Control of Calcium Entry Signals in B Cells

STIM-Orai钙信号通路在控制生长，转录， 在许多细胞类型中，包括免疫系统、肌肉、神经、皮肤和 血STIM蛋白与奥赖通道一起发挥作用，产生钙库操纵的钙离子内流（SOCE），这是一种基本的钙离子内流。 控制大多数细胞类型的关键信号机制。STIM和奥赖蛋白的遗传缺陷是 在许多免疫细胞中表现突出。我们的具体目标是了解动态分子 在内质网中的STIM Ca 2+传感器蛋白和奥赖之间发生的偶联机制 质膜中的Ca 2+通道，介导Ca 2+进入信号产生的关键机制 在所有的细胞。我们的研究利用了我们开发的一些重要的新分子探针，并依赖于 我们最近提出的关于STIM和 奥赖蛋白。这项工作集中在（a）STIM蛋白上活性位点的分子功能，（B）STIM蛋白上活性位点的分子功能。 奥赖通道的内在激活机制。使用包括B细胞在内的模型细胞系统， 研究和操纵SOCE的机制，我们的具体目标是：1：确定SOCE的分子机制， STIM蛋白偶联界面的基础：我们的目标是了解 STIM 1和STIM 2以及新的剪接变体STIM 2.1被激活并与奥赖通道相互作用， 产生Ca 2+信号。使用新的STIM衍生的荧光分子探针和高 分辨率FRET成像和生物物理测量，我们挑战目前的模型，涉及展开的 STIM蛋白，并测试STIM和奥赖之间的更简单的门控相互作用。2：确定分子 奥赖通道功能和STIM蛋白门控的基础：虽然奥赖通道的结构现在已经被 可以理解，通道的分子排列和导致通道开放的重排， STIM未知。我们已经开发了关键的新工具，用于研究奥赖的激活和功能 渠道这包括模拟STIM激活开放状态的Orai 1通道的分子修饰 这是以前从未被研究过的。目标3：了解组织和功能 Ca 2+进入偶联复合物：STIM和奥赖蛋白功能性相互作用的ER-PM连接 一直是备受关注却鲜有了解的话题我们的研究是针对一个假设， STIM 1能够在ER-PM连接处聚集Orai 1通道，我们研究了Ca 2+信号如何在ER-PM连接处聚集。 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 提供了重要的基本了解的关键耦合接口之间的STIM和奥赖蛋白质。使用 B细胞模型的建立对理解B细胞的功能和发育具有重要意义， 关于控制主要免疫性疾病（包括原代B细胞）机制的重要基本信息 缺乏症、淋巴增生性病症如慢性淋巴细胞性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.