Zhang Xuexin, Gueguinou Maxime, Trebak Mohamed
The identification of Orai and STIM proteins has opened up new avenues of research in the field of receptor-regulated calcium signaling. The ligation of phospholipase C (PLC)-coupled receptors can activate either the common tore-perated alcium ntry (SOCE) pathway or tore-ndependent alcium ntry (SICE) pathway. The representative conductance of the SOCE pathway is the alcium elease-ctivated alcium (CRAC) channel encoded by Orai (CRACM) proteins. The SICE pathway biophysical manifestation is currents activated by arachidonic acid (AA) or the AA metabolite leukotriene C (LTC) and termed rachidonate-egulated or TC-egulated alcium (ARC/LRC) current encoded by channels composed of both Orai1 and Orai3 proteins. About three decades ago, Putney first proposed the capacitative Ca entry model (subsequently known as SOCE) [1]. Orai1 protein, the pore forming subunit of the CRAC channel, was discovered almost simultaneously by three groups in 2006 [2–4]. The Orai family of channels contains three different proteins (Orai1, 2, and 3) encoded by independent genes [5]. A large number of agonists can act on G protein-coupled receptors (GPCRs) to activate PLC. PLC hydrolyzes phosphatidylinositol-4,5-bisphopshate (PIP) into diacylglycerol (DAG) and inositol-1,4,5-trisphosphate (IP) [6]. The latter binds to IP receptors (IPR) on the membrane of the endoplasmic reticulum (ER), resulting in Ca store emptying. The action of ER Ca store emptying causes stromal interaction molecule 1 (STIM1), a calcium sensor to lose Ca from its N-terminal low affinity EF hand located in the lumen of the ER [7,8]. This causes STIM1 to aggregate and to move to highly specialized areas where the ER comes close to the plasma membrane to physically trap and interact with Orai1 channels and activate Ca entry [8]. STIM1 has one homologue, STIM2, which mediates Orai1 channel activation under resting conditions in the absence of agonist stimulation [9]. In most cells studied so far, SOCE is mediated by STIM1 and Orai1 proteins [10]. However, we reported Orai3-mediated SOCE in a subset of estrogen receptor positive breast cancer cells [11–13]. The ARC channel and its role in calcium signaling have been first reported and intensely studied by Shuttleworth and colleagues [14–17]. This group identified and characterized a conductance in HEK293 cells activated by relatively low exogenous concentrations of arachidonic acid or by low concentrations of a muscarinic agonist [18]. Polyunsaturated fatty acids were described as poor activators of these channels, and mono unsaturated or saturated fatty acids were ineffective [19]. Unlike a number of channels of the ransient eceptor otential anonical (TRPC3/6/7) family [20,21], ARC channels are not activated by high concentrations of DAG (100 μM) [19]. Mignen and colleagues showed that despite many similarities with CRAC currents present in the same cell type studied, ARC channels possess distinct pharmacological characteristics and biophysical properties [18]. For instance, unlike CRAC channels, ARC channels do not show the typical fast a-ependent nactivation (CDI), are not inhibited by a reduction in extracellular pH from 7.2 to 6.7, and are insensitive to 2-aminoethoxydiphenyl borate (2-APB) [18,22]. As is the case with CRAC channels [34,35], the absence of divalent cations in the extracellular recording medium induces the permeability of ARC channels to monovalent cations, such as Na [23]. However, this monovalent macroscopic current has different characteristics from those observed for CRAC channels especially from the perspective of their depotentiation and permeability. By blocking monovalent currents by increasing extracellular calcium concentrations as a relative measure of selectivity of calcium channels, Mignen and colleagues proposed that ARC channels have high Ca selectivity and are 50 times more Ca-selective than CRAC channels [18,22]. These authors argued that ARC channels are the predominant calcium channels activated when cells are stimulated with low concentrations of agonists that induce repetitive calcium oscillations [24]. Using an M3 muscarinic receptor-expressing HEK293 cells and murine parotid and pancreatic acinar cells, they reported the activation of ARC channels mediating intracellular calcium oscillations by low concentrations (0.2–1 μM) of carbachol [25]. In the same cells they described the activation of the AA-producing enzyme, phospholipase A2 type IV, upon stimulation with low concentrations of carbachol. Earlier work by the Shuttleworth group suggested that the pharmacological inhibition of PLA2 with isotetrandrine blocks the activation of ARC channels, while the pharmacological inhibition of the lipoxygenase and cyclooxygenase pathways had no effect on ARC activation [14,26], indicating that AA is produced by receptor-mediated activation of PLA2 and that AA processing into downstream metabolites is not required for ARC channel activation. After identification of STIM and Orai proteins, Shuttleworth and colleagues showed that both Orai1 and Orai3 are required for ARC channel activation [27], in addition to the minor pool of STIM1 located in the plasma membrane [28]. More recent work from our laboratory identified a SICE channel in primary aortic vascular smooth muscle cells (VSMC). We found that this conductance is activated by AA, but AA metabolism into LTC by the enzymatic activity of LTC synthase (LTCS) provided a more robust activation of these channels; LTC acts intracellularly when applied through the patch pipette but not extracellularly when added to the bath solution. We named this channel TC-egulated alcium (LRC) channel [23,29–31]. Collectively, our data in VSMC showed that receptor activation causes production of AA through sequential activation of PLC and DAG lipase and that AA metabolism by 5-lipooxygenase and LTCS into LTC is required for LRC channel activation [29,31]. A molecular knockdown on LTC synthase (LTCS) abrogated receptor-mediated LRC channel activation (using the PAR1 agonist thrombin), while direct application of LTC through the patch pipette robustly activated LRC currents. The biophysical properties of LRC channels were identical to those of ARC channels, prompting us to undertake a side by side comparison in VSMC and HEK293 cells to determine whether these two conductances are mediated by the same or by different cellular pools of STIM and Orai proteins [23]. Briefly, using protein knockdown, pharmacological inhibitors, and a nonmetabolizable form of AA, we found that regardless of the cell type considered (HEK293 cells or VSMC), ARC and LRC currents are the manifestation of the same channel that can be activated by AA but is more robustly activated by LTC [23]. We also found that in both cell types, ARC/LRC currents depended on Orai1, Orai3, and STIM1 [23,29], but unlike findings from the Shuttleworth group, we were able to rescue ARC/LRC activity in HEK293 cells and VSMC with expressed STIM1 constructs that do not traffic to the plasma membrane when using Fura-2 calcium imaging and perforated patch recording in intact cells but not in whole-cell recordings. These results suggest a facilitatory role for PM-STIM1 in ARC/LRC channel activation [23]. Orai1 exists in two variants generated through alternative translation-initiation of the Orai1 mRNA: a longer Orai1α form contains an additional N-terminal (NT) 63 amino acids upstream of the conserved start site of a shorter Orai1β [32]. A study from our group showed that while Orai1α and Orai1β are interchangeable for forming CRAC channels, only Orai1α can support ARC/LRC channels by forming a unique heteromeric channel with Orai3. Studies by the Shuttleworth group were performed before Orai1α variant was discovered; it is therefore unclear which Orai1 subtype was used [33]. We also showed that a specific interaction of STIM1 second C-terminal (CT) coiled-coil (CC2) with Orai3 CT region is required for LRC channel activation by LTC [31]. In summary, the SICE pathway appears to be mediated by one channel entity. In succeeding text, we will refer to this channel as either ARC or LRC, depending on whether we are referring to experiments that used either AA or LTC to activate this conductance. ARC/LRC channels are encoded by Orai1 and Orai3 and regulated by STIM1. There are two major points of contention between our findings and those of the Shuttleworth group: (1) the requirement for AA metabolic conversion into LTC and (2) the cellular pool of STIM1 required for ARC/LRC activation, that is, ER-resident versus PM-resident STIM1.
Orai和STIM蛋白的鉴定为受体调节钙信号领域开辟了新的研究途径。磷脂酶C（PLC）偶联受体的结合可激活常见的储存-操作性钙内流（SOCE）途径或储存-非依赖性钙内流（SICE）途径。SOCE途径的代表性电导是由Orai（CRACM）蛋白编码的钙释放激活钙（CRAC）通道。SICE途径的生物物理表现是由花生四烯酸（AA）或AA代谢物白三烯C4（LTC4）激活的电流，称为由Orai1和Orai3蛋白组成的通道编码的花生四烯酸调节或LTC4调节钙（ARC/LRC）电流。大约三十年前，Putney首次提出了容量性钙内流模型（随后称为SOCE）[1]。CRAC通道的孔形成亚基Orai1蛋白于2006年几乎同时被三个研究小组发现[2-4]。Orai通道家族包含由独立基因编码的三种不同蛋白质（Orai1、2和3）[5]。大量激动剂可作用于G蛋白偶联受体（GPCR）以激活PLC。PLC将磷脂酰肌醇-4,5-二磷酸（PIP2）水解为二酰基甘油（DAG）和肌醇-1,4,5-三磷酸（IP3）[6]。后者与内质网（ER）膜上的IP受体（IPR）结合，导致钙储存排空。ER钙储存排空的作用使基质相互作用分子1（STIM1），一种钙传感器，从其位于ER腔内的N端低亲和力EF手失去钙[7,8]。这导致STIM1聚集并移动到ER靠近质膜的高度特化区域，以物理方式捕获并与Orai1通道相互作用并激活钙内流[8]。STIM1有一个同源物STIM2，它在没有激动剂刺激的静息条件下介导Orai1通道的激活[9]。在迄今为止研究的大多数细胞中，SOCE由STIM1和Orai1蛋白介导[10]。然而，我们报道了在一部分雌激素受体阳性乳腺癌细胞中存在Orai3介导的SOCE[11-13]。ARC通道及其在钙信号中的作用首先由Shuttleworth及其同事报道并进行了深入研究[14-17]。该小组在HEK293细胞中鉴定并表征了一种电导，该电导可由相对低浓度的外源性花生四烯酸或低浓度的毒蕈碱激动剂激活[18]。多不饱和脂肪酸被描述为这些通道的弱激活剂，单不饱和或饱和脂肪酸则无效[19]。与许多瞬时受体电位非典型（TRPC3/6/7）家族的通道不同[20,21]，ARC通道不会被高浓度的DAG（100μM）激活[19]。Mignen及其同事表明，尽管与同一细胞类型中存在的CRAC电流有许多相似之处，但ARC通道具有独特的药理学特性和生物物理特性[18]。例如，与CRAC通道不同，ARC通道不表现出典型酌快速钙依赖性失活（CDI），不受细胞外pH从7.2降至6.7的抑制，并且对2-氨基乙氧基二苯硼酸盐（2-APB）不敏感[18,22]。与CRAC通道的情况一样[34,35]，细胞外记录介质中缺乏二价阳离子会诱导ARC通道对单价阳离子（如Na+）的通透性[23]。然而，这种单价宏观电流具有与CRAC通道观察到的不同特性。特别是从它们的去增强作用和通透性的角度来看。通过增加细胞外钙浓度来阻断单价电流作为钙通道选择性的相对测量方法，Mignen及其同事提出ARC通道具有高钙选择性，并且比CRAC通道的钙选择性高50倍[18,22]。这些作者认为，当细胞用低浓度激动剂刺激诱导重复性钙振荡时，ARC通道是主要被激活的钙通道[24]。使用表达M3毒蕈碱受体的HEK293细胞以及小鼠腮腺和胰腺腺泡细胞，他们报道了低浓度（0.2-1μM）卡巴胆碱激活ARC通道介导细胞内钙振荡[25]。在同一细胞中，他们描述了低浓度卡巴胆碱刺激后产生AA的酶，IV型磷脂酶A2的激活。Shuttleworth小组早期的工作表明，用异粉防己碱对PLA2的药理学抑制会阻断ARC通道的激活，而对脂氧合酶和环氧化酶途径的药理学抑制对ARC激活没有影响[14,26]，这表明AA是由受体介导的PLA2激活产生的，并且AA加工成下游代谢物对于ARC通道激活不是必需的。在鉴定出STIM和Orai蛋白后，Shuttleworth及其同事表明，除了位于质膜中的少量STIM1外，ARC通道激活还需要Orai1和Orai3[27,28]。我们实验室最近的工作在原代主动脉血管平滑肌细胞（VSMC）中鉴定出一种SICE通道。我们发现这种电导由AA激活，但AA通过LTC合酶（LTCS）的酶活性代谢为LTC4能更有力地激活这些通道；LTC4通过膜片吸管施加时在细胞内起作用，但添加到浴液中时在细胞外不起作用。我们将此通道命名为LTC4调节钙（LRC）通道[23,29-31]。我们在VSMC中的数据总体表明，受体激活通过PLC和DAG脂酶的顺序激活导致AA产生，并且AA通过5-脂氧合酶和LTCS代谢为LTC4是LRC通道激活所必需的[29,31]。对LTC合酶（LTCS）的分子敲低消除了受体介导的LRC通道激活（使用PAR1激动剂凝血酶），而通过膜片吸管直接施加LTC4可有力地激活LRC电流。LRC通道的生物物理特性与ARC通道相同，这促使我们在VSMC和HEK293细胞中进行并排比较，以确定这两种电导是由相同还是不同的STIM和Orai蛋白细胞池介导的[23]。简而言之，使用蛋白敲低、药理学抑制剂和AA的不可代谢形式，我们发现无论考虑哪种细胞类型（HEK293细胞或VSMC），ARC和LRC电流都是同一通道的表现，该通道可由AA激活，但LTC4能更有力地激活它[23]。我们还发现，在这两种细胞类型中，ARC/LRC电流依赖于Orai1、Orai3和STIM1[23,29]，但与Shuttleworth小组的发现不同，当在完整细胞中使用Fura-2钙成像和穿孔膜片记录而不是全细胞记录时，我们能够用不转运到质膜的表达STIM1构建体挽救HEK293细胞和VSMC中的ARC/LRC活性。这些结果表明质膜STIM1在ARC/LRC通道激活中起促进作用[23]。Orai1存在通过Orai1mRNA的可变翻译起始产生的两种变体：较长的Orai1α形式在较短的Orai1β保守起始位点上游包含额外的N端（NT）63个氨基酸[32]。我们小组的一项研究表明，虽然Orai1α和Orai1β在形成CRAC通道方面是可互换的，但只有Orai1α可以通过与Orai3形成独特的异源通道来支持ARC/LRC通道。Shuttleworth小组的研究是在发现Orai1α变体之前进行的；因此不清楚使用的是哪种Orai1亚型[33]。我们还表明，LTC4激活LRC通道需要STIM1的第二个C端（CT）卷曲螺旋（CC2）与Orai3CT区域的特异性相互作用[31]。总之，SICE途径似乎由一个通道实体介导。在后续文本中，我们将根据我们所提及的是使用AA还是LTC4激活该电导的实验，将此通道称为ARC或LRC。ARC/LRC通道由Orai1和Orai3编码并受STIM1调节。我们的发现与Shuttleworth小组的发现之间存在两个主要争议点：（1）AA代谢转化为LTC4的必要性，以及（2）ARC/LRC激活所需的STIM1细胞池，即内质网驻留型与质膜驻留型STIM1。
