InsP₃receptors and Orai channels in pancreatic acinar cells: co-localization and its consequences

Orai1 proteins have been recently identified as subunits of SOCE (store-operated Ca2? entry) channels. In primary isolated PACs (pancreatic acinar cells), Orai1 showed remarkable co-localization and co-immunoprecipitation with all three subtypes of IP?Rs (InsP? receptors). The co-localization between Orai1 and IP?Rs was restricted to the apical part of PACs. Neither co-localization nor co-immunoprecipitation was affected by Ca2? store depletion. Importantly we also characterized Orai1 in basal and lateral membranes of PACs. The basal and lateral membranes of PACs have been shown previously to accumulate STIM1 (stromal interaction molecule 1) puncta as a result of Ca2? store depletion. We therefore conclude that these polarized secretory cells contain two pools of Orai1: an apical pool that interacts with IP?Rs and a basolateral pool that interacts with STIM1 following the Ca2? store depletion. Experiments on IP?R knockout animals demonstrated that the apical Orai1 localization does not require IP?Rs and that IP?Rs are not necessary for the activation of SOCE. However, the InsP?-releasing secretagogue ACh (acetylcholine) produced a negative modulatory effect on SOCE, suggesting that activated IP?Rs could have an inhibitory effect on this Ca2? entry mechanism.     

Are Orai1 and Orai3 channels more important than calcium influx for cell proliferation?

Transformed and tumoral cells share the characteristic of being able to proliferate even when external calcium concentration is very low. We have investigated whether Human Embryonic Kidney 293 cells, human hepatoma cell Huh-7 and HeLa cells were able to proliferate when kept 72 h in complete culture medium without external calcium. Our data showed that cell proliferation rate was similar over a range of external calcium concentration (2 μM to 1.8 mM). Incubation in the absence of external calcium for 72 h had no significant effect on endoplasmic reticulum (ER) Ca^2 + contents but resulted in a significant decrease in cytosolic free calcium concentration in all 3 cell types. Cell proliferation rates were dependent on Orai1 and Orai3 expression levels in HEK293 and HeLa cells. Silencing Orai1 or Orai3 resulted in a 50% reduction in cell proliferation rate. Flow cytometry analysis showed that Orai3 induced a small but significant increase in cell number in G₂/M phase. RO-3306, a cdk-1 inhibitor, induced a 90% arrest in G₂/M reversible in less than 15 min. Our data showed that progression through G₂/M phase after release from RO-3306-induced cell cycle arrest was slower in both Orai1 and Orai3 knock-downs. Overexpressing Orai1, Orai3 and the dominant negative non-permeant mutants E106Q-Orai1 and E81Q-Orai3 induced a 50% increase in cell proliferation rate in HEK293 cells. Our data clearly demonstrated that Orai1 and Orai3 proteins are more important than calcium influx to control cell proliferation in some cell lines and that this process is probably independent of I_CRAC and I_arc.     

Role of Trpc channels, Stim1 and Orai1 in PGF(2α)-induced calcium signaling in NRK fibroblasts

Normal rat kidney (NRK) fibroblasts exhibit growth-dependent changes in electrophysiological properties and intracellular calcium dynamics. The transition from a quiescent state to a density-arrested state results in altered calcium entry characteristics. This coincides with modulation of the expression of the genes encoding the calcium channels Trpc1, Trpc6 and Orai1, and of the intracellular calcium sensor Stim1. In the present study we have used gene selective short hairpin (sh) RNAs against these various genes to investigate their role in (a) capacitative store-operated calcium entry (SOCE); (b) non-capacitative OAG-induced receptor-operated calcium entry (ROCE); and (c) prostaglandin F(2α) (PGF(2α))-induced Ca(2+)-oscillations in NRK fibroblasts. Intracellular calcium measurements revealed that knockdown of the genes encoding Trpc1, Orai1 and Stim1 each caused a significant reduction of SOCE in NRK cells, whereas knockdown of the gene encoding Trpc6 reduced only the OAG-induced ROCE. Furthermore, our data show that knockdown of the genes encoding Trpc1, Orai1 and Stim1, but not Trpc6, substantially reduced the frequency (up to 60%) of PGF(2α)-induced Ca(2+) oscillations in NRK cells. These results indicate that in NRK cells distinct calcium channels control the processes of SOCE, ROCE and PGF(2α)-induced Ca(2+) oscillations.     

钙库调控的钙通道Orai1体外研究方法的建立

目的:建立钙通道Orai1的体外研究方法。方法:利用脂质体重组技术,将体外纯化的Orai1蛋白重组到脂质体膜上,利用蔗糖密度梯度离心来检测其重组效率及Orai1蛋白在脂质体膜上的结构,并利用钙染料Fura-2检测脂质体内钙离子的释放。结果:成功制备了脂质体及体外纯化了GST-Orai1融合蛋白,蔗糖密度梯度离心结果证明GST-Orai1蛋白成功重组到脂质体上,以及Orai1蛋白以多聚体的形式定位在脂质体膜上。钙离子释放实验证明脂质体内钙离子包装完好,可用于后续Orai1钙通道的功能研究。结论:利用脂质体重组技术建立了一种新的Orai1的研究方法,能够更直接有效地研究其功能及其活化机制。     

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.     

Histamine regulates cyclooxygenase 2 gene activation through Orai1-mediated NFκB activation in lung cancer cells

Histamine, an important chemical mediator, has been shown to regulate inflammation and allergic responses. Stimulation of histamine receptors results in a significant increase in cytoplasmic Ca²⁺, which could be mediated by inositol trisphosphate (IP₃)-dependent store-operated Ca²⁺ channels (SOC). However, the link between histamine-mediated signaling and activation of inflammatory genes such as cyclooxygenase 2 (COX-2) is still unknown. Our study indicated that the COX-2 protein was highly expressed in human lung cancer cells. Following stimulation with 10 μM of histamine, both store-operated Ca²⁺ entry (SOCE) and COX-2 gene expression were evoked. Histamine-mediated COX-2 activation can be prevented by 2-APB and SKF-96365, SOC channel inhibitors. In addition, deletion analysis of the COX-2 promoter suggested that the region between −80 bp and −250 bp, which contains NFκB binding sites, is the key element for histamine-mediated signaling. Knocking down ORAI1, one of the essential molecules of store-operated calcium channels, attenuated histamine-mediated COX-2 expression and NFκB activation. These results indicated that ORAI1-mediated NFκB activation was an important signaling pathway, responsible for transmitting histamine signals that trigger inflammatory reactions.     

Store-operated Ca2+ Entry Mediated by Orai1 and TRPC1 Participates to Insulin Secretion in Rat β-Cells

Store-operated Ca²⁺ channels (SOCs) are voltage-independent Ca²⁺ channels activated upon depletion of the endoplasmic reticulum Ca²⁺ stores. Early studies suggest the contribution of such channels to Ca²⁺ homeostasis in insulin-secreting pancreatic β-cells. However, their composition and contribution to glucose-stimulated insulin secretion (GSIS) remains unclear. In this study, endoplasmic reticulum Ca²⁺ depletion triggered by acetylcholine (ACh) or thapsigargin stimulated the formation of a ternary complex composed of Orai1, TRPC1, and STIM1, the key proteins involved in the formation of SOCs. Ca²⁺ imaging further revealed that Orai1 and TRPC1 are required to form functional SOCs and that these channels are activated by STIM1 in response to thapsigargin or ACh. Pharmacological SOCs inhibition or dominant negative blockade of Orai1 or TRPC1 using the specific pore mutants Orai1-E106D and TRPC1-F562A impaired GSIS in rat β-cells and fully blocked the potentiating effect of ACh on secretion. In contrast, pharmacological or dominant negative blockade of TRPC3 had no effect on extracellular Ca²⁺ entry and GSIS. Finally, we observed that prolonged exposure to supraphysiological glucose concentration impaired SOCs function without altering the expression levels of STIM1, Orai1, and TRPC1. We conclude that Orai1 and TRPC1, which form SOCs regulated by STIM1, play a key role in the effect of ACh on GSIS, a process that may be impaired in type 2 diabetes.     

Crystal Structure of Calmodulin Binding Domain of Orai1 in Complex with Ca2+?Calmodulin Displays a Unique Binding Mode

Orai1 is a plasma membrane protein that in its tetrameric form is responsible for calcium influx from the extracellular environment into the cytosol in response to interaction with the Ca²⁺-depletion sensor STIM1. This is followed by a fast Ca²⁺·calmodulin (CaM)-dependent inhibition, resulting from CaM binding to an Orai1 region called the calmodulin binding domain (CMBD). The interaction between Orai1 and CaM at the atomic level remains unknown. Here, we report the crystal structure of a CaM·Orai1-CMBD complex showing one CMBD bound to the C-terminal lobe of CaM, differing from other CaM-target protein complexes, in which both N- and C-terminal lobes of CaM (CaM-N and CaM-C) are involved in target binding. Orai1-CMBD binds CaM-C mainly through hydrophobic interactions, primarily involving residue Trp⁷⁶ of Orai1-CMBD, which interacts with the hydrophobic pocket of CaM-C. However, NMR data, isothermal titration calorimetry data, and pulldown assays indicated that CaM-N and CaM-C both can bind Orai1-CMBD, with CaM-N having ∼4 times weaker affinity than CaM-C. Pulldown assays of a Orai1-CMBD(W76E) mutant, gel filtration chromatography data, and NOE signals indicated that CaM-N and CaM-C can each bind one Orai1-CMBD. Thus our studies support an unusual, extended 1:2 binding mode of CaM to Orai1-CMBDs, and quantify the affinity of Orai1 for CaM. We propose a two-step mechanism for CaM-dependent Orai1 inactivation initiated by binding of the C-lobe of CaM to the CMBD of one Orai1 followed by the binding of the N-lobe of CaM to the CMBD of a neighboring Orai1.     

STIM1 and Orai1: novel targets for vascular diseases?

The past five years have witnessed the discovery of the endoplasmic reticulum calcium (Ca²⁺) sensor STIM1 and the plasma membrane Ca²⁺ channel Orai1 as the bona fide molecular components of the store-operated Ca²⁺ entry (SOCE) and the Ca²⁺ release-activated Ca²⁺ current (I _CRAC). It has been known for two decades that SOCE and I _CRAC are required for lymphocyte activation as evidenced by severe immunodeficient phenotypes in patients lacking I _CRAC. In recent years however, studies have uncovered expression of STIM1 and Orai1 proteins in various tissues and described additional roles for these proteins in physiological functions and pathophysiological conditions. Here, we will summarize novel findings pertaining to the role of STIM1 and Orai1 in the vascular system and discuss their potential use as targets in the therapy of vascular disease.     

Molecular dynamics studies on conformational thermodynamics of Orai1–calmodulin complex

Molecular understanding of bio-macromolecular binding is a challenging task due to large sizes of the molecules and presence of variety of interactions. Here, we study the molecular mechanism of calmodulin (CaM) binding to Orai1 that regulates Ca²⁺-dependent inactivation process in eukaryotic cells. Although experimental observations indicate that Orai1 binds to the C-terminal of Ca²⁺-loaded CaM, it is not decisive if N-domain of CaM interacts with Orai1. We address the issue of interaction of different domains of CaM with Orai1 using conformational thermodynamic changes, computed from histograms of dihedral angles over simulated trajectories of CaM, CaM-binding domain of Orai1 and complexes of CaM with Orai1. The changes for all residues of both C and N terminal domains of CaM upon Orai1 binding are compared. Our analysis shows that Orai1binds to both C-terminal and N-terminal domains of CaM, indicating 1:2 stoichiometry. The Orai1 binding to N-terminal domain of CaM is less stable than that to the C-terminal domain. The binding residues are primarily hydrophobic. These observations are in qualitative agreement to the experiments. The conformational thermodynamic changes thus provide a useful computational tool to provide atomic details of interactions in bio-macromolecular binding.     

Molecular Determinants of the Coupling between STIM1 and Orai Channels: DIFFERENTIAL ACTIVATION OF Orai1�C3 CHANNELS BY A STIM1 COILED-COIL MUTANT*

STIM1 and Orai1 have been reported to interact upon store depletion culminating in Ca²⁺ release-activated Ca²⁺ current activation. Recently, the essential region has been identified within the STIM1 C terminus that includes the second coiled-coil domain C-terminally extended by ∼50 amino acids and exhibits a strong binding to the Orai1 C terminus. Based on the homology within the Orai family, an analogous scenario might be assumed for Orai2 as well as Orai3 channels as both are activated in a similar STIM1-dependent manner. A combined approach of electrophysiology and Foerster resonance energy transfer microscopy uncovered a general mechanism in the communication of STIM1 with Orai proteins that involved the conserved putative coiled-coil domains in the respective Orai C terminus and the second coiled-coil motif in the STIM1 C terminus. A coiled-coil single mutation in the Orai1 C terminus abrogated communication with the STIM1 C terminus, whereas an analogous mutation in Orai2 and Orai3 still allowed for their moderate activation. However, increasing coiled-coil probability by a gain of function deletion in Orai1 or by generating an Orai1-Orai3 chimera containing the Orai3 C terminus recovered stimulation to a similar extent as with Orai2/3. At the level of STIM1, decreasing probability of the second coiled-coil domain by a single mutation within the STIM1 C terminus abolished activation of Orai1 but still enabled partial stimulation of Orai2/3 channels. A double mutation within the second coiled-coil motif of the STIM1 C terminus fully disrupted communication with all three Orai channels. In aggregate, the impairment in the overall communication between STIM1 and Orai channels upon decreasing probabilities of either one of the putative coiled-coil domains in the C termini might be compatible with the concept of their functional, heteromeric interaction.Store-operated Ca²⁺ entry is a key to cellular regulation of short term responses such as contraction and secretion as well as long term processes like proliferation and cell growth (1). The prototypic and best characterized store-operated channel is the Ca²⁺ release-activated Ca²⁺ (CRAC)⁵ channel (2–6). However, its molecular components have remained elusive until 4 years ago; the STIM1 (stromal interacting molecule 1) (7, 8) and later on Orai1 (9–11) have been identified as the two limiting components for CRAC activation. STIM1 is an ER-located Ca²⁺ sensor, and store depletion triggers its aggregation into punctae close to the plasma membrane, resulting in stimulation of CRAC currents (12, 13). Its N terminus is located in the ER lumen and contains an EF-hand Ca²⁺-binding motif, which senses the ER Ca²⁺ level, and a sterile α-motif, which is suggested to mediate homomeric STIM1 aggregation (14–16). In the cytosolic STIM1 C terminus, two coiled-coil regions overlapping with the ezrin-radixin-moesin-like domain and a lysine-rich region are essential for CRAC activation (14, 17, 18). Three recent studies have independently identified the ezrin-radixin-moesin domain as the essential Orai activating domain, named SOAR (STIM1 Orai-activating region) (20) which represents so far the shortest active fragment, OASF (Orai-activating small fragment) (21) or CAD (CRAC-activating domain) (22), which includes the second, more C terminally located coiled-coil domain and the following ∼55 amino acids. The latter amino acids are suggested to contain an additional cytosolic homomerization domain indispensable for OASF homomerization and Orai activation (21).The Orai family includes three highly Ca²⁺-selective ion channels (Orai1–3) that locate to the plasma membrane, and each protein contains four predicted transmembrane segments with cytosolic N and C termini (10). All three Orai proteins possess a conserved putative coiled-coil domain in the C terminus (23, 24), whereas only the N terminus of Orai1 consists of a proline/arginine-rich region (25). Orai1 has been assumed to act in concert with STIM1 (10, 27)-activating inward Ca²⁺ currents after store depletion. The two other members of the Orai family, Orai2 and Orai3, display similar but smaller store-operated inward Ca²⁺ currents when co-expressed with STIM1 with distinct inactivation profiles, permeability properties, and 2-aminoethoxydiphenyl borate sensitivity (28–32). Recently, we have provided evidence for a store depletion-induced, dynamic coupling of STIM1 to Orai1 that involves the putative coiled-coil domain in the C terminus of Orai1 (33). Furthermore, the C terminus of STIM1, in particular the essential cytosolic region 344–442 as narrowed down by SOAR, OASF, and CAD (20–22), has been established as the key fragment for CRAC as well as Orai1 activation, because its expression alone, without the necessity to deplete ER store, is sufficient for constitutive current activation (18, 32, 33). These fragments SOAR, OASF, and CAD when co-expressed with Orai1 (20–22) exhibit enhanced plasma membrane localization in comparison with the complete STIM1 C terminus in the presence of Orai1. Specificity of interaction of SOAR to the Orai1 C terminus has been shown by its disruption (20) employing the Orai1 L273S mutant (33). Park et al. (22) have provided additional, conclusive evidence for a direct binding by combining multiple biochemical approaches demonstrating CAD interaction with Orai1.This study focused specifically on the role of the putative coiled-coil domains of STIM1 as well as Orai proteins in their coupling. Coiled-coils generally function as protein-protein interaction sites with the ability of dynamic protein assembly and disassembly (35–37). We suggest the C-terminal, putative coiled-coil domains in all three Orai proteins and the second coiled-coil motif of STIM1 as essential for STIM1/Orai communication. Moreover, the single point coiled-coil STIM1 L373S mutant allowed for differential activation of Orai channels partially stimulating Orai2 as well as Orai3 but not Orai1.     

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。     

钙通道蛋白Orai1在骨肉瘤细胞转移中的作用研究

目的:骨肉瘤是一种常见的恶性骨肿瘤,恶性程度高,往往在早期就会发生远隔器官的转移,从而导致骨肉瘤的预后非常差。Orai1是一类定位于细胞膜,介导钙离子内流的受体依赖性钙通道蛋白。大量研究发现钙通道蛋白Orai1过表达于多种肿瘤细胞,并对维持肿瘤细胞粘附、侵袭、迁移等恶性表型有非常重要的作用。然而,钙通道蛋白Orai1是否参与了骨肉瘤的转移过程,目前未见相关报道。本研究的目的是探究钙通道蛋白Orai1是否在骨肉瘤转移过程中的发挥作用。方法:利用合成的靶向Orai1的小干扰RNA(Orai1 si RNA)片段,转染至人骨肉瘤细胞系Saos-2细胞。在Saos-2细胞中抑制Orai1的表达。采用细胞黏附实验、细胞划痕实验和细胞Transwell实验检测骨肉瘤细胞的黏附、迁移及侵袭等肿瘤细胞转移能力;Western-blot实验检测Saos-2细胞的中黏着斑激酶(FAK)和桩蛋白(Paxillin)的表达水平和磷酸化水平。结果:靶向Orai1 si RNA瞬时转染至骨肉瘤细胞系Saos-2细胞后,Saos-2细胞中Orai1蛋白表达水平和m RNA转录水平均显著下降。并且,在Saos-2细胞中抑制Orai1表达后,Saos-2细胞的黏附能力、迁移能力、及侵袭能力均显著下降。进一步研究发现,在Saos-2细胞中抑制Orai1表达后,Saos-2细胞的FAK和Paxillin磷酸化水平明显下降。结论:Orai1可以促进骨肉瘤细胞的黏附、迁移和侵袭,增加黏着斑的形成,从而促进骨肉瘤的转移。因此,深入研究钙通道蛋白Orai1调控骨肉瘤转移的分子机制,可为骨肉瘤转移的治疗提供新的新方向和新策略。     

Store-operated Ca2+ entry and Ca2+ responses to hypothalamic releasing hormones in anterior pituitary cells from Orai1−/− and heptaTRPC knockout mice

Store operated Ca²⁺ entry (SOCE) is the most important Ca²⁺ entry pathway in non-excitable cells. However, SOCE can also play a pivotal role in excitable cells such as anterior pituitary (AP) cells. The AP gland contains five different cell types that release six major AP hormones controlling most of the entire endocrine system. AP hormone release is modulated by Ca²⁺ signals induced by different hypothalamic releasing hormones (HRHs) acting on specific receptors in AP cells. TRH and LHRH both induce Ca²⁺ release and Ca²⁺ entry in responsive cells while GHRH and CRH only induce Ca²⁺ entry. SOCE has been shown to contribute to Ca²⁺ responses induced by TRH and LHRH but no molecular evidence has been provided. Accordingly, we used AP cells isolated from mice devoid of Orai1 channels (noted as Orai1−/− or Orai1 KO mice) and mice lacking expression of all seven canonical TRP channels (TRPC) from TRPC1 to TRPC7 (noted as heptaTRPC KO mice) to investigate contribution of these putative channel proteins to SOCE and intracellular Ca²⁺ responses induced by HRHs. We found that thapsigargin-evoked SOCE is lost in AP cells from Orai1−/− mice but unaffected in cells from heptaTRPC KO mice. Conversely, while spontaneous intracellular Ca²⁺-oscillations related to electrical activity were not affected in the Orai1−/− mice, these responses were significantly reduced in heptaTRPC KO mice. We also found that Ca²⁺ entry induced by TRH and LHRH is decreased in AP cells isolated from Orai1−/−. In addition, Ca²⁺ responses to several HRHs, particularly TRH and GHRH, are decreased in the heptaTRPC KO mice. These results indicate that expression of Orai1, and not TRPC channel proteins, is necessary for thapsigargin-evoked SOCE and is required to support Ca²⁺ entry induced by TRH and LHRH in mouse AP cells. In contrast, TRPC channel proteins appear to contribute to spontaneous Ca²⁺-oscillations and Ca²⁺ responses induced by TRH and GHRH. We conclude that expression of Orai1 and TRPC channels proteins may play differential and significant roles in AP physiology and endocrine control.     

Glu¹⁰⁶ in the Orai1 pore contributes to fast Ca²⁺-dependent inactivation and pH dependence of Ca²⁺ release-activated Ca²⁺ (CRAC) current

FCDI (fast Ca2?-dependent inactivation) is a mechanism that limits Ca2? entry through Ca2? channels, including CRAC (Ca2? release-activated Ca2?) channels. This phenomenon occurs when the Ca2? concentration rises beyond a certain level in the vicinity of the intracellular mouth of the channel pore. In CRAC channels, several regions of the pore-forming protein Orai1, and STIM1 (stromal interaction molecule 1), the sarcoplasmic/endoplasmic reticulum Ca2? sensor that communicates the Ca2? load of the intracellular stores to Orai1, have been shown to regulate fast Ca2?-dependent inactivation. Although significant advances in unravelling the mechanisms of CRAC channel gating have occurred, the mechanisms regulating fast Ca2?-dependent inactivation in this channel are not well understood. We have identified that a pore mutation, E106D Orai1, changes the kinetics and voltage dependence of the ICRAC (CRAC current), and the selectivity of the Ca2?-binding site that regulates fast Ca2?-dependent inactivation, whereas the V102I and E190Q mutants when expressed at appropriate ratios with STIM1 have fast Ca2?-dependent inactivation similar to that of WT (wild-type) Orai1. Unexpectedly, the E106D mutation also changes the pH dependence of ICRAC. Unlike WT ICRAC, E106D-mediated current is not inhibited at low pH, but instead the block of Na? permeation through the E106D Orai1 pore by Ca2? is diminished. These results suggest that Glu1?? inside the CRAC channel pore is involved in co-ordinating the Ca2?-binding site that mediates fast Ca2?-dependent inactivation.     

Thapsigargin activates Ca²+ entry both by store-dependent, STIM1/Orai1-mediated, and store-independent, TRPC3/PLC/PKC-mediated pathways in human endothelial cells

The ER Ca2+ sensor STIM1 and the Ca2+ channel Orai1 are key players in store-operated Ca2+ entry (SOCE). In addition, channels from the TRPC family were also shown to be engaged during SOCE, while their precise implication remains controversial. In this study, we investigated the molecular players involved in SOCE triggered by the SERCA pump inhibitor thapsigargin in an endothelial cell line, the EA.hy926. siRNA directed against STIM1 or Orai1 reduced Ca2+ entry by about 50-60%, showing that a large part of the entry is independent from these proteins. Blocking the PLC or the PKC pathway completely abolished thapsigargin-induced Ca2+ entry in cells depleted from STIM1 and/or Orai1. The phorbol ester PMA or the DAG analog OAG restored the Ca2+ entry inhibited by PLC blockers, showing an involvement of PLC/PKC pathway in SOCE. Using pharmacological inhibitors or siRNA revealed that the PKCeta is required for Ca2+ entry, and pharmacological inhibition of the tyrosine kinase Src also reduced Ca2+ entry. TRPC3 silencing diminished the entry by 45%, while the double STIM1/TRPC3 invalidation reduced Ca2+ entry by more than 85%. Hence, in EA.hy926 cells, TG-induced Ca2+ entry results from the activation of the STIM1/Orai1 machinery, and from the activation of TRPC3.     

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.     

TRPC1与Orai1的相互作用参与人脐静脉内皮细胞钙敏感受体介导的钙内流及一氧化氮生成

本研究旨在探讨Ca~(2+)感受蛋白经典瞬时感受器电位蛋白1(canonical transient receptor potential channel 1,TRPC1)与钙释放激活钙通道调节分子1(calcium release-activated calcium modulator 1,Orai1)的相互作用在人脐静脉内皮细胞(human umbilical vein endothelial cells,HUVECs)钙敏感受体(Ca-sensing receptor,Ca R)介导的Ca~(2+)内流和一氧化氮(NO)生成中的作用。取2~3代HUVECs,单独或联合用Ca R激动剂精胺[Spermine,激活钙库活化的钙通道(store-operated calcium channels,SOC)和受体活化的钙通道(receptor-operated calcium channels,ROC)]、ROC模拟剂12-O-十四烷酰佛波醇-13-乙酸酯(TPA)+Ca R负性变构调节剂Calhex231(阻断SOC,激活ROC)、PKC抑制剂Ro31-8220(激活SOC,阻断ROC)以及经典型PKCs和PKCμ抑制剂Go6967(激活SOC,阻断ROC)处理。用免疫荧光技术检测HUVECs中TRPC1和Orai1的蛋白表达、共定位;用免疫共沉淀法检测TRPC1和Orai1之间的相互作用模式;随后用TRPC1和Orai1干扰质粒联合转染HUVECs,采用荧光探针同时检测HUVECs中[Ca~(2+)]i和NO生成的变化。结果显示,HUVECs中TRPC1和Orai1蛋白表达共定位于胞膜。与Spermine+Ca~(2+)组相比,Calhex231+TPA+Spermine+Ca~(2+)组、Ro31-8220+Spermine+Ca~(2+)组和Go6976+Spermine+Ca~(2+)组HUVECs中定位于胞浆中的TRPC1、Orai1蛋白表达均显著降低,二者的结合也减少。免疫共沉淀结果显示,Calhex231+TPA+Spermine+Ca~(2+)组、Ro31-8220+Spermine+Ca~(2+)组和Go6976+Spermine+Ca~(2+)组TRPC1/Orai1和Orai1/TRPC1相对比值百分数均明显低于对照组以及Spermine+Ca~(2+)组(均P0.05),表明Orai1和TRPC1相互作用均减弱。联合转染质粒敲低TRPC1和Orai1显著降低Spermine+Ca~(2+)组、Calhex231+TPA+Spermine+Ca~(2+)组、Ro31-8220+Spermine+Ca~(2+)组和Go6976+Spermine+Ca~(2+)组的HUVECs中[Ca~(2+)]i和NO生成。以上结果提示,TRPC1与Orai1以二元复合物的形式激活SOC和ROC,介导Ca~(2+)内流及NO生成。     

A Highlights from MBoC Selection: Single-molecule analysis of diffusion and trapping of STIM1 and Orai1 at endoplasmic reticulum–plasma membrane junctions

Following endoplasmic reticulum (ER) Ca²⁺ depletion, STIM1 and Orai1 complexes assemble autonomously at ER–plasma membrane (PM) junctions to trigger store-operated Ca²⁺ influx. One hypothesis to explain this process is a diffusion trap in which activated STIM1 diffusing in the ER becomes trapped at junctions through interactions with the PM, and STIM1 then traps Orai1 in the PM through binding of its calcium release-activated calcium activation domain. We tested this model by analyzing STIM1 and Orai1 diffusion using single-particle tracking, photoactivation of protein ensembles, and Monte Carlo simulations. In resting cells, STIM1 diffusion is Brownian, while Orai1 is slightly subdiffusive. After store depletion, both proteins slow to the same speeds, consistent with complex formation, and are confined to a corral similar in size to ER–PM junctions. While the escape probability at high STIM:Orai expression ratios is <1%, it is significantly increased by reducing the affinity of STIM1 for Orai1 or by expressing the two proteins at comparable levels. Our results provide direct evidence that STIM-Orai complexes are trapped by their physical connections across the junctional gap, but also reveal that the complexes are surprisingly dynamic, suggesting that readily reversible binding reactions generate free STIM1 and Orai1, which engage in constant diffusional exchange with extrajunctional pools.     

Human ether à-gogo K(+) channel 1 (hEag1) regulates MDA-MB-231 breast cancer cell migration through Orai1-dependent calcium entry

Breast cancer (BC) has a poor prognosis due to its strong metastatic ability. Accumulating data present ether à go‐go (hEag1) K⁺ channels as relevant player in controlling cell cycle and proliferation of non‐invasive BC cells. However, the role of hEag1 in invasive BC cells migration is still unknown. In this study, we studied both the functional expression and the involvement in cell migration of hEag1 in the highly metastatic MDA‐MB‐231 human BC cells. We showed that hEag1 mRNA and proteins were expressed in human invasive ductal carcinoma tissues and BC cell lines. Functional activity of hEag1 channels in MDA‐MB‐231 cells was confirmed using astemizole, a hEag1 blocker, or siRNA. Blocking or silencing hEag1 depolarized the membrane potential and reduced both Ca²⁺ entry and MDA‐MB‐231 cell migration without affecting cell proliferation. Recent studies have reported that Ca²⁺ entry through Orai1 channels is required for MDA‐MB‐231 cell migration. Down‐regulation of hEag1 or Orai1 reduced Ca²⁺ influx and cell migration with similar efficiency. Interestingly, no additive effects on Ca²⁺ influx or cell migration were observed in cells co‐transfected with sihEag1 and siOrai1. Finally, both Orai1 and hEag1 are expressed in invasive breast adenocarcinoma tissues and invaded metastatic lymph node samples (LNM⁺). In conclusion, this study is the first to demonstrate that hEag1 channels are involved in the serum‐induced migration of BC cells by controlling the Ca²⁺ entry through Orai1 channels. hEag1 may therefore represent a potential target for the suppression of BC cell migration, and thus prevention of metastasis development. J. Cell. Physiol. 227: 3837–3846, 2012. © 2012 Wiley Periodicals, Inc.