Orai1 mediates the interaction between STIM1 and hTRPC1 and regulates the mode of activation of hTRPC1-forming Ca2+ channels

Orai1 and hTRPC1 have been presented as essential components of store-operated channels mediating highly Ca(2+) selective I(CRAC) and relatively Ca(2+) selective I(SOC), respectively. STIM1 has been proposed to communicate the Ca(2+) content of the intracellular Ca(2+) stores to the plasma membrane store-operated Ca(2+) channels. Here we present evidence for the dynamic interaction between endogenously expressed Orai1 and both STIM1 and hTRPC1 regulated by depletion of the intracellular Ca(2+) stores, using the pharmacological tools thapsigargin plus ionomycin, or by the physiological agonist thrombin, independently of extracellular Ca(2+). In addition we report that Orai1 mediates the communication between STIM1 and hTRPC1, which is essential for the mode of activation of hTRPC1-forming Ca(2+) permeable channels. Electrotransjection of cells with anti-Orai1 antibody, directed toward the C-terminal region that mediates the interaction with STIM1, and stabilization of an actin cortical barrier with jasplakinolide prevented the interaction between STIM1 and hTRPC1. Under these conditions hTRPC1 was no longer involved in store-operated calcium entry but in diacylglycerol-activated non-capacitative Ca(2+) entry. These findings support the functional role of the STIM1-Orai1-hTRPC1 complex in the activation of store-operated Ca(2+) entry.     

Orai1 and STIM reconstitute store-operated calcium channel function

The two membrane proteins, STIM1 and Orai1, have each been shown to be essential for the activation of store-operated channels (SOC). Yet, how these proteins functionally interact is not known. Here, we reveal that STIM1 and Orai1 expressed together reconstitute functional SOCs. Expressed alone, Orai1 strongly reduces store-operated Ca(2+) entry (SOCE) in human embryonic kidney 293 cells and the Ca(2+) release-activated Ca(2+) current (I(CRAC)) in rat basophilic leukemia cells. However, expressed along with the store-sensing STIM1 protein, Orai1 causes a massive increase in SOCE, enhancing the rate of Ca(2+)entry by up to 103-fold. This entry is entirely store-dependent since the same coexpression causes no measurable store-independent Ca(2+) entry. The entry is completely blocked by the SOC blocker, 2-aminoethoxydiphenylborate. Orai1 and STIM1 coexpression also caused a large gain in CRAC channel function in rat basophilic leukemia cells. The close STIM1 homologue, STIM2, inhibited SOCE when expressed alone but coexpressed with Orai1 caused substantial constitutive (store-independent) Ca(2+) entry. STIM proteins are known to mediate Ca(2+) store-sensing and endoplasmic reticulum-plasma membrane coupling with no intrinsic channel properties. Our results revealing a powerful gain in SOC function dependent on the presence of both Orai1 and STIM1 strongly suggest that Orai1 contributes the PM channel component responsible for Ca(2+) entry. The suppression of SOC function by Orai1 overexpression likely reflects a required stoichiometry between STIM1 and Orai1.     

Capacitative and non-capacitative signaling complexes in human platelets

Discharge of the intracellular Ca(2+) stores activates Ca(2+) entry through store-operated channels (SOCs). Since the recent identification of STIM1 and STIM2, as well as the Orai1 homologs, Orai2 and Orai3, the protein complexes involved in Ca(2+) signaling needs re-evaluation in native cells. Using real time PCR combined with Western blotting we have found the expression of the three Orai isoforms, STIM1, STIM2 and different TRPCs in human platelets. Depletion of the intracellular Ca(2+) stores with thapsigargin, independently of changes in cytosolic Ca(2+) concentration, enhanced the formation of a signaling complex involving STIM1, STIM2, Orai1, Orai2 and TRPC1. Furthermore, platelet treatment with the dyacylglicerol analog 1-oleoyl-2-acetyl-sn-glycerol (OAG) resulted in specific association of Orai3 with TRPC3. Treatment of platelets with arachidonic acid enhanced the association between Orai1 and Orai3 in human platelets and overexpression of Orai1 and Orai3 in HEK293 cells increased arachidonic acid-induced Ca(2+) entry. These results indicate that Ca(2+) store depletion results in the formation of exclusive signaling complexes involving STIM proteins, as well as Orai1, Orai2 and TRPC1, but not Orai3, which seems to be involved in non-capacitative Ca(2+) influx in human platelets.     

Functional requirement for Orai1 in store-operated TRPC1-STIM1 channels

Orai1 and TRPC1 have been proposed as core components of store-operated calcium release-activated calcium (CRAC) and store-operated calcium (SOC) channels, respectively. STIM1, a Ca(2+) sensor protein in the endoplasmic reticulum, interacts with and mediates store-dependent regulation of both channels. We have previously reported that dynamic association of Orai1, TRPC1, and STIM1 is involved in activation of store-operated Ca(2+) entry (SOCE) in salivary gland cells. In this study, we have assessed the molecular basis of TRPC1-SOC channels in HEK293 cells. We report that TRPC1+STIM1-dependent SOCE requires functional Orai1. Thapsigargin stimulation of cells expressing Orai1+STIM1 increased Ca(2+) entry and activated typical I(CRAC) current. STIM1 alone did not affect SOCE, whereas expression of Orai1 induced a decrease. Expression of TRPC1 induced a small increase in SOCE, which was greatly enhanced by co-expression of STIM1. Thapsigargin stimulation of cells expressing TRPC1+STIM1 activated a non-selective cation current, I(SOC), that was blocked by 1 microm Gd(3+) and 2-APB. Knockdown of Orai1 decreased endogenous SOCE as well as SOCE with TRPC1 alone. siOrai1 also significantly reduced SOCE and I(SOC) in cells expressing TRPC1+STIM1. Expression of R91WOrai1 or E106QOrai1 induced similar attenuation of TRPC1+STIM1-dependent SOCE and I(SOC), whereas expression of Orai1 with TRPC1+STIM1 resulted in SOCE that was larger than that with Orai1+STIM1 or TRPC1+STIM1 but not additive. Additionally, Orai1, E106QOrai1, and R91WOrai1 co-immunoprecipitated with similar levels of TRPC1 and STIM1 from HEK293 cells, and endogenous TRPC1, STIM1, and Orai1 were co-immunoprecipitated from salivary glands. Together, these data demonstrate a functional requirement for Orai1 in TRPC1+STIM1-dependent SOCE.     

Role of STIM1/Orai1-mediated store-operated Ca²⁺ entry in airway smooth muscle cell proliferation

Hyperplasia of airway smooth muscle cells (ASMCs) is a characteristic change of chronic asthma patients. However, the underlying mechanisms that trigger this process are not yet completely understood. Store-operated Ca(2+) (SOC) entry (SOCE) occurs in response to the intracellular sarcoplasma reticulum (SR)/endoplasmic reticulum (ER) Ca(2+) store depletion. SOCE plays an important role in regulating Ca(2+) signaling and cellular responses of ASMCs. Stromal interaction molecule (STIM)1 has been proposed as an ER/SR Ca(2+) sensor and translocates to the ER underneath the plasma membrane upon depletion of the ER Ca(2+) store, where it interacts with Orai1, the molecular component of SOC channels, and brings about SOCE. STIM1 and Orai1 have been proved to mediate SOCE of ASMCs. In this study, we investigated whether STIM1/Orai1-mediated SOCE is involved in rat ASMC proliferation. We found that SOCE was upregulated during ASMC proliferation accompanied by a mild increase of STIM1 and a significant increase of Orai1 mRNA expression, whereas the proliferation of ASMCs was partially inhibited by the SOC channel blockers SKF-96365, NiCl(2), and BTP-2. Suppressing the mRNA expression of STIM1 or Orai1 with specific short hairpin RNA resulted in the attenuation of SOCE and ASMC proliferation. Moreover, after knockdown of STIM1 or Orai1, the SOC channel blocker SKF-96365 had no inhibitory effect on the proliferation of ASMCs anymore. These results suggested that STIM1/Orai1-mediated SOCE is involved in ASMC proliferation.     

Temperature-dependent STIM1 activation induces Ca²+ influx and modulates gene expression

Intracellular Ca(2+) is essential for diverse cellular functions. Ca(2+) entry into many cell types including immune cells is triggered by depleting endoplasmic reticulum (ER) Ca(2+), a process termed store-operated Ca(2+) entry (SOCE). STIM1 is an ER Ca(2+) sensor. Upon Ca(2+) store depletion, STIM1 clusters at ER-plasma membrane junctions where it interacts with and gates Ca(2+)-permeable Orai1 ion channels. Here we show that STIM1 is also activated by temperature. Heating cells caused clustering of STIM1 at temperatures above 35 °C without depleting Ca(2+) stores and led to Orai1-mediated Ca(2+) influx as a heat off-response (response after cooling). Notably, the functional coupling of STIM1 and Orai1 is prevented at high temperatures, potentially explaining the heat off-response. Additionally, physiologically relevant temperature shifts modulate STIM1-dependent gene expression in Jurkat T cells. Therefore, temperature is an important regulator of STIM1 function.     

Canonical transient receptor potential 5 channel in conjunction with Orai1 and STIM1 allows Sr2+ entry, optimal influx of Ca2+, and degranulation in a rat mast cell line

Degranulation of mast cells in response to Ag or the calcium mobilizing agent, thapsigargin, is dependent on emptying of intracellular stores of Ca(2+) and the ensuing influx of external Ca(2+), also referred to as store-operated calcium entry. However, it is unlikely that the calcium release-activated calcium channel is the sole mechanism for the entry of Ca(2+) because Sr(2+) and other divalent cations also permeate and support degranulation in stimulated mast cells. In this study we show that influx of Ca(2+) and Sr(2+) as well as degranulation are dependent on the presence of the canonical transient receptor potential (TRPC) channel protein TRPC5, in addition to STIM1 and Orai1, as demonstrated by knock down of each of these proteins by inhibitory RNAs in a rat mast cell (RBL-2H3) line. Overexpression of STIM1 and Orai1, which are known to be essential components of calcium release-activated calcium channel, allows entry of Ca(2+) but not Sr(2+), whereas overexpression of STIM1 and TRPC5 allows entry of both Ca(2+) and Sr(2+). These and other observations suggest that the Sr(2+)-permeable TRPC5 associates with STIM1 and Orai1 in a stoichiometric manner to enhance entry of Ca(2+) to generate a signal for degranulation.     

STIM1L is a new actin-binding splice variant involved in fast repetitive Ca2+ release

Cytosolic Ca(2+) signals encoded by repetitive Ca(2+) releases rely on two processes to refill Ca(2+) stores: Ca(2+) reuptake from the cytosol and activation of a Ca(2+) influx via store-operated Ca(2+) entry (SOCE). However, SOCE activation is a slow process. It is delayed by >30 s after store depletion because stromal interaction molecule 1 (STIM1), the Ca(2+) sensor of the intracellular stores, must form clusters and migrate to the membrane before being able to open Orai1, the plasma membrane Ca(2+) channel. In this paper, we identify a new protein, STIM1L, that colocalizes with Orai1 Ca(2+) channels and interacts with actin to form permanent clusters. This property allowed the immediate activation of SOCE, a characteristic required for generating repetitive Ca(2+) signals with frequencies within seconds such as those frequently observed in excitable cells. STIM1L was expressed in several mammalian tissues, suggesting that many cell types rely on this Ca(2+) sensor for their Ca(2+) homeostasis and intracellular signaling.     

Role of lipid rafts in the interaction between hTRPC1, Orai1 and STIM1

Store-operated Ca2+ entry (SOCE) is a mechanism regulated by the filling state of the intracellular Ca2+ stores that requires the participation of the Ca2+ sensor STIM1, which communicates the Ca2+ content of the stores to the plasma membrane Ca2+-permeable channels. We have recently reported that Orai1 mediates the communication between STIM1 and the Ca2+ channel hTRPC1. This event is important to confer hTRPC1 store depletion sensitivity, thus supporting the functional role of the STIM1-Orai1-hTRPC1 complex in the activation of SOCE. Here we have explored the relevance of lipid rafts in the formation of the STIM1-Orai1-hTRPC1 complex and the activation of SOCE. Disturbance of lipid raft domains, using methyl-beta-cyclodextrin, reduces the interaction between endogenously expressed Orai1 and both STIM1 and hTRPC1 upon depletion of the intracellular Ca2+ stores and attenuates thapsigargin-evoked Ca2+ entry. These findings suggest that TRPC1, Orai1 and STIM1 form a heteromultimer associated with lipid raft domains and regulated by the intracellular Ca2+ stores.     

Orai1 mediates store-operated Ca2+ entry during fertilization in mammalian oocytes

The presence of the store-operated Ca(2+) entry channel Orai1 and its function in signal transduction during fertilization have been investigated in mammalian oocytes using the pig as a model. RT-PCR cloning and sequence analysis revealed that Orai1 is expressed in the oocytes with a coding sequence of 921bp. After indirect immunocytochemistry or the overexpression of EGFP-tagged Orai1, the fluorescent signal was present primarily in the cell cortex consistent with plasma membrane localization of the protein. Western blot and real-time PCR results showed that Orai1 expression decreases during oocyte maturation; this is associated with the oocytes gaining the ability to generate a large Ca(2+) influx after store depletion. Downregulation of Orai1 expression by siRNA microinjection blocked Ca(2+) influx after store depletion and subsequent Ca(2+) add-back; the Ca(2+) oscillations induced by the fertilizing sperm were also inhibited in oocytes with downregulated Orai1 levels. At the same time, overexpression of Orai1 in the oocytes also modified store-operated Ca(2+) entry and had an inhibitory effect on the fertilization Ca(2+) signal. The abnormal Ca(2+) signaling due to Orai1 downregulation had a strong negative impact on subsequent embryo development. Co-overexpression of Orai1 and STIM1 on the other hand, led to a dramatic increase in Ca(2+) entry after store depletion. The findings indicate that Orai1 is a plasma membrane-resident Ca(2+) channel that is responsible for mediating Ca(2+) entry after the mobilization of intracellular Ca(2+) in oocytes. Orai1 and a functional store-operated Ca(2+) entry pathway are required to maintain the Ca(2+) oscillations at fertilization and to support proper embryo development.     

Location and function of STIM1 in the activation of Ca2+ entry signals

Store-operated channels (SOCs) mediate Ca(2+) entry signals in response to endoplasmic reticulum (ER) Ca(2+) depletion in most cells. STIM1 senses decreased ER luminal Ca(2+) through its EF-hand Ca(2+)-binding motif and aggregates in near-plasma membrane (PM) ER junctions to activate PM Orai1, the functional SOC. STIM1 is also present in the PM, although its role there is unknown. STIM1-mediated coupling was examined using the stable EF20 HEK293 cell line expressing the STIM1-D76A/E87A EF-hand mutant (STIM1(EF)) deficient in Ca(2+) binding. EF20 cells were viable despite constitutive Ca(2+) entry, allowing study of SOC activation without depleting ER Ca(2+). STIM1(EF) was exclusively in stable near-PM junctions, 3.5-fold larger than formed with STIM1(WT). STIM(EF)-expressing cells had normal ER Ca(2+) levels but substantially reduced ER Ca(2+) leak. Expression of antiapoptotic Bcl-2 proteins (BCl-2, MCL-1, BCL-XL) were increased 2-fold in EF20 cells, probably reflecting survival of EF20 cells but not accounting for decreased ER Ca(2+) leak. Surface biotinylation and streptavidin pull-down of cells expressing STIM1(WT) or STIM1(EF) revealed strong PM interactions of both proteins. Although surface expression of STIM1(WT) was clearly detectable, STIM1(EF) was undetectable at the cell surface. Thus, the Ca(2+) binding-defective STIM1(EF) mutant exists exclusively in aggregates within near-PM junctions but, unlike STIM1(WT), is not trafficked to the PM. Although not inserted in the PM, external application of a monoclonal anti-N-terminal STIM1 antibody blocked constitutive STIM(EF)-mediated Ca(2+) entry, but only in cells expressing endogenous STIM1(WT) and not in DT40 STIM1 knock-out cells devoid of STIM(WT). This suggests that PM-STIM1 may play a regulatory role in SOC activation.     

Visualisation and identification of the interaction between STIM1s in resting cells

Store-operated Ca(2+) channels are a major Ca(2+) entry pathway in nonexcitable cells, which drive various essential cellular functions. Recently, STIM1 and Orai proteins have been identified as the major molecular components of the Ca(2+) release-activated Ca(2+) (CRAC) channel. As the key subunit of the CRAC channel, STIM1 is the ER Ca(2+) sensor and is essential for the recruitment and activation of Orai1. However, the mechanisms in transmission of information of STIM1 to Orai1 still need further investigation. Bimolecular fluorescence complementation (BiFC) is one of the most advanced and powerful tools for studying and visualising protein-protein interactions in living cells. We utilised BiFC and acceptor photobleaching fluorescence resonance energy transfer (FRET) experiments to visualise and determine the state of STIM1 in the living cells in resting state. Our results demonstrate that STIM1 exists in an oligomeric form in resting cells and that rather than the SAM motif, it is the C-terminus (residues 233-474) of STIM1 that is the key domain for the interaction between STIM1s. The STIM1 oligomers (BiFC-STIM1) and wild-type STIM1 colocalised and had a fibrillar distribution in resting conditions. Depletion of ER Ca(2+) stores induced BiFC-STIM1 distribution to become punctate, an effect that could be prevented or reversed by 2-APB. After depletion of the Ca(2+) stores, BiFC-STIM1 has the ability to form puncta that colocalise with wild-type STIM1 or Orai1 near the plasma membrane. Our data also indicate that the function of BiFC-STIM1 was not altered compared with that of wild-type STIM1.     

How strict is the correlation between STIM1 and Orai1 expression, puncta formation, and ICRAC activation?

Stromal interaction molecule 1 (STIM1) and Orai1 have been identified as crucial elements of the store-operated Ca(2+) entry (SOCE) pathway, but the mechanism of their functional interaction remains controversial. It is now well established that, upon depletion of the stores, both molecules can accumulate and colocalize in specific areas (puncta) where the endoplasmic reticulum comes in close proximity to the plasma membrane. Some models propose a direct interaction between STIM1 and Orai1 as the most straightforward mechanism for signal transduction from the stores to the plasma membrane. To test some of the predictions of a conformational coupling model, we assessed how tight the relationships are between STIM1 and Orai1 expression, puncta formation, and SOCE activation. Here we present evidence that STIM1 accumulates in puncta equally well in the presence or absence of Orai1 expression, that STIM1 accumulation is not sufficient for Orai1 accumulation in the same areas, and that normal Ca(2+) release-activated Ca(2+) current (I(CRAC)) can be activated in STIM1-deficient cells. These data challenge the idea of direct conformational coupling between STIM1 and Orai1 as a viable mechanism of puncta formation and SOCE activation and uncover greater complexity in their relationship, which may require additional intermediate elements.     

SARAF inactivates the store operated calcium entry machinery to prevent excess calcium refilling

Store operated calcium entry (SOCE) is a principal cellular process by which cells regulate basal calcium, refill intracellular Ca(2+) stores, and execute a wide range of specialized activities. STIM and Orai proteins have been identified as the essential components enabling the reconstitution of Ca(2+) release-activated Ca(2+) (CRAC) channels that mediate SOCE. Here, we report the molecular identification of SARAF as a negative regulator of SOCE. Using?heterologous expression, RNAi-mediated silencing and site directed mutagenesis combined with electrophysiological, biochemical and imaging techniques we show that SARAF is an endoplasmic reticulum membrane resident protein that associates with STIM to facilitate slow Ca(2+)-dependent inactivation of SOCE. SARAF plays a key role in shaping cytosolic Ca(2+) signals and determining the content of the major intracellular Ca(2+) stores, a role that is likely to be important in protecting cells from Ca(2+) overfilling.     

STIM和Orai在钙库操纵的钙内流途径及心脑血管疾病中的作用

钙库操纵的钙内流(SOCE)是调节钙离子(Ca2+)内流进入细胞最普遍的一种途径,它的通道称为钙库操纵的钙内流通道(SOC)。SOC存在于大多数非兴奋细胞和部分兴奋细胞上,近年来确定,STIM和Orai是组成SOC的两种主要蛋白质。本文就近年来对SOCE途径的机制,STIM和Orai不同亚型的结构、功能及在心脑血管疾病中的作用作一综述。     

Emerging perspectives in store-operated Ca2+ entry: roles of Orai, Stim and TRP

Depletion of intracellular Ca2+ stores induces Ca2+ influx across the plasma membrane through store-operated channels (SOCs). This store-operated Ca2+ influx is important for the replenishment of the Ca2+ stores, and is also involved in many signaling processes by virtue of the ability of intracellular Ca2+ to act as a second messenger. For many years, the molecular identities of particular SOCs, as well as the signaling mechanisms by which these channels are activated, have been elusive. Recently, however, the mammalian proteins STIM1 and Orai1 were shown to be necessary for the activation of store-operated Ca2+ entry in a variety of mammalian cells. Here we present molecular, pharmacological, and electrophysiological properties of SOCs, with particular focus on the roles that STIM1 and Orai1 may play in the signaling processes that regulate various pathways of store-operated entry.     

Overexpression of transient receptor potential canonical type 1 (TRPC1) alters both store operated calcium entry and depolarization-evoked calcium signals in C2C12 cells

When the intracellular calcium stores are depleted, a Ca(2+) influx is activated to refill these stores. This store-operated Ca(2+) entry (SOCE) depends on the cooperation of several proteins as STIM1, Orai1, and, possibly, TRPC1. To elucidate this role of TRPC1 in skeletal muscle, TRPC1 was overexpressed in C2C12 cells and SOCE was studied by measuring the changes in intracellular Ca(2+) concentration ([Ca(2+)](i)). TRPC1 overexpression significantly increased both the amplitude and the maximal rate-of-rise of SOCE. When YM-58483, an inhibitor of TRPC1 was used, these differences were eliminated, moreover, SOCE was slightly suppressed. A decrease in the expression of STIM1 together with the downregulation of SERCA was confirmed by Western-blot. As a consequence, a reduction in maximal Ca(2+) uptake rate and a higher resting [Ca(2+)](i) following the Ca(2+) transients evoked by 120mM KCl were detected. Morphological changes also accompanied the overexpression of TRPC1. Differentiation of the myoblasts started later, and the myotubes were thinner in TRPC1-overexpressing cultures. For these changes the observed decrease in the nuclear expression of NFAT1 could be responsible. Our results suggest that enhanced expression of TRPC1 increases SOCE and has a negative effect on the STIM1-Orai1 system, indicating an interaction between these proteins.     

Role of STIM and Orai proteins in the store-operated calcium signaling pathway

Ca(2+) signals are universal among cells in regulating a spectrum of cellular responses. Phospholipase C-coupled receptors activate two components of Ca(2+) signals--rapid Ca(2+) release from ER stores, followed by slower Ca(2+) entry from outside the cell. The coupling process between ER and PM to mediate this "store-operated" Ca(2+) entry process remained until recently a molecular mystery. The recent discovery of the necessity for STIM1 and Orai proteins in this process has provided crucial information on the coupling mechanism between stores and PM Ca(2+) entry. STIM1 is a single spanning membrane protein with an unpaired Ca(2+) binding EF-hand and appears to function as the sensor of ER luminal Ca(2+), and, through redistribution in the ER, transduces information directly to the PM. Orai1 is a tetra-spanning PM protein and functions as the highly Ca(2+)-selective channel in the PM that is gated through interactions with the store-activated ER Ca(2+) sensor. Recent evidence shows the two proteins together are necessary and sufficient for the function of store-operated Ca(2+) entry. However, many questions arise about how and where the interactions of the STIM1 and Orai1 proteins occur within cells. Here we discuss recent information and ideas about the coupling between these proteins that leads to store-operated channel activation.