CRAC CHANNEL COMPONENTS AND MOLECULAR BASIS OF STORE-OPERATED CALCIUM ENTRY

描述(由申请人提供)：通过钙释放激活钙(CRAC)通道的储存操作的钙内流，或SOCE，调节NFAT依赖的基因表达，这是淋巴细胞激活和产生针对抗原损伤的免疫反应所必需的。Orai(或CRACM)蛋白形成质膜(PM)CRAC通道的孔亚基，内质网(ER)驻留的基质相互作用蛋白(STIMS)作为钙离子感受器寡聚并移位到ER-PM连接激活CRAC通道。然而，活细胞内CRAC通道激活的最后步骤仍然难以捉摸。例如，直到最近，人们还认为内质网-PM连接中的STIM1-Orai1簇足以激活SOCE。然而，在缺乏我们最近发现的活性CRAC通道复合体的新的基本成分--α-可溶性神经营养因子附着蛋白(α-SNAP)的情况下，STIM1-Orai1簇在激活SOCE方面表现出强烈的缺陷。α-SNAP直接与STIM1和ORAI1结合，并调节CRAC通道簇中STIM1：ORAI1分子的比例；已知的CRAC通道活性和钙选择性的决定因素。此外，α-SNAP缺陷的小鼠原代CD4T淋巴细胞在SOCE、NFAT激活和细胞因子基因表达方面存在特异性缺陷。在AIM1中，我们建议测试这一假设，即α-SNAP促进STIM1内的结构变化，使STIM1-ORAI1簇能够获得活细胞中CRAC通道簇功能所需的最佳STIM1：ORAI1比率。为了验证我们的假设，我们将使用各种尖端的生化、结构和成像方法以及遗传工具。在目标2中，我们将结合新的超分辨率显微镜方法来构建野生型和α-SNAP耗竭细胞的ER-PM连接空间中CRAC通道大分子组装的三维分子结构。综上所述，我们将使用新的和高度跨学科的方法来获得急需的洞察力，以了解通过CRAC通道激活SOCE的分子机制。识别CRAC通道复合体的新的非冗余成分并详细了解它们的作用机制将对治疗自身免疫性和过敏性疾病的药物设计大有裨益。

DESCRIPTION (provided by applicant): Store-operated calcium entry, or SOCE, via calcium release activated calcium (CRAC) channels regulates NFAT dependent gene expression required for lymphocyte activation and generation of immune response against antigenic insults. Orai (or CRACM) proteins form the pore subunit of CRAC channels in the plasma membrane (PM), and endoplasmic reticulum (ER) resident stromal interaction proteins (Stims) act as store calcium sensors that oligomerize and translocate to ER-PM junctions to activate CRAC channels. However, the final steps involved in CRAC channel activation inside live cells have remained elusive. For instance, until recently it was believed that Stim1-Orai1 clustering in ER-PM junctions is sufficient to activate SOCE. However, in the absence of alpha-soluble NSF attachment protein (α-SNAP), a novel essential component of active CRAC channel complex that we have recently identified, Stim1-Orai1 clusters show a strong defect in activating SOCE. α-SNAP directly binds Stim1 as well as Orai1 and regulates the ratio of Stim1:Orai1 molecules within CRAC channel clusters; a known determinant of CRAC channel activity and calcium selectivity. Furthermore, α-SNAP deficient mouse primary CD4 T lymphocytes show a specific defect in SOCE, NFAT activation and cytokine gene expression. In Aim1, we propose to test the hypothesis that α-SNAP facilitates a structural change within Stim1 that enables the Stim1-Orai1 clusters to acquire optimal Stim1:Orai1 ratios required for the functionality of CRAC channel clusters in live cells. To test our hypothesis, we will use a variety of cutting edge biochemical, structural and imaging approaches along with genetic tools. In Aim 2, we will use a combination of novel super resolution microscopy approaches to construct the 3 dimensional molecular architecture of CRAC channel macromolecular assembly in ER-PM junctional space of wildtype and α-SNAP depleted cells. In summary, we will employ novel and highly interdisciplinary approaches to gain much needed insights into the molecular mechanism of SOCE activation via CRAC channels. Identification of new non-redundant components of the CRAC channel complex and detailed insights into their mechanism of action will be highly beneficial for drug design against autoimmune and allergic disorders.