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）通道的钙库操作钙进入或SOCE调节淋巴细胞活化和产生针对抗原损伤的免疫应答所需的NFAT依赖性基因表达。奥赖（或CRACM）蛋白形成质膜（PM）中CRAC通道的孔亚基，并且内质网（ER）驻留基质相互作用蛋白（Stim）充当储存钙传感器，其寡聚化并移位至ER-PM连接以激活CRAC通道。然而，涉及活细胞内CRAC通道激活的最后步骤仍然难以捉摸。例如，直到最近，人们才相信在ER-PM连接中的Stim 1-Orai 1聚类足以激活SOCE。然而，在缺乏α-可溶性NSF附着蛋白（α-SNAP）的情况下，我们最近发现的活性CRAC通道复合物的一种新的必需组分，Stim 1-Orai 1簇在激活SOCE方面表现出强烈的缺陷。α-SNAP直接结合Stim 1和Orai 1，并调节CRAC通道簇内Stim 1：Orai 1分子的比例;这是CRAC通道活性和钙选择性的已知决定因素。此外，α-SNAP缺陷的小鼠原代CD 4 T淋巴细胞在SOCE、NFAT活化和细胞因子基因表达方面显示出特异性缺陷。在Aim 1中，我们建议测试以下假设：α-SNAP促进Stim 1内的结构变化，使Stim 1-Orai 1簇能够获得活细胞中CRAC通道簇功能所需的最佳Stim 1：Orai 1比率。为了验证我们的假设，我们将使用各种尖端的生物化学，结构和成像方法沿着遗传工具。在目标2中，我们将使用新的超分辨率显微镜方法的组合来构建野生型和α-SNAP缺失细胞的ER-PM连接空间中的CRAC通道大分子组装体的三维分子结构。总之，我们将采用新的和高度跨学科的方法，以获得急需的见解SOCE激活通过CRAC通道的分子机制。鉴定CRAC通道复合物的新的非冗余组分并详细了解其作用机制将非常有益于针对自身免疫性和过敏性疾病的药物设计。

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.