Activation Mechanisms of Store-Operated Calcium Channels

商店操纵的钙通道的激活机制

• 批准号: 9070002
• 负责人: Murali Prakriya
• 金额: $ 29.22万
• 依托单位: NORTHWESTERN UNIVERSITY AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-06-01 至 2019-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9070002
• 关键词: Affect; Animals; Architecture; Binding; Biochemical; Biophysical Process; Blood Coagulation Disorders; Calcium Channel; Calmodulin; Catalytic Domain; Cell Proliferation; Cell Surface Receptors; Cell physiology; Cells; Chronic; Cysteine; Cytoplasmic Tail; Disease; Effector Cell; Electrophysiology (science); Endoplasmic Reticulum; Etiology; Exhibits; Experimental Designs; Fingerprint; Fluorescence Resonance Energy Transfer; GTP-Binding Proteins; Gap Junctions; Gene Expression; Genetic Transcription; Goals; Health; Human; Hypersensitivity; Immunologic Deficiency Syndromes; Inflammation; Inflammatory Bowel Diseases; Intervention; Ion Channel; Ions; Kinetics; Laboratories; Lead; Learning; Malignant Neoplasms; Maps; Membrane; Metals; Methods; Microscopy; Modeling; Molecular; Monitor; Motion; Muscle Weakness; Muscle relaxation phase; Mutagenesis; Mutation; Nature; Pharmaceutical Preparations; Physiological; Process; Property; Protein Engineering; Protein Tyrosine Kinase; Proteins; Regulation; Role; Rotation; STIM1 gene; Severe Combined Immunodeficiency; Side; Signal Transduction; Site; Stem cells; Stimulus; Structure; Sulfhydryl Reagents; Testing; Therapeutic; Thrombosis; Tissues; base; cell motility; cell type; cohort; conformational alteration; crosslink; drug discovery; fascinate; gain of function; genetic regulatory protein; human disease; insight; link protein; molecular rearrangement; patch clamp; programs; receptor coupling; response; sensor; therapeutic development; tool

 DESCRIPTION (provided by applicant): In many animal cells, stimulation of cell surface receptors coupled to G proteins or tyrosine kinases mobilizes Ca2+ influx through store-operated Ca2+ release-activated Ca2+ (CRAC) channels. The ensuing Ca2+ entry regulates a wide variety of effector cell responses including transcription, motility, and proliferation. CRAC channels exhibit a unique biophysical fingerprint characterized by exquisite Ca2+-selectivity, store-operated gating, and distinct pore properties, and serve as fascinating ion channels for understanding the biophysical mechanisms of ion permeation and gating. Moreover, because CRAC channels sit squarely at the nexus of the cellular Ca2+ signaling network in many cells, aberrant CRAC channel function is implicated in the etiology of several diseases including chronic inflammation, muscle weakness, and a severe combined immunodeficiency syndrome. Much has been learned in the last decade of the physiological roles of CRAC channels in different tissues and the cellular choreography of the CRAC channel activation process. Still, the molecular and structural basis of how depletion of ER Ca2+ stores regulates the opening of the CRAC channel pore continues to remain poorly understood. Opening of CRAC channels is governed through direct interactions between the pore-forming Orai proteins, and the ER Ca2+ sensor, STIM1, but how STIM1 binding transduces opening of the Orai1 channel pore remains unclear. Here, we propose a multi-pronged approach that will investigate several mechanistic aspects of the CRAC channel gating process, focusing on the molecular rearrangements in the CRAC channel pore and the conformational alterations in STIM1. We will employ an interdisciplinary experimental design for these studies that combines patch-clamp electrophysiology, cysteine mutagenesis, FRET microscopy, biochemical cross-linking, and protein engineering. Using the Orai1 protein as a prototypic CRAC channel, our goals are to: (1) test the hypothesis that opening of the CRAC channel pore occurs through rotation of the TM1 helix, thereby reorienting the hydrophobic V102 side-chains to remove an energy barrier, (2) elucidate the inner pore architecture and its role in regulating ion conduction in Orai1 channels, and (3) examine the conformational rearrangements in the critically important cytoplasmic region of STIM1 during activation and binding to Orai1. These studies will significantly advance our understanding of the molecular and structural mechanisms of CRAC channel activation and open new avenues for harnessing the therapeutic potential of CRAC channels for disease intervention.

 描述（由申请人提供）：在许多动物细胞中，刺激与G蛋白或酪氨酸激酶偶联的细胞表面受体可通过钙库操作的Ca2+释放激活的Ca2+（CRAC）通道动员Ca2+内流。随后的Ca2+进入调节多种效应细胞反应，包括转录、运动和增殖。CRAC通道表现出独特的生物物理指纹，其特征在于精致的Ca2+选择性，存储操作的门控和独特的孔特性，并作为迷人的离子通道，用于理解离子渗透和门控的生物物理机制。此外，由于CRAC通道直接位于许多细胞中细胞Ca 2+信号网络的连接处，因此异常的CRAC通道功能与多种疾病的病因有关，包括慢性炎症、肌肉无力和严重的联合免疫缺陷综合征。在过去的十年中，人们已经了解了CRAC通道在不同组织中的生理作用以及CRAC通道激活过程的细胞编排。尽管如此，ER Ca2+商店的消耗如何调节CRAC通道孔的开放的分子和结构基础仍然知之甚少。CRAC通道的开放通过成孔奥赖蛋白和ER Ca 2+传感器STIM 1之间的直接相互作用来控制，但STIM 1结合如何转导Orai 1通道孔的开放仍不清楚。在这里，我们提出了一个多管齐下的方法，将调查CRAC通道门控过程的几个机制方面，集中在CRAC通道孔的分子重排和STIM1的构象改变。我们将采用跨学科的实验设计，结合膜片钳电生理学，半胱氨酸诱变，FRET显微镜，生化交联和蛋白质工程的这些研究。使用Orai1蛋白作为原型CRAC通道，我们的目标是：（1）测试CRAC通道孔的打开通过TM 1螺旋的旋转发生的假设，从而重新定向疏水性V102侧链以去除能量屏障，（2）阐明内部孔结构及其在调节Orai 1通道中的离子传导中的作用，以及（3）检测STIM 1在活化和结合Orai 1期间的关键重要胞质区域中的构象重排。这些研究将显著推进我们对CRAC通道激活的分子和结构机制的理解，并为利用CRAC通道的治疗潜力进行疾病干预开辟新的途径。