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The lymphatic functions in maintaining lymph transport, and immune surveillance can be impaired by infections and inflammation, thereby causing debilitating disorders, such as lymphedema and inflammatory bowel disease. Histamine is a key inflammatory mediator known to trigger vasodilation and vessel hyperpermeability upon binding to its receptors and evoking intracellular Ca2+([Ca2+]i) dynamics for downstream signal transductions. However, the exact molecular mechanisms beneath the [Ca2+]idynamics and the downstream cellular effects have not been elucidated in the lymphatic system. Here, we show that Ca2+release-activated Ca2+(CRAC) channels, formed by Orai1 and stromal interaction molecule 1 (STIM1) proteins, are required for the histamine-elicited Ca2+signaling in human dermal lymphatic endothelial cells (HDLECs). Blockers or antagonists against CRAC channels, phospholipase C, and H1R receptors can all significantly diminish the histamine-evoked [Ca2+]idynamics in lymphatic endothelial cells (LECs), while short interfering RNA-mediated knockdown of endogenous Orai1 or STIM1 also abolished the Ca2+entry upon histamine stimulation in LECs. Furthermore, we find that histamine compromises the lymphatic endothelial barrier function by increasing the intercellular permeability and disrupting vascular endothelial-cadherin integrity, which is remarkably attenuated by CRAC channel blockers. Additionally, the upregulated expression of inflammatory cytokines, IL-6 and IL-8, after histamine stimulation was abolished by silencing Orai1 or STIM1 with RNAi in LECs. Taken together, our data demonstrated the essential role of CRAC channels in mediating the [Ca2+]isignaling and downstream endothelial barrier and inflammatory functions induced by histamine in the LECs, suggesting a promising potential to relieve histamine-triggered vascular leakage and inflammatory disorders in the lymphatics by targeting CRAC channel functions.

The lymphatic functions in maintaining lymph transport, and immune surveillance can be impaired by infections and inflammation, thereby causing debilitating disorders, such as lymphedema and inflammatory bowel disease. Histamine is a key inflammatory mediator known to trigger vasodilation and vessel hyperpermeability upon binding to its receptors and evoking intracellular Ca

Cardiac myosin binding protein C (cMyBP-C) is a phosphorylation-dependent force regulator and plays an important role in controlling myosin and actin dynamic interaction. Point-mutations of cMyBP-C that interfere with cMyBP-C threonine/serine phosphorylation resulted in hypertrophic cardiomyopathy and cardiac failure. However, it remains largely unknown how cMyBP-C tyrosine phosphorylation is regulated during cardiac hypertrophy and heart failure. Integrins are receptors of extracellular matrix and are the sensors of cardiac mechanical stretch. Focal adhesion kinase (FAK) plays an essential role in integrin-initiated signal transduction and regulates multiple cellular functions in various types of cells including cardiomyocytes. To identify the regulatory mechanism of cMyBP-C tyrosine phosphorylation during cardiac hypertrophy, we examined the effect of FAK on phosphorylation of cMyBP-C. Immunoprecipitation analysis showed that FAK and cMyBP-C are associated within the intact mouse heart. Results from our mutagenesis experiments demonstrated that the FAK kinase domain was required for FAK to associate with cMyBP-C. Our data also documented that the FAK Y397 site is required for FAK and cMyBP-C association. Importantly, overexpression dominant active Src Y527F with FAK significantly enhanced cMyBP-C phosphorylation. Interestingly, overexpression of cMyBP-C inhibited FAK phosphorylation. Taken together, cMyBP-C is one of effectors of Src/FAK complex in cardiomyocyte.

Store-operated calcium entry (SOCE) is critical for numerous calcium-related processes. The activation of SOCE requires engagement between stromal interaction molecule 1 (STIM1) molecules on the endoplasmic reticulum and Ca2+ release-activated Ca2+ channel (CRAC) Orai on the plasma membrane. However, the molecular details of their interactions remain elusive. Here, we analyzed STIM1-Orai interactions using synthetic peptides derived from the N- and C-termini of Orai channels (Orai-NT and Orai-CT, respectively) and purified fragments of STIM1. The binding of STIM1 to Orai-NT is hydrophilic-based, whereas binding to the Orai-CT is mostly hydrophobic. STIM1 decreases its affinity for Orai-CT when Orai-NT is present, supporting a stepwise interaction. Orai3-CT exhibits stronger binding to STIM1 than Orai1-CT, largely due to the shortness of one helical turn. The role of newly identified residues was confirmed by co-immunoprecipitation and calcium imaging using full-length molecules. Our results provide important insight into CRAC channel gating by STIM1.

Blood-brain barrier (BBB) breakdown and the associated microvascular hyperpermeability followed by brain edema are hallmark features of several brain pathologies, including traumatic brain injuries (TBI). Recent studies indicate that pro-inflammatory cytokine interleukin-1β (IL-1β) that is up-regulated following traumatic injuries also promotes BBB dysfunction and hyperpermeability, but the underlying mechanisms are not clearly known. The objective of this study was to determine the role of calpains in mediating BBB dysfunction and hyperpermeability and to test the effect of calpain inhibition on the BBB following traumatic insults to the brain. In these studies, rat brain microvascular endothelial cell monolayers exposed to calpain inhibitors (calpain inhibitor III and calpastatin) or transfected with calpain-1 siRNA demonstrated attenuation of IL-1β-induced monolayer hyperpermeability. Calpain inhibition led to protection against IL-1β-induced loss of zonula occludens-1 (ZO-1) at the tight junctions and alterations in F-actin cytoskeletal assembly. IL-1β treatment had no effect on ZO-1 gene (tjp1) or protein expression. Calpain inhibition via calpain inhibitor III and calpastatin decreased IL-1β-induced calpain activity significantly (p < 0.05). IL-1β had no detectable effect on intracellular calcium mobilization or endothelial cell viability. Furthermore, calpain inhibition preserved BBB integrity/permeability in a mouse controlled cortical impact model of TBI when studied using Evans blue assay and intravital microscopy. These studies demonstrate that calpain-1 acts as a mediator of IL-1β-induced loss of BBB integrity and permeability by altering tight junction integrity, promoting the displacement of ZO-1, and disorganization of cytoskeletal assembly. IL-1β-mediated alterations in permeability are neither due to the changes in ZO-1 expression nor cell viability. Calpain inhibition has beneficial effects against TBI-induced BBB hyperpermeability.

Store-operated Ca2+ entry (SOCE) mediated by STIM1 and Orai1 is crucial for Ca2+ signaling and homeostasis in most cell types. 2-Aminoethoxydiphenyl borate (2-APB) is a well-described SOCE inhibitor, but its mechanisms of action remain largely elusive. Here, we show that 2-APB does not affect the dimeric state of STIM1, but enhances the intramolecular coupling between the coiled-coil 1 (CC1) and STIM-Orai-activating region (SOAR) of STIM1, with subsequent reduction in the formation of STIM1 puncta in the absence of Orai1 overexpression. 2-APB also inhibits Orai1 channels, directly inhibiting Ca2+ entry through the constitutively active, STIM1-independent Orai1 mutants, Orai1-P245T and Orai1-V102A. When unbound from STIM1, the constitutively active Orai1-V102C mutant is not inhibited by 2-APB. Thus, we used Orai1-V012C as a tool to examine whether 2-APB can also inhibit the coupling between STIM1 and Orai1. We reveal that the functional coupling between STIM1 and Orai1-V102C is inhibited by 2-APB. This inhibition on coupling is indirect, arising from 2-APB's action on STIM1, and it is most likely mediated by functional channel residues in the Orai1 N-terminus. Overall, our findings on this two-site inhibition mediated by 2-APB provide new understanding on Orai1-activation by STIM1, important to future drug design.

Specialized junctional sites that connect the plasma membrane (PM) and endoplasmic reticulum (ER) play critical roles in controlling lipid metabolism and Ca2+ signaling1–4. Store operated Ca2+ entry mediated by dynamic STIM1-ORAI1 coupling represents a classical molecular event occurring at ER-PM junctions, but the protein composition and how previously-unrecognized protein regulators facilitate this process remain ill-defined. Using a combination of spatially-restricted biotin-labelling in situ coupled with mass spectrometry5, 6 and a secondary screen based on bimolecular fluorescence complementation7, we mapped the proteome of intact ER-PM junctions in living cells without disrupting their architectural integrity. Our approaches lead to the discovery of an ER-resident multi-transmembrane protein that we call STIMATE (STIM-activating enhancer, encoded by TMEM110) as a positive regulator of Ca2+ influx in vertebrates. STIMATE physically interacts with STIM1 to promote STIM1 conformational switch. Genetic depletion of STIMATE substantially reduces STIM1 puncta formation at ER-PM junctions and suppresses the Ca2+-NFAT signaling. Our findings enable further genetic studies to elucidate the function of STIMATE in normal physiology and disease, and set the stage to uncover more uncharted functions of hitherto underexplored ER-PM junctions.

The shear stress applied to lymphatic endothelial cells (LEC) by lymph flow changes dramatically under normal conditions as well as in response to disease conditions and immune reactions. In general, LEC are known to regulate the contraction frequency and strength of lymphatic pumping in response to shear stress. Intracellular calcium concentration ([Ca2+]i) is an important factor that regulates lymphatic contraction characteristics. In this study, we measured changes in the [Ca2+]iunder different shear stress levels and determined the source of this calcium signal. Briefly, human dermal LEC were cultured in custom-made microchannels for 3 days before loading with 2 µM fura-2 AM, a ratiometric calcium dye to measure [Ca2+]i. Step changes in shear stress resulted in a rapid increase in [Ca2+]ifollowed by a gradual return to the basal level and sometimes below the initial baseline (45.2 ± 2.2 nM). The [Ca2+]ireached a peak at 126.2 ± 5.6 nM for 10 dyn/cm2stimulus, whereas the peak was only 71.8 ± 5.4 nM for 1 dyn/cm2stimulus, indicating that the calcium signal depends on the magnitude of shear stress. Removal of the extracellular calcium from the buffer or pharmocological blockade of calcium release-activated calcium (CRAC) channels significantly reduced the peak [Ca2+]i, demonstrating a role of extracellular calcium entry. Inhibition of endoplasmic reticulum (ER) calcium pumps showed the importance of intracellular calcium stores in the initiation of this signal. In conclusion, we demonstrated that the shear-mediated calcium signal is dependent on the magnitude of the shear and involves ER store calcium release and extracellular calcium entry.

Background Cdc42 is a member of the Rho GTPase family and functions as a molecular switch in regulating cytoskeleton remodeling and cell polarity establishment. Inactivating Cdc42 in cardiomyocytes resulted in embryonic lethality with heart developmental defects, including ventricular septum defects and thin ventricle wall syndrome. Findings In this study, we have generated a Cdc42 cardiomyocyte knockout mouse line by crossing Cdc42/flox mice with myosin light chain 2a (MLC2a)-Cre mice. We found that the deletion of Cdc42 in embryonic cardiomyocytes resulted in an underdeveloped right ventricle. Microarray analysis and real-time PCR data analysis displayed that the deletion of Cdc42 decreased dHand expression level. In addition, we found evaginations in the ventricle walls of Cdc42 knockout hearts. Conclusion We concluded that Cdc42 plays an essential role in right ventricle growth.

Homotypic membrane fusion of the endoplasmic reticulum (ER) is mediated by a class of dynamin-like GTPases known as atlastin (ATL). Depletion of or mutations in ATL cause an unbranched ER morphology and hereditary spastic paraplegia (HSP), a neurodegenerative disease characterized by axon shortening in corticospinal motor neurons and progressive spasticity of the lower limbs. How ER shaping is linked to neuronal defects is poorly understood. Here, we show that dominant-negative mutants of ATL1 in PC-12 cells inhibit nerve growth factor (NGF)-induced neurite outgrowth. Overexpression of wild-type or mutant ATL1 or depletion of ATLs alters ER morphology and affects store-operated calcium entry (SOCE) by decreasing STIM1 puncta formation near the plasma membrane upon calcium depletion of the ER. In addition, blockage of the STIM1-Orai pathway effectively abolishes neurite outgrowth of PC-12 cells stimulated by NGF. These results suggest that SOCE plays an important role in neuronal regeneration, and mutations in ATL1 may cause HSP, partly by undermining SOCE.

Neural crest cells (NCCs) are physically responsible for craniofacial skeleton formation, pharyngeal arch artery remodeling and cardiac outflow tract septation during vertebrate development. Cdc42 (cell division cycle 42) is a Rho family small GTP-binding protein that works as a molecular switch to regulate cytoskeleton remodeling and the establishment of cell polarity. To investigate the role of Cdc42 in NCCs during embryonic development, we deleted Cdc42 in NCCs by crossing Cdc42 flox mice with Wnt1-cre mice. We found that the inactivation of Cdc42 in NCCs caused embryonic lethality with craniofacial deformities and cardiovascular developmental defects. Specifically, Cdc42 NCC knockout embryos showed fully penetrant cleft lips and short snouts. Alcian Blue and Alizarin Red staining of the cranium exhibited an unfused nasal capsule and palatine in the mutant embryos. India ink intracardiac injection analysis displayed a spectrum of cardiovascular developmental defects, including persistent truncus arteriosus, hypomorphic pulmonary arteries, interrupted aortic arches, and right-sided aortic arches. To explore the underlying mechanisms of Cdc42 in the formation of the great blood vessels, we generated Wnt1Cre-Cdc42-Rosa26 reporter mice. By beta-galactosidase staining, a subpopulation of Cdc42-null NCCs was observed halting in their migration midway from the pharyngeal arches to the conotruncal cushions. Phalloidin staining revealed dispersed, shorter and disoriented stress fibers in Cdc42-null NCCs. Finally, we demonstrated that the inactivation of Cdc42 in NCCs impaired bone morphogenetic protein 2 (BMP2)-induced NCC cytoskeleton remodeling and migration. In summary, our results demonstrate that Cdc42 plays an essential role in NCC migration, and inactivation of Cdc42 in NCCs impairs craniofacial and cardiovascular development in mice.

In T lymphocytes, Ca2+ release–activated Ca2+ (CRAC) channels composed of Orai1 subunits trigger Ag-induced gene expression and cell proliferation through the NFAT pathway. We evaluated the requirement of CRAC channel function for lymphocyte homing using expression of a dominant-negative Orai1-E106A mutant to suppress Ca2+ signaling. To investigate homing and motility of human lymphocytes in immunocompromised mouse hosts, we transferred human lymphocytes either acutely or after stable engraftment after a second transfer from the same blood donor. Human and mouse lymphocyte homing was assessed, and cells were tracked within lymph nodes (LNs) by two-photon microscopy. Our results demonstrate that human T and B lymphocytes home into and migrate within the LNs of immunocompromised NOD.SCID mice similar to murine lymphocytes. Human T and B cells colocalized in atrophied or reconstituted mouse LNs, where T cells migrated in a random walk at velocities of 9–13 μm/min and B cells at 6 μm/min. Expression of Orai1-E106A inhibited CRAC channel function in human and mouse T cells, and prevented homing from high endothelial venules into murine LNs. Ca2+ signals induced by CCL21 were also inhibited in T cells expressing Orai1-E106A. With CRAC channels inhibited, the high-affinity form of LFA-1 failed to become active, and T cells failed to migrate across endothelial cells in a transwell model. These results establish a requirement for CRAC channel–mediated Ca2+ influx for T cell homing to LNs mediated by high-affinity integrin activation and chemokine-induced transendothelial migration.

Cdc42 is a member of the Rho GTPase family and functions as a molecular switch in regulating cell migration, proliferation, differentiation and survival. However, the role of Cdc42 in heart development remains largely unknown. To determine the function of Cdc42 in heart formation, we have generated a Cdc42 cardiomyocyte knockout (CCKO) mouse line by crossing Cdc42 flox mice with myosin light chain (MLC) 2a-Cre mice. The inactivation of Cdc42 in embryonic cardiomyocytes induced lethality after embryonic day 12.5. Histological analysis of CCKO embryos showed cardiac developmental defects that included thin ventricular walls and ventricular septum defects. Microarray and real-time PCR data also revealed that the expression level of p21 was significantly increased and cyclin B1 was dramatically decreased, suggesting that Cdc42 is required for cardiomyocyte proliferation. Phosphorylated Histone H3 staining confirmed that the inactivation of Cdc42 inhibited cardiomyocytes proliferation. In addition, transmission electron microscope studies showed disorganized sarcomere structure and disruption of cell-cell contact among cardiomyocytes in CCKO hearts. Accordingly, we found that the distribution of N-cadherin/β-Catenin in CCKO cardiomyocytes was impaired. Taken together, our data indicate that Cdc42 is essential for cardiomyocyte proliferation, sarcomere organization and cell-cell adhesion during heart development.

Stim and Orai proteins comprise the molecular machinery of Ca 2+ release-activated Ca 2+ (CRAC) channels. As an approach toward understanding the gating of Orai1 channels, we investigated effects of selected mutations at two conserved sites in the first transmembrane segment (TM1): arginine 91 located near the cytosolic end of TM1 and glycine 98 near the middle of TM1. Orai1 R91C, when coexpressed with STIM1, was activated normally by Ca 2+ -store depletion. Treatment with diamide, a thiol-oxidizing agent, induced formation of disulfide bonds between R91C residues in adjacent Orai1 subunits and rapidly blocked STIM1-operated Ca 2+ current. Diamide-induced blocking was reversed by disulfide bond-reducing agents. These results indicate that R91 forms a very narrow part of the conducting pore at the cytosolic side. Alanine replacement at G98 prevented STIM1-induced channel activity. Interestingly, mutation to aspartate (G98D) or proline (G98P) caused constitutive channel activation in a STIM1-independent manner. Both Orai1 G98 mutants formed a nonselective Ca 2+ -permeable conductance that was relatively resistant to block by Gd 3+ . The double mutant R91W/G98D was also constitutively active, overcoming the normal inhibition of channel activity by tryptophan at the 91 position found in some patients with severe combined immunodeficiency (SCID), and the double mutant R91C/G98D was resistant to diamide block. These data suggest that the channel pore is widened and ion selectivity is altered by mutations at the G98 site that may perturb α-helical structure. We propose distinct functional roles for G98 as a gating hinge and R91 as part of the physical gate at the narrow inner mouth of the channel.

Marginal zone (MZ) B cells of the spleen and B1 cells, termed innate-like B cells, differ from follicular B cells by their attenuated Ca2+ mobilization, fast antibody secretion, and increased cell adhesion. We identified and characterized Mzb1 as an endoplasmic reticulum-localized and B cell-specific protein that was most abundantly expressed in MZ B and B1 cells. Knockdown of Mzb1 in MZ B cells increased Ca2+ mobilization and nuclear NFAT transcription factor localization, but reduced lipopolysaccharide-induced antibody secretion and integrin-mediated cell adhesion. Conversely, ectopic expression of an Lck-Mzb1 transgene in peripheral T cells resulted in attenuated Ca2+ mobilization and augmented integrin-mediated cell adhesion. In addition to its interaction with the substrate-specific chaperone Grp94, Mzb1 augmented the function of the oxidoreductase ERp57 in favoring the expression of integrins in their activated conformation. Thus, Mzb1 helps to diversify peripheral B cell functions by regulating Ca2+ stores, antibody secretion, and integrin activation.

Cdc42 is a member of the Rho GTPase family and functions as a molecular switch in regulating cell migration, proliferation, differentiation and survival. However, the role of Cdc42 in heart development remains largely unknown. To determine the function of Cdc42 in heart formation, we have generated a Cdc42 cardiomyocyte knockout (CCKO) mouse line by crossing Cdc42 flox mice with myosin light chain (MLC) 2a-Cre mice. The inactivation of Cdc42 in embryonic cardiomyocytes induced lethality after embryonic day 12.5. Histological analysis of CCKO embryos showed cardiac developmental defects that included thin ventricular walls and ventricular septum defects. Microarray and real-time PCR data also revealed that the expression level of p21 was significantly increased and cyclin B1 was dramatically decreased, suggesting that Cdc42 is required for cardiomyocyte proliferation. Phosphorylated Histone H3 staining confirmed that the inactivation of Cdc42 inhibited cardiomyocytes proliferation. In addition, transmission electron microscope studies showed disorganized sarcomere structure and disruption of cell-cell contact among cardiomyocytes in CCKO hearts. Accordingly, we found that the distribution of N-cadherin/β-Catenin in CCKO cardiomyocytes was impaired. Taken together, our data indicate that Cdc42 is essential for cardiomyocyte proliferation, sarcomere organization and cell-cell adhesion during heart development.

Store-operated Ca2+ entry mediated by STIM1 and ORAI1 constitutes one of the major Ca2+ entry routes in mammalian cells. The molecular choreography of STIM1–ORAI1 coupling is initiated by endoplasmic reticulum (ER) Ca2+ store depletion with subsequent oligomerization of the STIM1 ER-luminal domain, followed by its redistribution towards the plasma membrane to gate ORAI1 channels. The mechanistic underpinnings of this inside-out Ca2+ signalling were largely undefined. By taking advantage of a unique gain-of-function mutation within the STIM1 transmembrane domain (STIM1-TM), here we show that local rearrangement, rather than alteration in the oligomeric state of STIM1-TM, prompts conformational changes in the cytosolic juxtamembrane coiled-coil region. Importantly, we further identify critical residues within the cytoplasmic domain of STIM1 (STIM1-CT) that entail autoinhibition. On the basis of these findings, we propose a model in which STIM1-TM reorganization switches STIM1-CT into an extended conformation, thereby projecting the ORAI-activating domain to gate ORAI1 channels., Depletion of calcium from intracellular stores induces interaction between the endoplasmic reticulum STIM1 protein and the plasma membrane ORAI1 channel that facilitates cellular calcium entry. Here Ma et al. characterize a STIM1 gain-of-function mutant and propose a conformational switch that controls ORAI1 gating.

Recent RNA interference screens have identified several proteins that are essential for store-operated Ca2+ influx and Ca2+ release-activated Ca2+ (CRAC) channel activity in Drosophila and in mammals, including the transmembrane proteins Stim (stromal interaction molecule)1,2 and Orai3–5. Stim probably functions as a sensor of luminal Ca2+ content and triggers activation of CRAC channels in the surface membrane after Ca2+ store depletion1,6. Among three human homologues of Orai (also known as olf186-F), ORAI1 on chromosome 12 was found to be mutated in patients with severe combined immunodeficiency disease, and expression of wild-type Orai1 restored Ca2+ influx and CRAC channel activity in patient T cells3. The overexpression of Stim and Orai together markedly increases CRAC current5,7–9. However, it is not yet clear whether Stim or Orai actually forms the CRAC channel, or whether their expression simply limits CRAC channel activity mediated by a different channel-forming subunit. Here we show that interaction between wild-type Stim and Orai, assessed by co-immunoprecipitation, is greatly enhanced after treatment with thapsigargin to induce Ca2+ store depletion. By site-directed mutagenesis, we show that a point mutation from glutamate to aspartate at position 180 in the conserved S1–S2 loop of Orai transforms the ion selectivity properties of CRAC current from being Ca2+-selective with inward rectification to being selective for monovalent cations and outwardly rectifying. A charge-neutralizing mutation at the same position (glutamate to alanine) acts as a dominant-negative non-conducting subunit. Other charge-neutralizing mutants in the same loop express large inwardly rectifying CRAC current, and two of these exhibit reduced sensitivity to the channel blocker Gd3+. These results indicate that Orai itself forms the Ca2+-selectivity filter of the CRAC channel.

Recent studies by our group and others demonstrated a required and conserved role of Stim in store-operated Ca 2+ influx and Ca 2+ release-activated Ca 2+ (CRAC) channel activity. By using an unbiased genome-wide RNA interference screen in Drosophila S2 cells, we now identify 75 hits that strongly inhibited Ca 2+ influx upon store emptying by thapsigargin. Among these hits are 11 predicted transmembrane proteins, including Stim , and one, olf186-F , that upon RNA interference-mediated knockdown exhibited a profound reduction of thapsigargin-evoked Ca 2+ entry and CRAC current, and upon overexpression a 3-fold augmentation of CRAC current. CRAC currents were further increased to 8-fold higher than control and developed more rapidly when olf186-F was cotransfected with Stim . olf186-F is a member of a highly conserved family of four-transmembrane spanning proteins with homologs from Caenorhabditis elegans to human. The endoplasmic reticulum (ER) Ca 2+ pump sarco-/ER calcium ATPase (SERCA) and the single transmembrane-soluble N -ethylmaleimide-sensitive (NSF) attachment receptor (SNARE) protein Syntaxin5 also were required for CRAC channel activity, consistent with a signaling pathway in which Stim senses Ca 2+ depletion within the ER, translocates to the plasma membrane, and interacts with olf186-F to trigger CRAC channel activity.

As the sole Ca2+ entry mechanism in a variety of non-excitable cells, store-operated calcium (SOC) influx is important in Ca2+ signalling and many other cellular processes1,2,3. A calcium-release-activated calcium (CRAC) channel in T lymphocytes is the best-characterized SOC influx channel4,5,6 and is essential to the immune response, sustained activity of CRAC channels being required for gene expression and proliferation7,8,9,10. The molecular identity and the gating mechanism of SOC and CRAC channels have remained elusive. Previously we identified Stim and the mammalian homologue STIM1 as essential components of CRAC channel activation in Drosophila S2 cells and human T lymphocytes11. Here we show that the expression of EF-hand mutants of Stim or STIM1 activates CRAC channels constitutively without changing Ca2+ store content. By immunofluorescence, EM localization and surface biotinylation we show that STIM1 migrates from endoplasmic-reticulum-like sites to the plasma membrane upon depletion of the Ca2+ store. We propose that STIM1 functions as the missing link between Ca2+ store depletion and SOC influx, serving as a Ca2+ sensor that translocates upon store depletion to the plasma membrane to activate CRAC channels.

Serotonin (5HT) is a pivotal signaling molecule that modulates behavioral and endocrine responses to diverse chemical and physical stimuli. We report cell-specific regulation of 5HT biosynthesis by transient receptor potential V(TRPV) ion channels in C. elegans. Mutations in the TRPV genes osm-9 or ocr-2 dramatically downregulate the expression of the gene encoding the 5HT synthesis enzyme tryptophan hydroxylase(tph-1) in the serotonergic chemosensory neurons ADF, but neither the mutation nor the double mutation of both channel genes affects other types of serotonergic neurons. The TRPV genes are expressed in the ADF neurons but not in other serotonergic neurons, and act cell-autonomously to regulate a neuron-specific transcription program. Whereas in olfactory neurons OSM-9 and OCR-2 function is dependent on ODR-3 Gα, the activity of ODR-3 or two other Gα proteins expressed in the ADF neurons is not required for upregulating tph-1 expression, thus the TRPV ion channels in different neurons may be regulated by different mechanisms. A gain-of-function mutation in CaMKII UNC-43 partially suppresses the downregulation of tph-1 in the TRPV mutants, thus CaMKII may be an effector of the TRPV signaling. Mutations in the TRPV genes cause worms developmentally arrest at the Dauer stage. This developmental defect is due in part to reduced 5HT inputs into daf-2/insulin neuroendocrine signaling.

Histamine is an important immunomodulator involved in allergic reactions and inflammatory responses. In endothelial cells, histamine induces Ca(2+) mobilization by releasing Ca(2+) from the endoplasmic reticulum and eliciting Ca(2+) entry across the plasma membrane. Herein, we show that histamine-evoked Ca(2+) entry in human umbilical vein endothelial cells (HUVECs) is sensitive to blockers of Ca(2+) release-activated Ca(2+) (CRAC) channels. RNA interference against STIM1 or Orai1, the activating subunit and the pore-forming subunit of CRAC channels, respectively, abolishes this histamine-evoked Ca(2+) entry. Furthermore, overexpression of dominant-negative CRAC channel subunits inhibits while co-expression of both STIM1 and Orai1 enhances histamine-induced Ca(2+) influx. Interestingly, gene silencing of STIM1 or Orai1 also interrupts the activation of calcineurin/nuclear factor of activated T-cells (NFAT) pathway and the production of interleukin 8 triggered by histamine in HUVECs. Collectively, these results suggest a central role of STIM1 and Orai1 in mediating Ca(2+) mobilization linked to inflammatory signaling of endothelial cells upon histamine stimulation.

Cdc42 is a Ras-related GTPase that plays an important role in the regulation of a range of cellular functions, including cell migration, proliferation, and survival. Consistent with its critical functions in vitro, the inactivation of Cdc42 in mice has been shown to result in embryonic lethality at embryonic day 6.5 (E6.5) before blood vessel formation. To determine the role of Cdc42 in new blood vessel formation, we have generated vascular endothelial cell (EC)-specific Cdc42 knockout mice by crossing Cdc42(flox/flox) mice with Tie2-Cre mice. The deletion of Cdc42 in ECs caused embryonic lethality with vasculogenesis and angiogenesis defects. We observed that Cdc42 is critical for EC migration and survival but not for cell cycle progression. Moreover, we found that the inactivation of Cdc42 in ECs decreased the level of vascular endothelial growth factor receptor 2 (VEGFR2) protein on the EC surface and promoted the production of a 75-kDa membrane-associated C-terminal VEGFR2 fragment. Using cultured primary mouse ECs and human umbilical vein ECs, we have demonstrated that the deletion of Cdc42 increased ADAM17-mediated VEGFR2 shedding. Notably, inhibition of ADAM17 or overexpression of VEGFR2 can partially reverse Cdc42 deletion-induced EC apoptosis. These data indicate that Cdc42 is essential for VEGFR2-mediated signal transduction in blood vessel formation.

The entry of extracellular Ca(2+), which is mediated by Ca(2+) release-activated Ca(2+) (CRAC) channels, is essential for T cell activation and the normal functioning of other immune cells. Although the molecular components of CRAC channels, the Orai1 pore-forming subunit and the STIM1-activating subunit have been recently identified, the gating mechanism by which Orai1 channels conduct Ca(2+) entry upon Orai1-STIM1 interaction following Ca(2+) store release remains elusive. Herein, we show that C-terminal truncations or point mutations prevented Orai1 from binding to STIM1 and subsequent channel opening. In contrast, an Orai1 mutant with an N-terminal truncation interacted with but failed to be activated by STIM1. Moreover, Orai1 channels with C-terminal disruption, but not N-terminal truncation, could be gated by fused functional domains of STIM1. Interestingly, the channel activities of Orai1 mutants carrying either an N-terminal or a C-terminal truncation were restored by a methionine mutation at the putative gating hinge, the conserved Gly-98 site in the first transmembrane segment (TM1) of Orai1. Collectively, these results support a stepwise gating mechanism of STIM1-operated Orai1 channels; the initial binding between STIM1 and the C terminus of Orai1 docks STIM1 onto the N terminus of Orai1 to initiate conformational changes of the pore-lining TM1 helix of Orai1, leading to the opening of the channel.

Specialized junctional sites that connect the plasma membrane (PM) and endoplasmic reticulum (ER) play critical roles in controlling lipid metabolism and calcium signaling. Store-operated calcium entry mediated by dynamic STIM1-ORAI1 coupling constitutes a classical molecular event occurring at ER-PM junctions, but the protein composition and how previously-unrecognized protein regulators facilitate calcium influx at this particular subcellular compartment remain poorly defined. By taking advantage of a spatially-restricted biotin-labeling technique coupled with mass spectrometry, we systematically mapped the proteome of intact ER-PM junctions in living cells while maintaining their architectural integrity. Using a secondary screen based on bimolecular fluorescence complementation, we further discovered a novel multi-transmembrane protein that we call STIMATE (STIM-activating enhancer, encoded by TMEM110) as a positive regulator of calcium influx in vertebrates. STIMATE colocalizes with STIM1 to the ER and promotes STIM1 conformational switch through physical contact with the STIM1 juxtamembrane coiled coil domain. This represents a novel regulatory mechanism to modulate STIM1-dependent calcium flux in mammalian cells. Genetic depletion of STIMATE substantially reduces STIM1 puncta formation at ER-PM junctions and suppresses the calcium-NFAT signaling. To noninvasively manipulate membrane contact sites, we also created a novel tool to reversibly control the formation of ER-PM junctions by harnessing the power of light. This optogenetic tool makes it possible for future screening of protein/chemical regulators that are involved in maintaining ER-PM junctions. Our findings not only afford an initial view on the protein composition of intact ER-PM junctions under physiological conditions, but also provide a solid framework to uncover more uncharted functions of hitherto underexplored ER-PM junctions. Increasing our comprehension of knowledge on this elusive and intriguing subcellular structure will lead to better design of therapeutic strategies to treat human disorders associated with aberrant lipid metabolism and calcium signaling.

Rationale: The endoplasmic reticulum (ER) is a major intracellular Ca 2+ store in endothelial cells (ECs). The Ca 2+ concentration in the ER greatly contributes to the generation of Ca 2+ signals that regulate endothelial functions. Many proteins, including stromal interaction molecule 1/2 (STIM1/2), Orai1/2/3, and sarcoplasmic/endoplasmic reticulum Ca 2+ -ATPase 3 (SERCA3), are involved in the ER Ca 2+ refilling after store depletion in ECs. Objective: This study is designed to examine the role of Ca 2+ in the ER in coronary endothelial dysfunction in diabetes. Methods and Results: Mouse coronary ECs (MCECs) isolated from diabetic mice exhibited (1) a significant decrease in the Ca 2+ mobilization from the ER when the cells were treated by SERCA inhibitor, and (2) significant downregulation of STIM1 and SERCA3 protein expression in comparison to the controls. Overexpression of STIM1 restored (1) the increase in cytosolic Ca 2+ concentration due to Ca 2+ leak from the ER in diabetic MCECs, (2) the Ca 2+ concentration in the ER, and (3) endothelium-dependent relaxation that was attenuated in diabetic coronary arteries. Conclusions: Impaired ER Ca 2+ refilling in diabetic MCECs, due to the decrease in STIM1 protein expression, attenuates endothelium-dependent relaxation in diabetic coronary arteries, while STIM1 overexpression has a beneficial and therapeutic effect on coronary endothelial dysfunction in diabetes.

Sustained calcium entry through Calcium Release-Activated Calcium (CRAC) channels is essential for T cell activation and proliferation via sensing the depletion of endoplasmic reticulum (ER) calcium-store by STIM1 subunits and the opening of pore-forming Orai1 subunits. However, the molecular mechanism underlying this process remains elusive. Using gain-of-function human Orai1 point mutants with Aspartate and Proline substitutions on a conserved Glycine residue (98) in the middle of transmembrane segment (TM) 1, we have previously suggested that the putative conformational change at this G98 site (gating-hinge) is a key step toward channel opening. Herein, we found in the mutation studies that the elongated side-chains at this 98-site could constitutively gate the corresponding channels through a conserved “bent-hinge” mechanism without affecting permeation pathway. We further demonstrated in Orai1-G98X mutants that the spontaneous opening of Orai1 channels is independent of STIM1 and store content. A truncated Orai1-G98X mutant without both intracellular N- and C- termini exhibited similar store-independent calcium entry as full-length Orai1-G98X mutants, indicating that Orai1 TM region is fully capable of and responsible for pore-forming and channel assembling. Moreover, truncated Orai3 proteins lacking both N- and C- termini remained responsive to 2-aminoethyl diphenylborinate stimulation, but not to store-depletion, indicating the structural requirement for channel assembling and pore formation. These results provide molecular details of the assembling and gating mechanisms of CRAC channels and support the Glycine “gating-hinge” hypothesis.

Publisher Summary Store-operated Ca2+ (SOC) channels in the plasma membrane (PM) open when Ca2+ is depleted within the lumen of the endoplasmic reticulum (ER). SOC channels can be highly selective Ca2+ channels that are, under physiological conditions, impermeable to monovalent cations such as sodium or potassium; or Ca2+-permeable but relatively non-selective cation channels. The former are known as Ca2+-release-activated Ca2+ (CRAC) channels, which mediate sustained Ca2+ signaling in T cells in response to antigen presentation. This chapter summarizes biophysical properties of the CRAC channel in relation to the molecular physiology of STIM and Orai coupling. It begins by describing the biophysical properties of the CRAC channel. Ion channel functional properties are generally characterized in terms of gating (modes of opening and closing), ion selectivity, single-channel conductance, modulation, and pharmacology. Under this, it considers STIM as the ER Ca2+ sensor and STIM as the messenger to the plasma membrane. Furthermore, it discusses how Orai forms the Ca2+-selective pore of the CRAC channel. Finally, it takes up the phenomenon of STIM-induced activation of Orai channels.

Background— Excessive proliferation of pulmonary artery smooth muscle cells (PASMCs) plays an important role in the development of idiopathic pulmonary arterial hypertension (IPAH), whereas a rise in cytosolic Ca 2+ concentration triggers PASMC contraction and stimulates PASMC proliferation. Recently, we demonstrated that upregulation of the TRPC6 channel contributes to proliferation of PASMCs isolated from IPAH patients. This study sought to identify single-nucleotide polymorphisms (SNPs) in the TRPC6 gene promoter that are associated with IPAH and have functional significance in regulating TRPC6 activity in PASMCs. Methods and Results— Genomic DNA was isolated from blood samples of 237 normal subjects and 268 IPAH patients. Three biallelic SNPs, −361 (A/T), −254(C/G), and −218 (C/T), were identified in the 2000-bp sequence upstream of the transcriptional start site of TRPC6. Although the allele frequencies of the −361 and −218 SNPs were not different between the groups, the allele frequency of the −254(C→G) SNP in IPAH patients (12%) was significantly higher than in normal subjects (6%; P 2+ influx in PASMCs of IPAH patients harboring the −254G allele. Conclusions— These results suggest that the −254(C→G) SNP may predispose individuals to an increased risk of IPAH by linking abnormal TRPC6 transcription to nuclear factor-κB, an inflammatory transcription factor.

Ca2+-release-activated Ca2+(CRAC) channels underlie sustained Ca2+signalling in lymphocytes and numerous other cells after Ca2+liberation from the endoplasmic reticulum (ER). RNA interference screening approaches identified two proteins, Stim and Orai, that together form the molecular basis for CRAC channel activity. Stim senses depletion of the ER Ca2+store and physically relays this information by translocating from the ER to junctions adjacent to the plasma membrane, and Orai embodies the pore of the plasma membrane calcium channel. A close interaction between Stim and Orai, identified by co-immunoprecipitation and by Förster resonance energy transfer, is involved in the opening of the Ca2+channel formed by Orai subunits. Most ion channels are multimers of pore-forming subunits surrounding a central channel, which are preassembled in the ER and transported in their final stoichiometry to the plasma membrane. Here we show, by biochemical analysis after cross-linking in cell lysates and intact cells and by using non-denaturing gel electrophoresis without cross-linking, that Orai is predominantly a dimer in the plasma membrane under resting conditions. Moreover, single-molecule imaging of green fluorescent protein (GFP)-tagged Orai expressed in Xenopus oocytes showed predominantly two-step photobleaching, again consistent with a dimeric basal state. In contrast, co-expression of GFP-tagged Orai with the carboxy terminus of Stim as a cytosolic protein to activate the Orai channel without inducing Ca2+store depletion or clustering of Orai into punctae yielded mostly four-step photobleaching, consistent with a tetrameric stoichiometry of the active Orai channel. Interaction with the C terminus of Stim thus induces Orai dimers to dimerize, forming tetramers that constitute the Ca2+- selective pore. This represents a new mechanism in which assembly and activation of the functional ion channel are mediated by the same triggering molecule. ©2008 Macmillan Publishers Limited. All rights reserved.

We evaluated currents induced by expression of human homologs of Orai together with STIM1 in human embryonic kidney cells. When co-expressed with STIM1, Orai1 induced a large inwardly rectifying Ca2+-selective current with Ca2+-induced slow inactivation. A point mutation of Orai1 (E106D) altered the ion selectivity of the induced Ca2+ release-activated Ca2+ (CRAC)-like current while retaining an inwardly rectifying I-V characteristic. Expression of the C-terminal portion of STIM1 with Orai1 was sufficient to generate CRAC current without store depletion. 2-APB activated a large relatively nonselective current in STIM1 and Orai3 co-expressing cells. 2-APB also induced Ca2+ influx in Orai3-expressing cells without store depletion or co-expression of STIM1. The Orai3 current induced by 2-APB exhibited outward rectification and an inward component representing a mixed calcium and monovalent current. A pore mutant of Orai3 inhibited store-operated Ca2+ entry and did not carry significant current in response to either store depletion or addition of 2-APB. Analysis of a series of Orai1-3 chimeras revealed the structural determinant responsible for 2-APB-induced current within the sequence from the second to third transmembrane segment of Orai3. The Orai3 current induced by 2-APB may reflect a store-independent mode of CRAC channel activation that opens a relatively nonselective cation pore.

For efficient development of an immune response, T lymphocytes require long-lasting calcium influx through calcium release-activated calcium (CRAC) channels and the formation of a stable immunological synapse (IS) with the antigen-presenting cell (APC). Recent RNAi screens have identified Stim and Orai inDrosophilacells, and their corresponding mammalian homologs STIM1 and Orai1 in T cells, as essential for CRAC channel activation. Here, we show that STIM1 and Orai1 are recruited to the immunological synapse between primary human T cells and autologous dendritic cells. Both STIM1 and Orai1 accumulated in the area of contact between either resting or super-antigen (SEB)-pretreated T cells and SEB-pulsed dendritic cells, where they were colocalized with T cell receptor (TCR) and costimulatory molecules. In addition, imaging of intracellular calcium signaling in T cells loaded with EGTA revealed significantly higher Ca2+concentration near the interface, indicating Ca2+influx localized at the T cell/dendritic cell contact area. Expression of a dominant-negative Orai1 mutant blocked T cell Ca2+signaling but did not interfere with the initial accumulation of STIM1, Orai1, and CD3 in the contact zone. In activated T cell blasts, mRNA expression for endogenous STIM1 and all three human homologs of Orai was up-regulated, accompanied by a marked increase in Ca2+influx through CRAC channels. These results imply a positive feedback loop in which an initial TCR signal favors up-regulation of STIM1 and Orai proteins that would augment Ca2+signaling during subsequent antigen encounter.

Plasmid DNA purification from E. coli is a core technique for molecular cloning. Small scale purification (miniprep) from less than 5 ml of bacterial culture is a quick way for clone verification or DNA isolation, followed by further enzymatic reactions (polymerase chain reaction and restriction enzyme digestion). Here, we video-recorded the general procedures of miniprep through the QIAGEN's QIAprep 8 Miniprep Kit, aiming to introducing this highly efficient technique to the general beginners for molecular biology techniques. The whole procedure is based on alkaline lysis of E. coli cells followed by adsorption of DNA onto silica in the presence of high salt. It consists of three steps: 1) preparation and clearing of a bacterial lysate, 2) adsorption of DNA onto the QIAprep membrane, 3) washing and elution of plasmid DNA. All steps are performed without the use of phenol, chloroform, CsCl, ethidium bromide, and without alcohol precipitation. It usually takes less than 2 hours to finish the entire procedure.

Ca(2+) release-activated Ca(2+) (CRAC) channels, located in the plasma membrane, are opened upon release of Ca(2+) from intracellular stores, permitting Ca(2+) entry and sustained [Ca(2+)](i) signaling that replenishes the store in numerous cell types. This mechanism is particularly important in T lymphocytes of the immune system, providing the missing link in the signal transduction cascade that is initiated by T cell receptor engagement and leads to altered expression of genes that results ultimately in the production of cytokines and cell proliferation. In the past three years, RNA interference screens together with over-expression and site-directed mutagenesis have identified the triggering molecule (Stim) that links store depletion to CRAC channel-mediated Ca(2+) influx and the pore subunit (Orai) of the CRAC channel that allows highly selective entry of Ca(2+) ions into cells.

Store-operated Ca2+ (SOC) channels regulate many cellular processes, but the underlying molecular components are not well defined. Using an RNA interference (RNAi)-based screen to identify genes that alter thapsigargin (TG)-dependent Ca2+ entry, we discovered a required and conserved role of Stim in SOC influx. RNAi-mediated knockdown of Stim in Drosophila S2 cells significantly reduced TG-dependent Ca2+ entry. Patch-clamp recording revealed nearly complete suppression of the Drosophila Ca2+ release-activated Ca2+ (CRAC) current that has biophysical characteristics similar to CRAC current in human T cells. Similarly, knockdown of the human homologue STIM1 significantly reduced CRAC channel activity in Jurkat T cells. RNAi-mediated knock-down of STIM1 inhibited TG- or agonist-dependent Ca2+ entry in HEK293 or SH-SY5Y cells. Conversely, overexpression of STIM1 in HEK293 cells modestly enhanced TG-induced Ca 2+ entry. We propose that STIM1, a ubiquitously expressed protein that is conserved from Drosophila to mammalian cells, plays an essential role in SOC influx and may be a common component of SOC and CRAC channels. © The Rockefeller University Press.

A fundamental question in developmental neurobiology is how a common neurotransmitter is specified in different neuronal types?. We describe cell-specific regulation of the serotonergic phenotype by the C. elegans POU-transcription factor UNC-86. We show that unc-86 regulates particular aspects of the terminal neuronal identity in four classes of serotonergic neurons, but that the development of the ADF serotonergic neurons is regulated by an UNC-86-independent program. In the NSM neurons, the role of unc-86 is confined in late differentiation; the neurons are generated but do not express genes necessary for serotonergic neurotransmission. unc-86-null mutations affect the expression in NSM of tph-1, which encodes the serotonin synthetic enzyme tryptophan hydroxylase, and cat-1, which encodes a vesicular transporter that loads serotonin into synaptic vesicles, suggesting that unc-86 coordinately regulates serotonin synthesis and packaging. However, unc-86-null mutations do not impair the ability of NSM to reuptake serotonin released from the ADF serotonergic chemosensory neurons and this serotonin reuptake is sensitive to the serotonin reuptake block drugs imipramine and fluoxetine, demonstrating that serotonin synthesis and reuptake is regulated by distinct factors. The NSM neurons in unc-86-null mutants also display abnormal neurite outgrowth, suggesting a role of unc-86 in regulating this process as well.

Recent RNAi screens have identified Stim and Orai as critical components of the Ca2+ release-activated Ca2+ (CRAC) channel. Stim senses depletion of the Endoplasmic Reticulum (ER) Ca2+ store, translocates from the ER to junctions adjacent to the plasma membrane (PM), and activates Orai pore-forming channel subunits in the PM to open the CRAC channel. The Orai oligomerization interface was investigated by co-immunoprecipitation of N-and/or C-terminal Orai deletion mutants and expressed Orai N- and C-terminal fragments. The transmembrane core domain plays a predominant role in subunit assembly; a weaker interaction interface was identified at the N-terminal region. We analyzed the quaternary structure of the Orai subunit and showed by cross-linking, and by non-denaturing gel electrophoresis that Orai is predominantly a dimer under resting conditions with or without co-expression of Stim. Single-molecule imaging of GFP-tagged Orai expressed in Xenopus oocytes revealed predominantly two-step photo-bleaching, consistent with a dimeric basal state. In contrast, co-expression of GFP-tagged Orai with the C-terminus of Stim as a cytosolic protein to activate the Orai channel without inducing Ca2+ store depletion or clustering of Orai into punctae yielded predominantly four-step photobleaching, consistent with a tetrameric Orai stoichiometry of the active CRAC channel. Interaction of the Orai C-terminal coiled-coil domain (as shown by structure-disruptive mutations) with the C-terminus of Stim thus induces Orai dimers to dimerize, forming a tetramer that constitutes the Ca2+-selective pore. This represents a novel mechanism in which assembly and activation of the functional ion channel are mediated by the same triggering molecule and may reveal a new channel gating mechanism. New data will be presented on the Stim-Orai stoichiometry and activation mechanism.

Stim and Orai proteins comprise the molecular machinery of Ca2+ release-activated Ca2+ (CRAC) channels. In T-lymphocytes, Orai1 is the pore-forming subunit and STIM1 serves as the ER Ca2+-sensor that opens Orai1 following ER Ca2+-store depletion. We are investigating mutations within Orai1 TM1 that alter channel activity in transfected HEK cells. At the extracellular side of TM1, we and others had previously shown that E106 confers Ca2+ selectivity on the STIM1-operated Orai1 current. We now show that R91, the site of the R91W mutation that causes severe combined immune deficiency, forms a very narrow part of the conducting pore at the cytosolic side of TM1. Orai1 R91C when co-expressed with STIM1 was activated normally by Ca2+ store depletion. However, treatment with diamide, a thiol-oxidizing agent, induced formation of disulfide bonds between R91C residues in adjacent Orai1 subunits. Moreover, diamide rapidly blocked STIM1-operated Ca2+ current, and current recovered during treatment with a reducing agent. In the middle of TM1, mutation of a conserved glycine residue to alanine (G98A) prevented STIM1-induced channel activity. Interestingly, mutation to aspartate (G98D) caused constitutive channel activation in a STIM1-independent manner to form a non-selective Ca2+-permeable conductance that was relatively resistant to block by Gd3+ (310 nM Kd vs 7 nM in wild-type Orai1). Moreover, the double mutant R91WG98D was also constitutively active, overcoming the normal inhibition of channel activity by tryptophan at the 91 position; and the double mutant R91CG98D was resistant to diamide block. We tentatively propose that R91 forms the gate of the Orai1 channel at the narrow inner mouth of the channel, G98 functions as a gating-hinge, and E106 promotes selective conduction of Ca2+. The channel pore is widened and ion selectivity perturbed by a negative charge at the G98 site.