Ion Channels and Signaling Mechanisms in T Lymphocytes

描述（由申请人提供）：本提案的长期目标是了解T淋巴细胞中产生钙信号的分子和细胞机制。细胞内游离Ca 2+浓度的升高是允许T细胞通过增殖和获得功能能力来响应外来抗原的必需信号。Ca2+释放激活的Ca2+（CRAC）通道通过产生诱导T细胞活化基因所必需的持续Ca2+内流在该过程中起核心作用。CRAC通道是普遍存在的钙库操纵通道家族的原型成员，其响应于内质网（ER）中储存的Ca 2+的耗尽而打开。尽管研究了20多年，但将存储耗尽与通道激活联系起来的机制仍然是一个长期的谜。然而，最近STIM 1作为ER Ca2+传感器和Orai 1作为CRAC通道的亚基的鉴定已经催化了阐明CRAC通道激活机制的快速进展。存储耗尽导致STIM 1和Orai 1分别在ER和质膜内重新分布，使得它们最终积聚在ER与PM相距10 - 25 nm的区域（ER-PM结）中。它们在这些位点的相互作用导致CRAC通道和Ca2+内流的局部激活。虽然我们现在有了CRAC通道激活事件的概述，但还有许多关键事件尚未了解。这个建议的目标是阐明在分子细节的动态事件导致存储耗尽的CRAC通道激活复合物的形成。我们最近的研究表明，STIM 1的多聚化是触发CRAC通道激活的关键事件。该建议的第一个目的是确定STIM1和CRAC通道的亚基化学计量的存储耗尽之前和之后，在完整的细胞中使用单分子光漂白技术。定量双色成像将用于测量STIM 1和CRAC通道在ER-PM连接处共组装时的比例，以首次定量描述CRAC通道激活复合物的形成。第二个目标是集中在STIM 1和Orai 1的相互作用，并利用我们发现的STIM 1片段，强烈激活CRAC通道，而不支持Ca2+依赖性失活。通过截短和扩展这个域，我们将确定关键区域的STIM1参与激活和失活。将通过将纯化的蛋白质应用于CRAC通道来确定该结构域的效力，并且将在由纯化的蛋白质重构的Ca2+通量测定系统中测试其通过直接相互作用激活Orai 1的能力。第三个目的是应用单分子跟踪来解决STIM 1和Orai 1是否通过被动扩散移动，并与GFP光活化技术相结合，以揭示这些蛋白质在活化复合物内的周转。总之，拟议的实验将提供一个定量的基础，了解关键事件，链接存储耗尽CRAC通道激活。公共卫生相关性：该提案旨在了解控制钙通过T淋巴细胞离子通道进入的机制。抑制这一过程的突变会导致人类患者出现致命的严重联合免疫缺陷，表明钙进入是正常免疫系统功能和生存所必需的。更好地理解T细胞中钙进入的分子机制对于鉴定用于药物开发的新靶点是重要的，所述药物开发旨在增强具有受损免疫力的个体的免疫功能，或抑制免疫应答以治疗自身免疫性疾病（例如，糖尿病、狼疮、关节炎）或防止器官移植排斥。

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.