Ion Channels and Signaling Mechanisms in T Lymphocytes

项目主管/主要调查员（最后，第一，中间）：刘易斯，理查德S。 项目摘要 通过钙库操纵的钙通道（SOC）的信号传导对于许多生理过程是至关重要的，包括 免疫细胞活化和分化。因此，SOC功能的丧失直接导致致命的 严重的联合免疫缺陷综合征。SOCs通过消耗来自细胞内的Ca 2+而被激活。 内质网（ER），其导致ER Ca 2+传感器STIM 1在ER-血浆中积累 膜（PM）连接处，在那里它结合并激活Orai 1，Orai 1是Ca 2+释放的成孔亚基。 激活Ca 2+通道（CRAC），阻止Ca 2+进入细胞。 分子详细描述了控制Ca 2+通过CRAC通道流入的潜在机制。我们有 开发了许多新方法来解决这些问题，包括STIM 1的单分子跟踪 和Orai 1，用CRISPR/Cas9进行基因编辑以标记和诱变内源性蛋白质，串联 Orai 1的多联体，其允许CRAC通道的亚基选择性诱变，和单分子 FRET探测构象动力学在一个高度定义的体外系统。未来五年，我们将 应用这些方法来理解CRAC在三个方面的通道调节。首先，我们的目标是了解 天然STIM 1和Orai 1在ER-PM连接处的定位和相互作用的机制。几乎所有我们 关于SOC机制的已知是基于STIM 1和Orai 1的异源高水平过表达， 这可能会超越许多涉及少量辅助蛋白的重要调节机制。 我们将利用基因编辑技术来标记和修饰内源性STIM 1和Orai 1，并研究这些因素。 控制PM对STIM 1的初始捕获、Orai 1在结中的停留时间以及 STIM 1和Orai 1在天然连接处化学计量学和相互作用动力学。第二，我们将扩大 Ca 2+依赖性失活（CO 1）的机制研究，反馈抑制的主要机制 CRAC频道通过六聚体Orai 1多联体的亚基选择性诱变，我们将表征 STIM 1与II-III胞内环和Orai 1的选定孔残基的相互作用， 第三个也是主要的焦点将是确定COl的动态构象变化。 STIM 1和Orai 1激活的基础构象变化。通过测量单分子FRET， 标记的STIM 1和Orai 1在体外，我们将确定结构，保持STIM 1失活，以及如何， 在存储耗尽后重新排列以激活STIM 1。单分子方法将广泛应用于其他 STIM 1与Orai 1结合的化学计量、动力学和构象以及 导致Orai 1孔开放的构象变化和被认为是 调节活细胞中的STIM-Orai相互作用。这些研究有可能解决许多最重要的问题。 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 钙信号提供新的治疗自身免疫和免疫缺陷综合征。 相关性 钙池操纵的钙通道（SOC）对于激活免疫应答是必不可少的，并且它们的缺陷是免疫应答的重要组成部分。 手术会导致人类致命的免疫缺陷。本提案的短期目标是了解 调节SOC活性的机制，其长期目标是确定药物治疗的新靶点。 开发旨在增强免疫力以治疗免疫抑制性疾病，或抑制免疫抑制性疾病， 对抗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.