Ion Channels and Signaling Mechanisms in T Lymphocytes

项目总监J首席调查员(最后、第一、中间)：Lewis，Richard S. 项目总结 通过存储操作的钙通道(SOC)的信号在许多生理过程中是关键的，包括 免疫细胞的激活和分化。因此，SOC功能的丧失直接导致致命的 人类的严重联合免疫缺陷综合症。SOC是通过耗尽钙离子而激活的。 内质网(ER)，导致内质网钙传感器STIM1聚集在内质网血浆中 膜(PM)连接，在那里它结合和激活Orai1，它是钙释放的造孔亚单位- 激活的钙通道，triggeringCa2+entryintothecell.Ourlong-termgoalistounderstandin 分子详细说明了通过CRAC通道控制钙离子内流的潜在机制。我们有 开发了一些解决这些问题的新方法，包括对STIM1的单分子跟踪 和Orai1，使用CRISPR/Cas9进行基因编辑以标记和诱变内源蛋白，串联 允许CRAC通道亚单位选择性突变的Orai1级联体，以及单分子 FRET在一个高度明确的体外系统中探索构象动力学。在未来五年，我们将 应用这些方法在三个方面了解CRAC通道监管。首先，我们的目标是了解 天然STIM1和Orai1在内质网-质膜连接的定位和相互作用的机制。几乎我们所有人 对SOC机制的了解是基于STIM1和Orai1的异源高水平过表达， 这可能会推翻许多涉及低量辅助蛋白的重要调控机制。 我们将利用基因编辑技术来标记和修饰内源STIM1和Orai1，并研究其影响因素 控制PM对STIM1的初始捕获、Orai1在结点的停留时间以及 STIM1和Orai1在天然结上的化学计量和相互作用动力学。第二，我们将延长 反馈抑制的主要机制--钙依赖失活(COL)的机制研究 CRAC频道。通过对六聚体Orai1的亚基选择性突变，我们将表征 STIM1与Orai1胞内II-III环和部分孔残基的相互作用 Col的构象变化。第三个也是主要的焦点将是确定动态 STIM1和Orai1激活背后的构象变化。通过测量单分子FRET 在体外标记STIM1和Orai1，我们将确定保持STIM1不活跃的结构以及它们是如何 存储耗尽后重新排列以激活STIM1。单分子方法将广泛应用于其他 有关STIM1与Orai1结合的化学计量学、动力学和构象以及 导致Orai1孔开放的构象变化以及纯化的辅助蛋白对Orai1孔开放的影响 在活细胞中调节STIM-Orai相互作用。这些研究有可能解决许多最 与SOC激活相关的困难和重要问题，并可能提出新的调节策略 钙信号为自身免疫和免疫缺陷综合症提供新的治疗方法。 相关性 存储操作的钙通道(SOC)是激活免疫反应所必需的，而其缺陷 手术会导致人类致命的免疫缺陷。这项提议的短期目标是理解 调节SOC活动的机制，长期目标是确定新的药物靶点 旨在提高免疫力以治疗免疫抑制障碍或抑制免疫的研究进展 应对自身免疫性疾病或防止器官移植排斥反应。

Program DirectorJPrincipallnvestigator (Last, First, Middle): Lewis, Richard S. PROJECT SUMMARY Signaling through store-operated Ca2+ channels (SOCs) is critical for many physiological processes including immune cell activation and differentiation. Accordingly, the loss of SOC function leads directly to a lethal severe combined immunodeficiency syndrome in humans. SOCs are activated by the depletion of Ca2+ from the endoplasmic reticulum (ER), which causes the ER Ca2+sensor STIM1 to accumulate at ER-plasma membrane (PM) junctions where it binds and activates Orai1, the pore-forming subunit of the Ca2+release- activatedCa2+(CRAC)channel,triggeringCa2+entryintothecell.Ourlong-termgoalistounderstandin molecular detail the underlying mechanisms that control Ca2+ influx through CRAC channels. We have developed a number of new approaches to tackle these issues, including single-molecule tracking of STIM1 and Orai1, gene editing with CRISPR/Cas9 to label and mutagenize endogenous proteins, tandem concatemers of Orai1 that allow subunit-selective mutagenesis of the CRAC channel, and single-molecule FRET to probe conformational dynamics in a highly defined in vitro system. Over the next five years, we will apply these approaches to understand CRAC channel regulation in three areas. First, we aim to understand the mechanisms of native STIM1 and Orai1 localization and interaction at ER-PM junctions. Nearly all we know about the SOC mechanism is based on heterologous high-level overexpression of STIM1 and Orai1, which is likely to override many important regulatory mechanisms involving low amounts of accessory proteins. We will exploit gene editing techniques to label and modify endogenous STIM1 and Orai1 and study the factors that control the initial trapping of STIM1 by the PM, the residence time of Orai1 in junctions, and the stOichiometry and interaction kinetics of STIM1 and Orai1 at native junctions. Second, we will extend mechanistic studies of Ca2+-dependent inactivation (COl), the predominant mechanism for feedback inhibition of CRAC channels. By subunit-selective mutagenesis of hexameric Orai1 concatemers we will characterize the interactions of STIM1 with the II-III intracellular loop and selected pore residues of Orai1 that drive conformational changes underlying COl. The third and major focus will be to identify the dynamic conformational changes that underlie activation of STIM1 and Orai1. By measuring single-molecule FRET of labeled STIM1 and Orai1 in vitro, we will identify the structures that keep STIM1 inactive and how they rearrange after store depletion to activate STIM1. The single-molecule approach will be widely applied to other questions such as the stoichiometry, dynamics and conformation of STIM1 binding to Orai1 as well as the conformational changes leading to Orai1 pore opening and the effects of purified accessory proteins thought to modulate STIM-Orai interactions in living cells. These studies have the potential to resolve many of the most difficult and important issues related to SOC activation, and may suggest new strategies for modulating calcium signals to provide new treatments for autoimmune and immunodeficiency syndromes. RELEVANCE Store-operated calcium channels (SOCs) are essential for activating the immune response, and defects in their operation cause a lethal immunodeficiency in humans. The short-term goal of this proposal is to understand the mechanisms that regulate SOC activity, with the long-term goal of identifying new targets for drug development aimed at enhancing immunity to treat immunosuppressive disorders, or inhibiting the immune response to combat autoimmune disease or prevent the rejection of organ transplants.