Calmodulin binding to the polybasic C-termini of STIM proteins involved in store-operated calcium entry

Translocation of STIM1 and STIM2 from the endoplasmic reticulum to the plasma membrane is a key step in store-operated calcium entry in the cell. We show by isothermal titration calorimetry that calmodulin binds in a calcium-dependent manner to the polybasic C-termini of STIM1 and STIM2, a region critical for their translocation to the plasma membrane ( K D < or = 1 microM in calcium). HSQC NMR spectroscopy shows this interaction is in the fast exchange regime. By binding STIM1 and STIM2, calmodulin may regulate store refilling, thereby ensuring the maintenance of its own action in intracellular signaling.     

STIM1 is necessary for store-operated calcium entry in turning growth cones

J. Neurochem. (2012) 122, 1155-1166. ABSTRACT: Coordinated calcium signalling is vital for neuronal growth cone function and axon pathfinding. Although store-operated calcium entry (SOCE) has been suggested to be an important source of calcium in growth cone navigation, the mechanisms that regulate calcium signalling, particularly the regulation of internal calcium stores within growth cones, are yet to be fully determined. Stromal Interaction Molecule 1 (STIM1) is a calcium-sensing protein localized in the endoplasmic reticulum membrane that interacts with Orai proteins in the plasma membrane to initiate SOCE and refilling of intracellular calcium stores. We hypothesize that STIM1- and Orai1/2-mediated SOCE are necessary for growth cone turning responses to extracellular guidance cues. We show that STIM1 and Orai reorganize into puncta upon store depletion and during growth cone turning with STIM1 localization biased towards the turning side (high calcium side) of the growth cone. Importantly, STIM1 knock-down perturbed growth cone turning responses to the guidance cues brain-derived neurotrophic factor and semaphorin-3a (Sema-3a), as well as abolishing Sema-3a-induced growth cone collapse. Furthermore, STIM1 knock-down abolished SOCE induced by brain-derived neurotrophic factor, but not Sema-3a. Our data suggest that STIM1 is essential for correct growth cone navigation, playing multiple roles in growth cone motility, including the activation of SOCE.     

Differential roles for STIM1 and STIM2 in store-operated calcium entry in rat neurons

The interaction between Ca(2+) sensors STIM1 and STIM2 and Ca(2+) channel-forming protein ORAI1 is a crucial element of store-operated calcium entry (SOCE) in non-excitable cells. However, the molecular mechanism of SOCE in neurons remains unclear. We addressed this issue by establishing the presence and function of STIM proteins. Real-time polymerase chain reaction from cortical neurons showed that these cells contain significant amounts of Stim1 and Stim2 mRNA. Thapsigargin (TG) treatment increased the amount of both endogenous STIM proteins in neuronal membrane fractions. The number of YFP-STIM1/ORAI1 and YFP-STIM2/ORAI1 complexes was also enhanced by such treatment. The differences observed in the number of STIM1 and STIM2 complexes under SOCE conditions and the differential sensitivity to SOCE inhibitors suggest their distinct roles. Endoplasmic reticulum (ER) store depletion by TG enhanced intracellular Ca(2+) levels in loaded with Fura-2 neurons transfected with YFP-STIM1 and ORAI1, but not with YFP-STIM2 and ORAI1, which correlated well with the number of complexes formed. Moreover, the SOCE inhibitors ML-9 and 2-APB reduced Ca(2+) influx in neurons expressing YFP-STIM1/ORAI1 but produced no effect in cells transfected with YFP-STIM2/ORAI1. Moreover, in neurons transfected with YFP-STIM2/ORAI1, the increase in constitutive calcium entry was greater than with YFP-STIM1/ORAI1. Our data indicate that both STIM proteins are involved in calcium homeostasis in neurons. STIM1 mainly activates SOCE, whereas STIM2 regulates resting Ca(2+) levels in the ER and Ca(2+) leakage with the additional involvement of STIM1.     

Structural and mechanistic insights into STIM1-mediated initiation of store-operated calcium entry

Stromal interaction molecule-1 (STIM1) activates store-operated Ca2+ entry (SOCE) in response to diminished luminal Ca2+ levels. Here, we present the atomic structure of the Ca2+-sensing region of STIM1 consisting of the EF-hand and sterile alpha motif (SAM) domains (EF-SAM). The canonical EF-hand is paired with a previously unidentified EF-hand. Together, the EF-hand pair mediates mutually indispensable hydrophobic interactions between the EF-hand and SAM domains. Structurally critical mutations in the canonical EF-hand, "hidden" EF-hand, or SAM domain disrupt Ca2+ sensitivity in oligomerization via destabilization of the entire EF-SAM entity. In mammalian cells, EF-SAM destabilization mutations within full-length STIM1 induce punctae formation and activate SOCE independent of luminal Ca2+. We provide atomic resolution insight into the molecular basis for STIM1-mediated SOCE initiation and show that the folded/unfolded state of the Ca2+-sensing region of STIM is crucial to SOCE regulation.     

STIM1 signalling controls store-operated calcium entry required for development and contractile function in skeletal muscle

It is now well established that stromal interaction molecule 1 (STIM1) is the calcium sensor of endoplasmic reticulum stores required to activate store-operated calcium entry (SOC) channels at the surface of non-excitable cells. However, little is known about STIM1 in excitable cells, such as striated muscle, where the complement of calcium regulatory molecules is rather disparate from that of non-excitable cells. Here, we show that STIM1 is expressed in both myotubes and adult skeletal muscle. Myotubes lacking functional STIM1 fail to show SOC and fatigue rapidly. Moreover, mice lacking functional STIM1 die perinatally from a skeletal myopathy. In addition, STIM1 haploinsufficiency confers a contractile defect only under conditions where rapid refilling of stores would be needed. These findings provide insight into the role of STIM1 in skeletal muscle and suggest that STIM1 has a universal role as an ER/SR calcium sensor in both excitable and non-excitable cells.     

The KSR2-calcineurin complex regulates STIM1-ORAI1 dynamics and store-operated calcium entry (SOCE)

Store-operated calcium entry (SOCE) is the predominant Ca²⁺ entry mechanism in nonexcitable cells and controls a variety of physiological and pathological processes. Although significant progress has been made in identifying the components required for SOCE, the molecular mechanisms underlying it are elusive. The present study provides evidence for a direct involvement of kinase suppressor of Ras 2 (KSR2) in SOCE. Using lymphocytes and fibroblasts from ksr2^−/− mice and shKSR2-depleted cells, we find that KSR2 is critical for the elevation of cytosolic Ca²⁺ concentration. Specifically, our results show that although it is dispensable for Ca²⁺-store depletion, KSR2 is required for optimal calcium entry. We observe that KSR2 deficiency affects stromal interaction molecule 1 (STIM1)/ORAI1 puncta formation, which is correlated with cytoskeleton disorganization. Of interest, we find that KSR2-associated calcineurin is crucial for SOCE. Blocking calcineurin activity impairs STIM1/ORAI1 puncta-like formation and cytoskeleton organization. In addition, we observe that calcineurin activity and its role in SOCE are both KSR2 dependent.     

Morphological and functional aspects of STIM1-dependent assembly and disassembly of store-operated calcium entry complexes

The SOCE (store-operated Ca2+ entry) pathway is a central component of cell signalling that links the Ca2+-filling state of the ER (endoplasmic reticulum) to the activation of Ca2+-permeable channels at the PM (plasma membrane). SOCE channels maintain a high free Ca2+ concentration within the ER lumen required for the proper processing and folding of proteins, and fuel the long-term cellular Ca2+ signals that drive gene expression in immune cells. SOCE is initiated by the oligomerization on the membrane of the ER of STIMs (stromal interaction molecules) whose luminal EF-hand domain switches from globular to an extended conformation as soon as the free Ca2+ concentration within the ER lumen ([Ca2+]ER) decreases below basal levels of ~500 μM. The conformational changes induced by the unbinding of Ca2+ from the STIM1 luminal domain promote the formation of higher-order STIM1 oligomers that move towards the PM and exposes activating domains in STIM1 cytosolic tail that bind to Ca2+ channels of the Orai family at the PM and induce their activation. Both SOCE and STIM1 oligomerization are reversible events, but whether restoring normal [Ca2+]ER levels is sufficient to initiate the deoligomerization of STIM1 and to control the termination of SOCE is not known. The translocation of STIM1 towards the PM involves the formation of specialized compartments derived from the ER that we have characterized at the ultrastructural level and termed the pre-cortical ER, the cortical ER and the thin cortical ER. Pre-cortical ER structures are thin ER tubules enriched in STIM1 extending along microtubules and located deep inside cells. The cortical ER is located in the cell periphery in very close proximity (8-11?nm) to the plasma membrane. The thin cortical ER consists of thinner sections of the cortical ER enriched in STIM1 and devoid of chaperones that appear to be specialized ER compartments dedicated to Ca2+ signalling.     

The distinct role of STIM1 and STIM2 in the regulation of store-operated Ca2+ entry and cellular function

Stromal interaction molecules STIM1 and STIM2 are endoplasmic reticulum (ER) Ca²⁺ sensors that initiate store-operated Ca ²⁺ entry (SOCE). The roles of STIM1-mediated SOCE in cancer biology have been highlighted in different types of cancer, but that of STIM2 is unknown. By the model of cervical cancer, here we focus on the cooperative regulation of SOCE by STIM proteins and their distinct roles in cellular function. Immunofluorescent stainings of surgical specimens of cervical cancer show that STIM1 and STIM2 are abundant in tumor tissues, but STIM1 is the major ER Ca ²⁺ sensor identified in the invasive front of cancer tissues. STIM1 or STIM2 overexpression in cervical cancer SiHa cells induces an upregulated SOCE. Regarding cellular function, STIM1 and STIM2 are necessary for cell proliferation, whereas STIM1 is the dominant ER Ca ²⁺ sensor involved in cell migration. During SOCE, STIM1 is aggregated and translocated towards the Orai1-containing plasma membrane in association with the microtubule plus-end binding protein EB1. In contrast, STIM2 is constitutively aggregated without significant trafficking or association with microtubules. These results show the distinct role of STIM1 and STIM2 in SOCE and cellular function of cervical cancer cells.     

Fluctuation in O-GlcNAcylation inactivates STIM1 to reduce store-operated calcium ion entry via down-regulation of Ser621 phosphorylation

Stromal interaction molecule 1 (STIM1) plays a pivotal role in store-operated Ca²⁺ entry (SOCE), an essential mechanism in cellular calcium signaling and in maintaining cellular calcium balance. Because O-GlcNAcylation plays pivotal roles in various cellular function, we examined the effect of fluctuation in STIM1 O-GlcNAcylation on SOCE activity. We found that both increase and decrease in STIM1 O-GlcNAcylation impaired SOCE activity. To determine the molecular basis, we established STIM1-knockout HEK293 (STIM1-KO-HEK) cells using the CRISPR/Cas9 system and transfected STIM1 WT (STIM1-KO-WT-HEK), S621A (STIM1-KO-S621A-HEK), or T626A (STIM1-KO-T626A-HEK) cells. Using these cells, we examined the possible O-GlcNAcylation sites of STIM1 to determine whether the sites were O-GlcNAcylated. Co-immunoprecipitation analysis revealed that Ser⁶²¹ and Thr⁶²⁶ were O-GlcNAcylated and that Thr⁶²⁶ was O-GlcNAcylated in the steady state but Ser⁶²¹ was not. The SOCE activity in STIM1-KO-S621A-HEK and STIM1-KO-T626A-HEK cells was lower than that in STIM1-KO-WT-HEK cells because of reduced phosphorylation at Ser⁶²¹. Treatment with the O-GlcNAcase inhibitor Thiamet G or O-GlcNAc transferase (OGT) transfection, which increases O-GlcNAcylation, reduced SOCE activity, whereas treatment with the OGT inhibitor ST045849 or siOGT transfection, which decreases O-GlcNAcylation, also reduced SOCE activity. Decrease in SOCE activity due to increase and decrease in O-GlcNAcylation was attributable to reduced phosphorylation at Ser⁶²¹. These data suggest that both decrease in O-GlcNAcylation at Thr⁶²⁶ and increase in O-GlcNAcylation at Ser⁶²¹ in STIM1 lead to impairment of SOCE activity through decrease in Ser⁶²¹ phosphorylation. Targeting STIM1 O-GlcNAcylation could provide a promising treatment option for the related diseases, such as neurodegenerative diseases.     

Sigma1 receptors inhibit store-operated Ca2+ entry by attenuating coupling of STIM1 to Orai1

Sigma1 receptors (σ1Rs) are expressed widely; they bind diverse ligands, including psychotropic drugs and steroids, regulate many ion channels, and are implicated in cancer and addiction. It is not known how σ1Rs exert such varied effects. We demonstrate that σ1Rs inhibit store-operated Ca²⁺ entry (SOCE), a major Ca²⁺ influx pathway, and reduce the Ca²⁺ content of the intracellular stores. SOCE was inhibited by expression of σ1R or an agonist of σ1R and enhanced by loss of σ1R or an antagonist. Within the endoplasmic reticulum (ER), σ1R associated with STIM1, the ER Ca²⁺ sensor that regulates SOCE. This interaction was modulated by σ1R ligands. After depletion of Ca²⁺ stores, σ1R accompanied STIM1 to ER–plasma membrane (PM) junctions where STIM1 stimulated opening of the Ca²⁺ channel, Orai1. The association of STIM1 with σ1R slowed the recruitment of STIM1 to ER–PM junctions and reduced binding of STIM1 to PM Orai1. We conclude that σ1R attenuates STIM1 coupling to Orai1 and thereby inhibits SOCE.     

The role of STIM1 in the receptor- and store-operated calcium influx in HEK293 cells

The possible role of STIM1 protein in the regulation of activity of receptor- and store-operated Ca²⁺ channels in non-excitable cells has been studied. Receptor- and store-operated Ca²⁺ influxes have been measured using the fluorescent method of detection of cytosolic Ca²⁺ concentration and the electrophysiological methods of whole-cell and single-channel current recordings in the control HEK293 cells and in HEK293 cells with suppressed expression of STIM1. The experiments have shown that STIM1 suppression results in a reduction of the amplitudes of both receptor- and store-operated inward calcium currents. The decrease of total Ca²⁺ influx of in response to an agonist or to passive depletion of calcium stores upon STIM1 suppression was due to the decrease or total absence of the activity of high-conductance channels I_max and non-selective channels I_ns in HEK293 cells. A decrease in the STIM1 amount also altered the activity regulation of low-conductance I_min channels that changed from exclusively agonist-operated into store-dependent channels in HEK293 cells.     

Overexpression of Orai1 and STIM1 proteins alters regulation of store-operated Ca2+ entry by endogenous mediators

Orai1 and STIM1 have been identified as the main determinants of the store-operated Ca²⁺ entry (SOCE). Their specific roles in SOCE and their molecular interactions have been studied extensively following heterologous overexpression or molecular knockdown and extrapolated to the endogenous processes in naïve cells. Using molecular and imaging techniques, we found that variation of expression levels of Orai1 or STIM1 can significantly alter expression and role of some endogenous regulators of SOCE. Although functional inhibition of Ca²⁺-independent phospholipase A₂ β (iPLA₂β or PLA2g6A), or depletion of plasma membrane cholesterol caused a dramatic loss of endogenous SOCE in HEK293 cells, these effects were attenuated significantly when either Orai1 or STIM1 were overexpressed. Molecular knockdown of iPLA₂β impaired SOCE in both control cells and cells overexpressing STIM1. We also discovered important cross-talk between expression of Orai1 and a specific plasma membrane variant of iPLA₂β but not STIM1. These data confirm the role of iPLA₂β as an essential mediator of endogenous SOCE and demonstrate that its physiological role can be obscured by Orai1 and STIM1 overexpression.     

Evidence for the interaction of peroxiredoxin-4 with the store-operated calcium channel activator STIM1 in liver cells

Ca²⁺ entry through store-operated Ca²⁺ channels (SOCs) in the plasma membrane (PM) of hepatocytes plays a central role in the hormonal regulation of liver metabolism. SOCs are composed of Orai1, the channel pore protein, and STIM1, the activator protein, and are regulated by hormones and reactive oxygen species (ROS). In addition to Orai1, STIM1 also interacts with several other intracellular proteins. Most previous studies of the cellular functions of Orai1 and STIM1 have employed immortalised cells in culture expressing ectopic proteins tagged with a fluorescent polypeptide such as GFP. Little is known about the intracellular distributions of endogenous Orai1 and STIM1. The aims are to determine the intracellular distribution of endogenous Orai1 and STIM1 in hepatocytes and to identify novel STIM1 binding proteins. Subcellular fractions of rat liver were prepared by homogenisation and differential centrifugation. Orai1 and STIM1 were identified and quantified by western blot. Orai1 was found in the PM (0.03%), heavy (44%) and light (27%) microsomal fractions, and STIM1 in the PM (0.09%), and heavy (85%) and light (13%) microsomal fractions. Immunoprecipitation of STIM1 followed by LC/MS or western blot identified peroxiredoxin-4 (Prx-4) as a potential STIM1 binding protein. Prx-4 was found principally in the heavy microsomal fraction. Knockdown of Prx-4 using siRNA, or inhibition of Prx-4 using conoidin A, did not affect Ca²⁺ entry through SOCs but rendered SOCs susceptible to inhibition by H₂O₂. It is concluded that, in hepatocytes, a considerable proportion of endogenous Orai1 and STIM1 is located in the rough ER. In the rough ER, STIM1 interacts with Prx-4, and this interaction may contribute to the regulation by ROS of STIM1 and SOCs.     

The elementary unit of store-operated Ca2+ entry: local activation of CRAC channels by STIM1 at ER-plasma membrane junctions

The activation of store-operated Ca(2+) entry by Ca(2+) store depletion has long been hypothesized to occur via local interactions of the endoplasmic reticulum (ER) and plasma membrane, but the structure involved has never been identified. Store depletion causes the ER Ca(2+) sensor stromal interacting molecule 1 (STIM1) to form puncta by accumulating in junctional ER located 10-25 nm from the plasma membrane (see Wu et al. on p. 803 of this issue). We have combined total internal reflection fluorescence (TIRF) microscopy and patch-clamp recording to localize STIM1 and sites of Ca(2+) influx through open Ca(2+) release-activated Ca(2+) (CRAC) channels in Jurkat T cells after store depletion. CRAC channels open only in the immediate vicinity of STIM1 puncta, restricting Ca(2+) entry to discrete sites comprising a small fraction of the cell surface. Orai1, an essential component of the CRAC channel, colocalizes with STIM1 after store depletion, providing a physical basis for the local activation of Ca(2+) influx. These studies reveal for the first time that STIM1 and Orai1 move in a coordinated fashion to form closely apposed clusters in the ER and plasma membranes, thereby creating the elementary unit of store-operated Ca(2+) entry.     

Relocalization of STIM1 for activation of store-operated Ca(2+) entry is determined by the depletion of subplasma membrane endoplasmic reticulum Ca(2+) store

STIM1 (stromal interacting molecule 1), an endoplasmic reticulum (ER) protein that controls store-operated Ca(2+) entry (SOCE), redistributes into punctae at the cell periphery after store depletion. This redistribution is suggested to have a causal role in activation of SOCE. However, whether peripheral STIM1 punctae that are involved in regulation of SOCE are determined by depletion of peripheral or more internal ER has not yet been demonstrated. Here we show that Ca(2+) depletion in subplasma membrane ER is sufficient for peripheral redistribution of STIM1 and activation of SOCE. 1 microM thapsigargin (Tg) induced substantial depletion of intracellular Ca(2+) stores and rapidly activated SOCE. In comparison, 1 nM Tg induced slower, about 60-70% less Ca(2+) depletion but similar SOCE. SOCE was confirmed by measuring I(SOC) in addition to Ca(2+), Mn(2+), and Ba(2+) entry. Importantly, 1 nM Tg caused redistribution of STIM1 only in the ER-plasma membrane junction, whereas 1 microM Tg caused a relatively global relocalization of STIM1 in the cell. During the time taken for STIM1 relocalization and SOCE activation, 1 nM Bodipy-fluorescein Tg primarily labeled the subplasma membrane region, whereas 1 microM Tg labeled the entire cell. The localization of Tg in the subplasma membrane region was associated with depletion of ER in this region and activation of SOCE. Together, these data suggest that peripheral STIM1 relocalization that is causal in regulation of SOCE is determined by the status of [Ca(2+)] in the ER in close proximity to the plasma membrane. Thus, the mechanism involved in regulation of SOCE is contained within the ER-plasma membrane junctional region.     

A single lysine in the N-terminal region of store-operated channels is critical for STIM1-mediated gating

Store-operated Ca(2+) entry is controlled by the interaction of stromal interaction molecules (STIMs) acting as endoplasmic reticulum ER Ca(2+) sensors with calcium release-activated calcium (CRAC) channels (CRACM1/2/3 or Orai1/2/3) in the plasma membrane. Here, we report structural requirements of STIM1-mediated activation of CRACM1 and CRACM3 using truncations, point mutations, and CRACM1/CRACM3 chimeras. In accordance with previous studies, truncating the N-terminal region of CRACM1 or CRACM3 revealed a 20-amino acid stretch close to the plasma membrane important for channel gating. Exchanging the N-terminal region of CRACM3 with that of CRACM1 (CRACM3-N(M1)) results in accelerated kinetics and enhanced current amplitudes. Conversely, transplanting the N-terminal region of CRACM3 into CRACM1 (CRACM1-N(M3)) leads to severely reduced store-operated currents. Highly conserved amino acids (K85 in CRACM1 and K60 in CRACM3) in the N-terminal region close to the first transmembrane domain are crucial for STIM1-dependent gating of CRAC channels. Single-point mutations of this residue (K85E and K60E) eliminate store-operated currents induced by inositol 1,4,5-trisphosphate and reduce store-independent gating by 2-aminoethoxydiphenyl borate. However, short fragments of these mutant channels are still able to communicate with the CRAC-activating domain of STIM1. Collectively, these findings identify a single amino acid in the N terminus of CRAC channels as a critical element for store-operated gating of CRAC channels.     

The C-terminal domain of TRPV4 is essential for plasma membrane localization

Many members of the TRP superfamily oligomerize in the ER before trafficking to the plasma membrane. For membrane localization of the non-selective cation channel TRPV4 specific domains in the N-terminus are required, but the role of the C-terminus in the oligomerization and trafficking process has been not determined until now. Therefore, the localization of recombinant TRPV4 in two cell models was analyzed: HaCaT keratinocytes that express TRPV4 endogenously were compared to CHO cells that are devoid of endogenous TRPV4. When deletions were introduced in the C-terminal domain three states of TRPV4 localization were defined: a truncated TRPV4 protein of 855 amino acids was exported to the plasma membrane like the full-length channel (871 aa) and was also functional. Mutants with a length of 828 to 844 amino acids remained in the ER of CHO cells, but in HaCaT cells plasma membrane localization was partially rescued by oligomerization with endogenous TRPV4. This was confirmed by coexpression of recombinant full-length TRPV4 together with these deletion mutants, which resulted in an almost complete plasma membrane localization of both proteins and significant FRET in the plasma membrane and the ER. All deletions upstream of amino acid 828 resulted in total ER retention that could not rescued by coexpression with the full-length protein. However, these deletion mutants did not impair export of full-length TRPV4, implying that no oligomerization took place. These data indicate that the C-terminus of TRPV4 is required for oligomerization, which takes place in the ER and precedes plasma membrane trafficking.     

The C-terminal domain of TRPV4 is essential for plasma membrane localization

Many members of the TRP superfamily oligomerize in the ER before trafficking to the plasma membrane. For membrane localization of the non-selective cation channel TRPV4 specific domains in the N-terminus are required, but the role of the C-terminus in the oligomerization and trafficking process has been not determined until now. Therefore, the localization of recombinant TRPV4 in two cell models was analyzed: HaCaT keratinocytes that express TRPV4 endogenously were compared to CHO cells that are devoid of endogenous TRPV4. When deletions were introduced in the C-terminal domain three states of TRPV4 localization were defined: a truncated TRPV4 protein of 855 amino acids was exported to the plasma membrane like the full-length channel (871 aa) and was also functional. Mutants with a length of 828 to 844 amino acids remained in the ER of CHO cells, but in HaCaT cells plasma membrane localization was partially rescued by oligomerization with endogenous TRPV4. This was confirmed by coexpression of recombinant full-length TRPV4 together with these deletion mutants, which resulted in an almost complete plasma membrane localization of both proteins and significant FRET in the plasma membrane and the ER. All deletions upstream of amino acid 828 resulted in total ER retention that could not rescued by coexpression with the full-length protein. However, these deletion mutants did not impair export of full-length TRPV4, implying that no oligomerization took place. These data indicate that the C-terminus of TRPV4 is required for oligomerization, which takes place in the ER and precedes plasma membrane trafficking.     

Transient receptor potential ankyrin-1 (TRPA1) modulates store-operated Ca2 + entry by regulation of STIM1-Orai1 association

TRPA1 is a non-selective Ca^2 + permeable channel located in the plasma membrane that functions as a cellular sensor detecting mechanical, chemical and thermal stimuli, being a component of neuronal, epithelial, blood and smooth muscle tissues. TRPA1 has been shown to influence a broad range of physiological processes that involve Ca^2 +-dependent signaling pathways. Here we report that TRPA1 is expressed in MEG01 but not in platelets at the protein level. MEG01 cells maturation induced by PMA results in attenuation of TRPA1 protein expression and enhances thapsigargin-evoked Ca^2 + entry without altering the release of Ca^2 + from intracellular stores. Inhibition of TRPA1 by HC-030031 results in enhancement of both thrombin- and thapsigargin-stimulated Ca^2 + entry. Co-immunoprecipitation experiments revealed that TRPA1 associates with STIM1, as well as Orai1, TRPC1 and TRPC6. Downregulation of TRPA1 expression by MEG01 maturation, as well as pharmacological inhibition of TRPA1 by HC-030031, results in enhancement of the association between STIM1 and Orai1. Altogether, these findings provide evidence for a new and interesting function of TRPA1 in cellular function associated to the regulation of agonist-induced Ca^2 + entry by the modulation of STIM1/Orai1 interaction.     

Function of a STIM1 homologue in C. elegans: evidence that store-operated Ca2+ entry is not essential for oscillatory Ca2+ signaling and ER Ca2+ homeostasis

1,4,5-trisphosphate (IP(3))-dependent Ca(2+) signaling regulates gonad function, fertility, and rhythmic posterior body wall muscle contraction (pBoc) required for defecation in Caenorhabditis elegans. Store-operated Ca(2+) entry (SOCE) is activated during endoplasmic reticulum (ER) Ca(2+) store depletion and is believed to be an essential and ubiquitous component of Ca(2+) signaling pathways. SOCE is thought to function to refill Ca(2+) stores and modulate Ca(2+) signals. Recently, stromal interaction molecule 1 (STIM1) was identified as a putative ER Ca(2+) sensor that regulates SOCE. We cloned a full-length C. elegans stim-1 cDNA that encodes a 530-amino acid protein with approximately 21% sequence identity to human STIM1. Green fluorescent protein (GFP)-tagged STIM-1 is expressed in the intestine, gonad sheath cells, and spermatheca. Knockdown of stim-1 expression by RNA interference (RNAi) causes sterility due to loss of sheath cell and spermatheca contractile activity required for ovulation. Transgenic worms expressing a STIM-1 EF-hand mutant that constitutively activates SOCE in Drosophila and mammalian cells are sterile and exhibit severe pBoc arrhythmia. stim-1 RNAi dramatically reduces STIM-1GFP expression, suppresses the EF-hand mutation-induced pBoc arrhythmia, and inhibits intestinal store-operated Ca(2+) (SOC) channels. However, stim-1 RNAi surprisingly has no effect on pBoc rhythm, which is controlled by intestinal oscillatory Ca(2+) signaling, in wild type and IP(3) signaling mutant worms, and has no effect on intestinal Ca(2+) oscillations and waves. Depletion of intestinal Ca(2+) stores by RNAi knockdown of the ER Ca(2+) pump triggers the ER unfolded protein response (UPR). In contrast, stim-1 RNAi fails to induce the UPR. Our studies provide the first detailed characterization of STIM-1 function in an intact animal and suggest that SOCE is not essential for certain oscillatory Ca(2+) signaling processes and for maintenance of store Ca(2+) levels in C. elegans. These findings raise interesting and important questions regarding the function of SOCE and SOC channels under normal and pathophysiological conditions.