Modulation of ORAI2 and ORAI3 Calcium Channels

项目总结/摘要 该提案的目的是了解奥赖钙通道对细胞和组织功能的贡献 健康和疾病。3种奥赖蛋白（ORAI 1、2、3）中的每一种在以下情况下形成钙通道： 在细胞中过度表达。ORAI 1是这些中最好的特征，并且编码钙释放激活的 钙（CRAC）。具有ORAI 1无效突变的患者由于免疫缺陷而患有严重的免疫缺陷。 免疫细胞中的钙离子流入。利用ORAI 1缺陷小鼠，我们发现ORAI 1是淋巴细胞增殖所必需的。 体内的功能和免疫应答以及抑制ORAI 1保护小鼠免受T细胞介导的 自身免疫与ORAI 1相反，ORAI 2和ORAI 3在体内的作用在很大程度上是未知的， 不清楚内源性表达的ORAI 2和ORAI 3是否有助于免疫或非免疫的功能， 细胞这种知识差距在很大程度上是由于缺乏特异性试剂，包括Orai 2和Orai 3敲除 小鼠和高亲和性抗体，分析健康人和正常人中奥赖蛋白的功能和表达， 疾病ORAI 3被证明介导某些癌细胞中的钙流入并促进其生长， 侵略性这些发现很有趣，但需要使用基因敲除小鼠进行体内验证。 问题研究关于ORAI 2在体内的作用几乎一无所知。使用新的Orai 2缺陷报告小鼠，我们 在许多免疫细胞和大脑中发现了ORAI 2的表达。在免疫细胞中，单独缺乏ORAI 2并不能 削弱钙内流，但其与ORAI 1一起缺失消除了钙内流并严重损害T细胞- 介导的免疫。ORAI 1和ORAI 2的这种协同作用提高了这两种同源物可以 在免疫细胞和潜在的其他细胞类型中形成异聚体通道。为了了解生理上的 内源性ORAI 1、2和3通道在体内的病理生理作用，我们需要条件性敲除 允许单个或多个奥赖基因的组织和细胞类型特异性缺失的小鼠。我们建议生成 条件Orai 2和Orai 3敲除小鼠，并将这些小鼠彼此杂交，并将Orai 1缺陷小鼠杂交， 产生双重和三重敲除小鼠以研究单个奥赖蛋白及其 在体内组织中的协同作用。条件性缺失是必要的，因为Orai 1的完全缺失是致命的， 因此，它与其他奥赖基因的联合缺失也是致命的。对奥赖信道的研究是 由于缺乏用于检测活细胞中奥赖蛋白的特异性抗体以测试其 表达、亚细胞定位和功能。我们最近产生了针对ORAI 1的抗体， 检测活细胞中的ORAI 1，但也抑制钙内流。我们建议开发特异性抗体， 研究其在健康和患病组织中的表达并抑制其功能。这些 新的工具将与其他实验室共享，并将提供重要的新的洞察生理和 奥赖通道的病理作用，目的是为开发治疗性 奥赖通道抑制剂。

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.