [Ca(2+)]i oscillations and, in turn, enhances c-fos gene expression in T lymphocytes.