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。     

内质网钙池操控钙内流研究进展

细胞内的内质网钙库清空所引发的钙内流是细胞钙信号的重要组成,介导胞外钙离子进入细胞内,并参与细胞内一系列广泛的生理过程。该过程主要由内质网上的钙离子感受器STIM蛋白和细胞膜上的Orai钙离子通道的所介导的。对钙库操控性钙内流的研究进展进行了讨论,并展望了未来的研究方向,以期为相关研究提供参考。     

内质网–细胞质膜连接区蛋白质组鉴定及对其中的STIMATE的功能研究

在细胞内存在着连接内质网和细胞膜的特殊连接部位,称之为内质网–细胞质膜连接区(endoplasmic reticulum-plasma membrane junction,ER-PM J)。ER-PM J对脂类代谢及钙信号传导等有重要的作用,主要由STIM1(stromal interaction molecule 1)和Orai1介导的钙库操纵的钙内流(store-operated calcium entry,SOCE)就发生在该部位。但由于技术手段的缺乏,人们对ER-PM连接区中参与调控SOCE的特异的蛋白质组成了解得还不清楚,因而相关研究一直进展缓慢。这里引入了抗坏血酸过氧化酶2(ascorbate peroxidase 2,APEX2)介导的活体生物素原位标记法,标记STIM1附近的蛋白质组,鉴定出了对钙内流起调节作用的STIM激活增强子STIMATE(STIM-activating enhancer),并对其作用机制进行了初步的探究。     

SOCE通路调控的钙超载与急性胰腺炎研究进展

急性胰腺炎(acute pancreatitis, AP)作为临床常见的急腹症,以胰蛋白酶过度激活引发的腺泡细胞及周围组织自身消化为主要特征。大量证据表明,持续钙超载导致腺泡细胞坏死及过度凋亡是AP的重要发病环节。钙库操纵的Ca~(2+)通道(store-operated calcium entry, SOCE)是引起包括胰腺腺泡细胞在内的非兴奋细胞钙超载的关键,而钙释放激活钙通道蛋白Orai作为SOCE信号通路的核心分子调节钙通道的开放状态。新近的研究证实,SOCE通路在调控胰腺腺泡细胞钙超载上发挥重要作用。该文拟对SOCE调控钙超载参与AP的研究进展做一综述。     

钙释放激活钙通道研究进展

库操纵的钙(Store Operated Calcium,SOC)进入参与许多重要Ca2+信号生理过程,如细胞分化和凋亡虽然SOC的许多生物物理特性被表述,但研究最清楚的是钙释放激活的钙(Ca2+ release-activated Ca2+,CRAC)通道.最近通过RNA干扰技术在果蝇和哺乳动物细胞上鉴定出CRAC通道的两个组成蛋白STIM1和Orail细胞静息时,STIM1均匀分布在内质网膜(ER)上.一当钙库耗竭,ER上STIM1会聚集迁移到细胞膜下,相比而言,Orail是一个形成CRAC通道孔的四次跨膜蛋白.有报道说STIM1作为ER上一个Ca2+感受器向细胞膜传导钙库耗竭信号.虽然钙库耗竭激活CRAC通道的过程在最近的研究中被定量描述为四个步骤,但还有很多细节仍然不清楚.如STIM1是如何感受钙库耗竭而导致其发生聚集的不清楚,又如STIM1是如何定位到细胞膜下又如何传导信息的不清楚,STIM1和Orai1直接到底是如何相互作用的等都有待进一步的研究.本文对CRAC通道的研究历史和最新进展进行了讨论.     

内质网上的蛋白质RCN2与STIM1-Orai1复合体相互作用

膜蛋白质Orail组成了一类被称为钙释放激活钙通道（CRAC）的离子通道,并且由相互作用的蛋白质STIM1作为其在内质网上的钙感受器．但是这类通道的调节机制还未研究透彻．通过串连亲和纯化STIM1-Orai1复合体,发现与之相互作用的内质网蛋白质RCN2．共聚焦显微术显示RCN2与STIM1在钙库排空前后完全共定位．对RCN2的EFhands结构突变体所作单细胞测钙,结果显示其对钙库操控通道电流特性有微弱影响．全内反射荧光显微术显示,RCN2以花环状围绕包围STIM1聚集堆,这提示RCN2在STIM1聚集中起到一种结构约束作用．     

血管平滑肌收缩的Ca^2＋信号调节机制

血管平滑肌细胞内Ca^2＋的浓度（[Ca^2＋]i）的变化及胞内收缩蛋白对Ca^2＋的敏感性是影响血管紧张的主要因素。研究表明细胞内Ca^2＋浓度的变化在血管平滑肌细胞的激活中发挥重要作用。在静息状态,细胞内的Ca^2＋浓度主要受膜电位的调节,同时,[Ca^2＋]i也可反馈调节膜电位。在平滑肌细胞内存在多种[Ca^2＋]i调节机制。本文概述了这些机制在调节血管平滑肌紧张中的作用,主要包括：[Ca^2＋]i在血管平滑肌收缩中的作用;环二磷酸腺苷（cADPR）在调节Ca^2＋释放中的作用;cADPR介导的肉桂碱受体的激活在调节平滑肌紧张度中的作用;血管平滑肌细胞的Ca^2＋闪烁和细胞膜Ca^2＋敏感性钾通道的激活;[Ca^2＋]i与膜电位之间的相互作用等。     

离子通道与人类遗传病

细胞膜离子通道结构和功能正常是细胞进行生理活动的基础,对离子通道功能具有决定性意义的特定位点的突变导致其开放、关闭或激活、失活功能异常,引起组织机能紊乱,形成各种遗传性疾病。本文从水通道蛋白,钙通道,钠通道,钾通道等多种通道蛋白引起的遗传病的现象以及机理做较深入的阐述。     

H_2O_2通过钙稳态失调诱导小鼠胚胎肝细胞凋亡

该文研究了体外培养肝细胞内钙离子浓度改变对细胞存活率、凋亡和增殖的影响。建立了H2O2诱导小鼠胚胎肝细胞损伤模型,CCK-8检测细胞存活率,Fura-2/AM负载检测细胞内[Ca2+]i;免疫荧光和Western blot分别检测STIM1和Orai1在细胞内的定位和含量;流式细胞术检测细胞凋亡;Brdu掺入检测细胞增殖。结果显示,H2O2刺激后细胞存活率降低为对照组的73%,凋亡细胞比例增加,增殖细胞数目显著减少,细胞内[Ca2+]i升高,STIM1和Orai1蛋白质水平增加,且STIM1可与Orai1蛋白质共定位。2-APB预处理组可以降低细胞内[Ca2+]i,减少STIM1和Orai1蛋白质表达水平,抑制STIM1和Orai1蛋白质的相互作用。结果表明,H2O2可通过影响细胞内钙离子稳态导致细胞凋亡。     

容量性钙内流通道的分子代表

细胞内钙库排空产生一种信号,诱导细胞膜上的钙库操纵的钙通道（SOC）开放,使Ca^2 由细胞外进入细胞内,称为容量性钙内流（CCE）,或钙释放激活的钙通道（CRAC）,可能由果蝇一过性受体电位（trp)和trp样（trpl)基因编码,钙库排空和通道开放之间的偶联机制不清,目前主要提出三种机制：（1）弥散信使;（2）蛋白质－蛋白质之间的相互作用;（3）囊泡分泌。本文综述了CCE的分子代表 ,可能机制及电生理表型。     

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.     

TRPC1, Orai1, and STIM1 in SOCE: Friends in tight spaces

Store-operated calcium entry (SOCE) is a ubiquitous Ca²⁺ entry pathway that is activated in response to depletion of ER-Ca²⁺ stores and critically controls the regulation of physiological functions in miscellaneous cell types. The transient receptor potential canonical 1 (TRPC1) is the first member of the TRPC channel subfamily to be identified as a molecular component of SOCE. While TRPC1 has been shown to contribute to SOCE and regulate various functions in many cells, none of the reported TRPC1-mediated currents resembled I_CRAC, the highly Ca²⁺-selective store-dependent current first identified in lymphocytes and mast cells. Almost a decade after the cloning of TRPC1 two proteins were identified as the primary components of the CRAC channel. The first, STIM1, is an ER-Ca²⁺ sensor protein involved in activating SOCE. The second, Orai1 is the pore-forming component of the CRAC channel. Co-expression of STIM1 and Orai1 generated robust I_CRAC. Importantly, STIM1 was shown to also activate TRPC1 via its C-terminal polybasic domain, which is distinct from its Orai1-activating domain, SOAR. In addition, TRPC1 function critically depends on Orai1-mediated Ca²⁺ entry which triggers recruitment of TRPC1 into the plasma membrane where it is then activated by STIM1. TRPC1 and Orai1 form discrete STIM1-gated channels that generate distinct Ca²⁺ signals and regulate specific cellular functions. Surface expression of TRPC1 can be modulated by trafficking of the channel to and from the plasma membrane, resulting in changes to the phenotype of TRPC1-mediated current and [Ca²⁺]_i signals. Thus, TRPC1 is activated downstream of Orai1 and modifies the initial [Ca²⁺]_i signal generated by Orai1 following store depletion. This review will summarize the important findings that underlie the current concepts for activation and regulation of TRPC1, as well as its impact on cell function.     

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.     

cAMP induces stromal interaction molecule 1 (STIM1) puncta but neither Orai1 protein clustering nor store-operated Ca2+ entry (SOCE) in islet cells

The events leading to the activation of store-operated Ca(2+) entry (SOCE) involve Ca(2+) depletion of the endoplasmic reticulum (ER) resulting in translocation of the transmembrane Ca(2+) sensor protein, stromal interaction molecule 1 (STIM1), to the junctions between ER and the plasma membrane where it binds to the Ca(2+) channel protein Orai1 to activate Ca(2+) influx. Using confocal and total internal reflection fluorescence microscopy, we studied redistribution kinetics of fluorescence-tagged STIM1 and Orai1 as well as SOCE in insulin-releasing β-cells and glucagon-secreting α-cells within intact mouse and human pancreatic islets. ER Ca(2+) depletion triggered accumulation of STIM1 puncta in the subplasmalemmal ER where they co-clustered with Orai1 in the plasma membrane and activated SOCE. Glucose, which promotes Ca(2+) store filling and inhibits SOCE, stimulated retranslocation of STIM1 to the bulk ER. This effect was evident at much lower glucose concentrations in α- than in β-cells consistent with involvement of SOCE in the regulation of glucagon secretion. Epinephrine stimulated subplasmalemmal translocation of STIM1 in α-cells and retranslocation in β-cells involving raising and lowering of cAMP, respectively. The cAMP effect was mediated both by protein kinase A and exchange protein directly activated by cAMP. However, the cAMP-induced STIM1 puncta did not co-cluster with Orai1, and there was no activation of SOCE. STIM1 translocation can consequently occur independently of Orai1 clustering and SOCE.     

Open Sesame: treasure in store-operated calcium entry pathway for cancer therapy

Store-operated Ca2+ entry(SOCE) controls intracellular Ca2+ homeostasis and regulates a wide range of cellular events including proliferation,migration and invasion.The discovery of STIM proteins as Ca2+ sensors and Orai proteins as Ca2+ channel pore forming units provided molecular tools to understand the physiological function of SOCE.Many studies have revealed the pathophysiological roles of Orai and STIM in tumor cells.This review focuses on recent advances in SOCE and its contribution to tumorigenesis.Altered Orai and/or STIM functions may serve as biomarkers for cancer prognosis,and targeting the SOCE pathway may provide a novel means for cancer treatment.     

Pore opening mechanism of CRAC channels

Three decades ago, James W. Putney Jr. conceptualized the idea of store-operated calcium entry (SOCE) to explain how depletion of endoplasmic reticulum (ER) Ca²⁺ stores evokes Ca²⁺ influx across the plasma membrane. Since the publication of this highly influential idea, it is now established that SOCE is universal among non-excitable and probably even many types of excitable cells, and contributes to numerous effector functions impacting immunity, muscle contraction, and brain function. The molecules encoding SOCE, the STIM and Orai proteins, are now known and our understanding of how this pathway is activated in response to ER Ca²⁺ store depletion has advanced significantly. In this review, we summarize the current knowledge of how Orai1 channels are activated by STIM1, focusing on recent work supporting a hydrophobic gating mechanism for the opening of the Orai1 channel pore.     

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.     

STIM1 long and STIM1 gate differently TRPC1 during store-operated calcium entry

During myogenesis, a long splice variant of STIM1, called STIM1L is getting expressed, while the level of STIM1 remains constant. Previous work demonstrated that STIM1L is more efficient in eliciting store-operated Ca²⁺ entry (SOCE), but no current analysis of the channel(s) activated by this new STIM1L isoform was performed until now. In this study, we investigate the ionic channel(s) activated by STIM1L and whether differences exist between the two STIM1 isoforms, using HEK-293 T cells as a model system. Our data show that STIM1 and STIM1L activate Orai1 channel but also the endogenously expressed TRPC1. The channel activation occurs in two steps, with first Orai1 activation followed, in a subset of cells, by TRPC1 opening. Remarkably, STIM1L more frequently activates TRPC1 and preferentially interacts with TRPC1. In low intracellular Ca²⁺ buffering condition, the frequency of TRPC1 opening increases significantly, strongly suggesting a Ca²⁺-dependent channel activation. The ability of STIM1L to open Orai1 appears decreased compared to STIM1, which might be explained by its stronger propensity towards TRPC1. Indeed, increasing the amount of STIM1L results in an enhanced Orai1 current. The role of endogenous TRPC1 in STIM1- and STIM1L-induced SOCE was confirmed by Ca²⁺ imaging experiments. Overall, our findings provide a detailed analysis of the channels activated by both STIM1 isoforms, revealing that STIM1L is more prone to open TRPC1, which might explain the larger SOCE elicited by this isoform.