Modulation of ORAI2 and ORAI3 Calcium Channels

项目摘要/摘要 这项建议的目的是了解ORAI钙通道对细胞和组织功能的贡献 在健康和疾病方面。3个ORAI蛋白(ORAI1、2、3)中的每一个在以下情况下形成钙通道 在细胞中过表达。ORAI1是其中最具特征的，它编码钙释放激活的 钙(CRAC)。ORAI1零突变患者由于受损而患有严重的免疫缺陷 免疫细胞内的钙离子内流。利用ORAI1缺陷小鼠，我们发现ORAI1对淋巴细胞是必不可少的 体内功能和免疫反应及抑制ORAI1对T细胞介导的小鼠的保护作用 自身免疫力。与ORAI1相比，ORAI2和ORAI3在体内的作用在很大程度上是未知的，而且它是 不清楚内源性表达的ORAI2和ORAI3是否参与免疫或非免疫功能 细胞。这种知识差距在很大程度上是由于缺乏包括Orai2和Orai3基因敲除在内的特定试剂 小鼠和高亲和力抗体分析ORAI蛋白在健康和健康人群中的功能和表达 疾病。ORAI3被证明在某些癌细胞中介导钙内流，并促进其生长和 侵犯性。这些发现很耐人寻味，但需要用体内的基因敲除小鼠来证实。 学习。关于ORAI2在体内的作用，人们几乎一无所知。使用新的Orai2缺陷报告小鼠，我们 在许多免疫细胞和大脑中发现了ORAI2的表达。在免疫细胞中，ORAI2的缺乏本身并不能 抑制钙内流，但其缺失与ORAI1一起取消钙内流，严重损害T细胞- 中介免疫。ORAI1和ORAI2的这种协同作用增加了两个同源物都可以 在免疫细胞和潜在的其他细胞类型中形成异构体通道。要了解生理学 以及内源性ORAI1、ORAI2和ORAI3通道在体内的病理生理作用我们需要条件基因敲除 允许组织和细胞类型特定的单个或多个ORAI基因缺失的小鼠。我们建议生成 有条件的Orai2和Orai3基因敲除小鼠，并将它们彼此杂交，以及Orai1缺陷小鼠 建立双基因敲除和三基因敲除小鼠以研究单个ORAI蛋白及其基因的功能 体内组织中的协同作用。条件删除是必要的，因为完全删除Orai1是致命的，并且 因此，它与其他Orai基因的联合缺失也将是致命的。对ORAI信道的研究 由于缺乏检测活细胞中ORAI蛋白的特异性抗体来测试其 表达、亚细胞定位和功能。我们最近产生了针对ORAI1的抗体，不仅 检测活细胞中的ORAI1，但也抑制钙内流。我们建议开发特异性抗体来对抗 ORAI2和ORAI3研究它们在健康和疾病组织中的表达，并抑制它们的功能。这些 新工具将与其他实验室共享，并将提供对生理学和 ORAI通道的病理作用，目的是为治疗的发展创造理论基础 Orai通道抑制剂。

Project Summary / Abstract The goal of this proposal is to understand the contribution of ORAI calcium channels to cell and tissue function in health and disease. Each of the 3 ORAI proteins (ORAI1, 2, 3) forms a calcium channel when overexpressed in cells. ORAI1 is the best characterized of these and encodes the calcium release-activated calcium (CRAC). Patients with null mutations in ORAI1 suffer from severe immunodeficiency due to impaired calcium influx in immune cells. Using ORAI1 deficient mice, we found that ORAI1 is essential for lymphocyte function and immune responses in vivo and inhibition of ORAI1 protects mice from T cell mediated autoimmunity. In contrast to ORAI1, the roles of ORAI2 and ORAI3 in vivo are largely unknown and it is unclear if endogenously expressed ORAI2 and ORAI3 contribute to the function of immune or non-immune cells. This knowledge gap is in large part due to a lack of specific reagents including Orai2 and Orai3 knockout mice and high affinity antibodies to analyze the function and expression of ORAI proteins in health and disease. ORAI3 was shown to mediate calcium influx in certain cancer cells and to promote their growth and invasiveness. These findings are intriguing, but need to be corroborated using knockout mice for in vivo studies. Almost nothing is known about the role of ORAI2 in vivo. Using new Orai2-deficient reporter mice, we found expression of ORAI2 in many immune cells and the brain. In immune cells, lack of ORAI2 alone does not impair calcium influx, but its deletion together with ORAI1 abolishes calcium influx and severely impairs T cell- mediated immunity. This cooperative role of ORAI1 and ORAI2 raises the possibility that both homologs can form heteromeric channels in immune cells and potentially other cell types. To understand the physiological and pathophysiological roles of endogenous ORAI1, 2 and 3 channels in vivo we need conditional knockout mice that allow tissue and cell type-specific deletion of single or multiple Orai genes. We propose to generate conditional Orai2 and Orai3 knockout mice and to cross these to each other and Orai1-deficient mice to generate double and triple knockout mice to investigate the function of individual ORAI proteins and their synergistic roles in tissues in vivo. Conditional deletion is essential as complete deletion of Orai1 is lethal and its combined deletion with other Orai genes would therefore be lethal, too. Research on ORAI channels is severely handicapped by a lack of specific antibodies for the detection of ORAI proteins in live cells to test their expression, subcellular localization and function. We recently generated antibodies against ORAI1 that not only detect ORAI1 in live cells but also inhibit calcium influx. We propose to develop specific antibodies against ORAI2 and ORAI3 to study their expression in healthy and diseased tissues and to inhibit their function. These new tools will be shared with other labs and will provide important new insight into the physiological and pathological roles of ORAI channels, with the goal of creating rationales for the development of therapeutic ORAI channel inhibitors.