Ion Channels and Signaling Mechanisms in T Lymphocytes

T 淋巴细胞中的离子通道和信号传导机制

• 批准号: 9238964
• 负责人: RICHARD S LEWIS
• 金额: $ 64.1万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-07-01 至 2022-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9238964
• 关键词: Area; Autoimmune Diseases; Autoimmune Process; Binding; CRISPR/Cas technology; Calcium Channel; Calcium Signaling; Cell membrane; Cells; Defect; Disease; Endoplasmic Reticulum; Feedback; Genes; Goals; Human; Immune Cell Activation; Immune response; Immunity; Immunologic Deficiency Syndromes; Immunosuppressive Agents; In Vitro; Ion Channel; Kinetics; Label; Measures; Molecular; Molecular Conformation; Mutagenesis; Physiological Processes; Proteins; Regulation; STIM1 gene; Severe Combined Immunodeficiency; Signal Transduction; Structure; System; T-Lymphocyte; Techniques; Time; base; combat; drug development; new therapeutic target; novel strategies; operation; organ transplant rejection; overexpression; prevent; programs; residence; sensor; single molecule; single-molecule FRET; stoichiometry

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.

项目总监JPrincipalIvestigator（最后，第一，中间）：Lewis, Richard S. 项目概要 通过钙池操纵的 Ca2+ 通道 (SOC) 发出的信号对于许多生理过程至关重要，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后果 人类严重联合免疫缺陷综合症。 SOC 通过消耗 Ca2+ 来激活 内质网 (ER)，导致 ER Ca2+ 传感器 STIM1 在 ER 血浆中积聚 膜 (PM) 连接处，它结合并激活 Orai1，Ca2+ 释放的孔形成亚基 激活Ca2+（CRAC）通道，触发Ca2+​​进入细胞。我们的长期目标是了解 分子详细描述了控制 Ca2+ 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 STIM1 的单分子追踪 和 Orai1，使用 CRISPR/Cas9 进行基因编辑以标记和诱变内源蛋白，串联 Orai1 的多联体允许 CRAC 通道的亚基选择性诱变，以及单分子 FRET 用于探测高度定义的体外系统中的构象动力学。未来五年，我们将 应用这些方法来了解三个领域的 CRAC 通道监管。首先，我们的目的是了解 ER-PM 连接处天然 STIM1 和 Orai1 定位和相互作用的机制。几乎我们所有人 了解 SOC 机制是基于 STIM1 和 Orai1 的异源高水平过度表达， 这可能会推翻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STIM1和Orai1并研究其影响因素 控制 PM 对 STIM1 的初始捕获、Orai1 在连接处的停留时间以及 STIM1 和 Orai1 在天然连接处的化学计量学和相互作用动力学。其次，我们将延长 Ca2+ 依赖性失活 (COl) 的机制研究，这是反馈​​抑制的主要机制 CRAC 通道数。通过六聚体 Orai1 串联体的亚基选择性诱变，我们将表征 STIM1 与 II-III 细胞内环以及驱动 Orai1 的选定孔残基的相互作用 COl 的构象变化。第三个也是主要重点是确定动态 STIM1 和 Orai1 激活的构象变化。通过测量单分子 FRET 在体外标记 STIM1 和 Orai1，我们将识别使 STIM1 保持非活性的结构以及它们如何 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领域 STIM1 与 Orai1 结合的化学计量、动力学和构象等问题以及 导致 Orai1 孔打开的构象变化以及纯化辅助蛋白的影响被认为 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棘手的问题 与 SOC 激活相关的困难和重要问题，可能会提出新的调节策略 钙信号为自身免疫和免疫缺陷综合征提供新的治疗方法。 关联 钙库操纵的钙通道 (SOC) 对于激活免疫反应至关重要，其缺陷 手术会导致人类致命的免疫缺陷。该提案的短期目标是了解 调节 SOC 活性的机制，长期目标是确定药物的新靶点 旨在增强免疫力以治疗免疫抑制疾病或抑制免疫的开发 对抗自身免疫性疾病或防止器官移植排斥的反应。