Ion Channels and Signaling Mechanisms in T Lymphocytes

描述(申请人提供)：这项建议的长期目标是了解在T淋巴细胞中产生钙信号的分子和细胞机制。细胞内游离钙离子浓度的升高是T细胞通过增殖和获得功能能力对外来抗原做出反应所必需的信号。钙释放激活的钙通道(CRAC)通过产生诱导T细胞激活基因所需的钙离子持续内流，在这一过程中发挥核心作用。CRAC通道是普遍存在的商店操作的通道家族中的典型成员，这些通道在内质网(ER)中存储的钙离子耗尽时开放。尽管经过了20多年的研究，但将存储枯竭与通道激活联系起来的机制仍然是一个长期存在的谜。然而，最近发现的STIM1是内质网钙离子感受器，Orai1是CRAC通道的一个亚单位，这促进了在阐明CRAC通道激活机制方面的快速进展。存储耗尽导致STIM1和Orai1分别在内质网和质膜内重新分布，从而它们最终聚集在ER距离PM 10-25 nm的区域(ER-PM连接)它们在这些部位的相互作用导致CRAC通道的局部激活和钙内流。尽管我们现在已经对CRAC通道激活背后的事件有了一个概要，但仍有许多关键事件没有被理解。这项建议的目的是从分子细节上阐明从储存耗尽到CRAC通道激活复合体形成的动态事件。我们最近的研究表明，STIM1的多聚体是触发CRAC通道激活的关键事件。这项建议的第一个目的是利用完整细胞中的单分子光漂白技术，确定STIM1和CRAC通道在存储耗尽前后的亚基化学计量比。定量双色成像将用于测量STIM1和CRAC通道在ER-PM连接处共组装时的比率，以首次定量描述CRAC通道激活复合体的形成。第二个目的是研究STIM1和Orai1的相互作用，并利用我们发现的STIM1片段强烈激活CRAC通道，而不支持钙依赖的失活。通过该结构域的截断和扩展，我们将确定STIM1参与激活和失活的关键区域。该结构域的效力将通过将纯化的蛋白应用于CRAC通道来确定，其通过直接相互作用激活Orai1的能力将在由纯化的蛋白重组的钙通量分析系统中进行测试。第三个目的是应用单分子跟踪来研究STIM1和Orai1是否通过被动扩散移动，并结合GFP光激活技术来揭示这些蛋白质在激活复合体中的周转。总之，所提出的实验将为理解将存储器耗尽与CRAC通道激活联系起来的关键事件提供量化基础。公共卫生相关性：这项建议试图了解T淋巴细胞通过离子通道控制钙进入的机制。抑制这一过程的突变会导致人类患者致命的严重联合免疫缺陷，表明钙离子进入是正常免疫系统功能和生存所绝对需要的。更好地了解T细胞内钙离子进入的分子机制对于确定新的药物开发靶点非常重要，这些药物旨在增强免疫功能低下的个体的免疫功能，或抑制免疫反应以治疗自身免疫性疾病(如糖尿病、狼疮、关节炎)或防止器官移植的排斥反应。

DESCRIPTION (provided by applicant): The long-term objective of this proposal is to understand the molecular and cellular mechanisms that generate calcium signals in T lymphocytes. The elevation of intracellular free Ca2+ concentration is a required signal that allows T cells to respond to foreign antigens by proliferating and acquiring functional abilities. The Ca2+ release-activated Ca2+ (CRAC) channel plays a central role in this process, by generating the sustained influx of Ca2+ necessary for induction of T cell activation genes. CRAC channels are the prototypic members of a ubiquitous family of store-operated channels that open in response to the depletion of the Ca2+ stored in the endoplasmic reticulum (ER). Despite more than 20 years of research, the mechanism that links store depletion to channel activation has remained a longstanding mystery. However, the recent identification of STIM1 as the ER Ca2+ sensor and Orai1 as a subunit of the CRAC channel has catalyzed rapid progress in elucidating the mechanism of CRAC channel activation. Store depletion causes STIM1 and Orai1 to redistribute within the ER and plasma membranes, respectively, so that they ultimately accumulate in regions (ER-PM junctions) where the ER is 10-25 nm from the PM. Their interaction at these sites leads to localized activation of CRAC channels and Ca2+ influx. Although we now have an outline of events underlying CRAC channel activation, there are many critical events that are not understood. The goal of this proposal is to elucidate in molecular detail the dynamic events leading from store depletion to the formation of the CRAC channel activation complex. Our recent studies suggest that multimerization of STIM1 is the key event that triggers CRAC channel activation. The first aim of this proposal is to determine the subunit stoichiometries of STIM1 and the CRAC channel before and after store depletion, using single-molecule photobleaching techniques in intact cells. Quantitative two-color imaging will be applied to measure the ratio of STIM1 and the CRAC channel as they co-assemble at ER-PM junctions, to provide the first quantitative description of the formation of the CRAC channel activation complex. The second aim is focused on the interaction of STIM1 and Orai1 and makes use of a fragment of STIM1 that we have found to activate CRAC channels strongly without supporting Ca2+-dependent inactivation. Through truncations and extensions of this domain we will identify the critical regions of STIM1 involved in activation and inactivation. The potency of this domain will be determined by applying purified protein to CRAC channels, and its ability to activate Orai1 through a direct interaction will be tested in a Ca2+ flux assay system reconstituted from purified proteins. The third aim applies single- molecule tracking to address whether STIM1 and Orai1 move by passive diffusion, and in combination with GFP photoactivation techniques, to reveal the turnover of these proteins within the activation complex. Together, the proposed experiments will provide a quantitative basis for understanding the critical events that link store depletion to CRAC channel activation. PUBLIC HEALTH RELEVANCE: This proposal seeks to understand the mechanism that controls calcium entry through ion channels in T lymphocytes. Mutations that inhibit this process lead to a lethal, severe combined immunodeficiency in human patients, showing that calcium entry is absolutely required for proper immune system function and survival. A better understanding of the molecular mechanism of calcium entry in T cells is important for identifying novel targets for drug development aimed at enhancing immune function in individuals with compromised immunity, or at inhibiting the immune response to treat autoimmune disease (e.g., diabetes, lupus, arthritis) or prevent the rejection of organ transplants.