Control of Calcium Entry Signals in B Cells

STIM-Orai钙信号通路在调控生长、转录、 多种细胞类型的分泌和发育，包括免疫系统、肌肉、神经、皮肤和 血。Stim蛋白与Orai通道一起作用于产生钙离子内流(SOCE)，这是一种基本的 对大多数细胞类型的控制至关重要的信号机制。STIM和Orai蛋白中的遗传缺陷是 突出表现在许多免疫细胞中。我们的具体目标是理解动态的分子 内质网中STIM钙离子感受器蛋白与Orai的偶联机制 质膜上的Ca~(2+)通道，这是调节Ca~(2+)进入信号产生的关键机制 在所有的牢房里。我们的研究利用了我们开发的许多重要的新分子探测器，并依赖于 我们最近就STIM和STIM之间的“耦合接口”提出了创新的机制理解 Orai蛋白质。这项工作的重点是(A)STIM蛋白活性部位的分子功能，(B) Orai通道的内在激活机制。使用模型细胞系统，包括B细胞，在其中我们可以 研究和操纵调节SOCE的机制，我们的具体目标是：1：确定分子 STIM蛋白偶联界面的基础：我们的目标是了解STIM蛋白偶联界面的分子机制 STIM1和STIM2以及一种新的剪接变体STIM2.1被激活，并与ORAI通道相互作用 产生钙离子信号。使用新的STIM衍生的荧光分子探针和高密度 分辨率FRET成像和生物物理测量，我们挑战一个当前的模型，涉及展开 STIM蛋白，并测试STIM和ORAI之间更简单的门控相互作用。2：确定分子 Orai通道功能的基础和STIM蛋白的门控：尽管Orai通道的结构现在是 理解，通道的分子排列和导致通道打开的重排 斯蒂姆是个未知数。我们已经开发了关键的新工具来研究Orai的激活和功能 频道。这包括模拟STIM激活的开放状态的Orai1通道的分子修饰 这条从未被研究过的航道。目标3：了解组织和职能 钙离子进入偶联复合体：STIM和Orai蛋白功能相互作用的内质网-质膜连接 一直是许多人密切关注的主题，但几乎没有人理解。我们的研究针对的是一个假设 STIM1能够聚集ER-PM连接内的Orai1通道，我们研究了钙信号是如何 以及它们如何激活B细胞中的细胞转录机制。通过这些目标，我们的研究 对STIM和Orai蛋白之间的关键偶联界面提供重要的基本理解。vbl.使用 B细胞模型的研究工作对于了解B细胞的功能和发育，提供 关于控制包括初级B细胞在内的主要免疫性疾病的机制的重要基本信息 缺乏，淋巴增生性疾病，如慢性淋巴细胞白血病，和自身免疫性疾病。

The STIM-Orai calcium signaling pathway plays a ubiquitous and central role in controlling growth, transcription, secretion, and development in many cell types including those of the immune system, muscle, nerve, skin, and blood. STIM proteins function with Orai channels to generate store-operated Ca2+ entry (SOCE), a fundamental signaling mechanism crucial to control of most cell types. Genetic defects in the STIM and Orai proteins are manifested prominently in many immune cells. Our specific aims are to understand the dynamic molecular coupling mechanism that occurs between the STIM Ca2+ sensor proteins in the endoplasmic reticulum and Orai Ca2+ channels in the plasma membrane, a mechanism crucial to mediating the generation of Ca2+ entry signals in all cells. Our studies utilize a number of important new molecular probes we have developed, and rest upon innovative mechanistic understanding we have recently presented on the “coupling interface” between STIM and Orai proteins. This work has focused on (a) the molecular functioning of the active site on STIM proteins, (b) the intrinsic activation mechanism of Orai channels. Using model cell systems including B cells in which we can study and manipulate the machinery mediating SOCE, our specific aims are: 1: To determine the molecular basis of the STIM protein coupling interface: Our goals are to understand the molecular mechanism by which STIM1 and STIM2 and a novel splice variant, STIM2.1, become activated and interact with Orai channels to generate Ca2+ signals. Using new STIM-derived fluorescent molecular probes and a combination of high resolution FRET imaging and biophysical measurements, we challenge a current model involving unfolding of STIM proteins, and test a simpler gating interaction between STIM and Orai. 2: To determine the molecular basis of Orai channel function and gating by STIM proteins: Although the structure of Orai channels is now understood, the molecular arrangement of the channel and rearrangement that leads to channel opening with STIM is unknown. We have developed critical new tools with which to study activation and function of Orai channels. This includes a molecular modification of the Orai1 channel that mimics the STIM-activated open state of the channel which has never before been studied. Aim 3: To understand the organization and function of the Ca2+ entry coupling complex: The ER-PM junction wherein STIM and Orai proteins functionally interact has been the subject of much scrutiny but little understanding. Our studies are directed toward a hypothesis that STIM1 is able to cluster Orai1 channels within the ER-PM junction, and we examine how Ca2+ signals can be generated and how they activate the cellular transcriptional machinery in B cells. Through these aims, our studies provide important basic understanding of the crucial coupling interface between STIM and Orai proteins. Using B cell models the work has particular significance to understanding B cell function and development, providing important basic information on mechanisms to control major immunological diseases including primary B cell deficiencies, lymphoproliferative disorders such as chronic lymphocytic leukemia, and autoimmune diseases.