3D Structure and Function of CRAC Channels

CRAC 通道的 3D 结构和功能

• 批准号: 8537945
• 负责人: Stephen Barstow Long
• 金额: $ 51.37万
• 依托单位: SLOAN-KETTERING INST CAN RESEARCH
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-09-01 至 2015-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8537945
• 关键词: 3-Dimensional; Address; Amino Acid Sequence Homology; Architecture; Binding; Biochemical; Biological Assay; Brain Infarction; Calcium; Calcium Channel; Calcium Signaling; Calcium ion; Cell membrane; Cloning; Complex; Crystallization; Data; Detergents; Development; Endoplasmic Reticulum; Engineering; Eukaryotic Cell; Event; Family member; Foundations; Genes; Goals; Human; Immune response; Immune system; In Vitro; Integral Membrane Protein; Ion Channel; Ions; Lipids; Mammary Neoplasms; Measures; Membrane; Membrane Proteins; Micelles; Molecular; Monoclonal Antibodies; Mutation; Neoplasm Metastasis; Pathway interactions; Patients; Platelet Activation; Play; Potassium Channel; Process; Proteins; Research; Roentgen Rays; Role; Second Messenger Systems; Severe Combined Immunodeficiency; Signal Transduction; Solutions; Structure; T-Cell Activation; Techniques; Therapeutic; Thrombosis; X-Ray Crystallography; base; improved; in vitro activity; inhibitor/antagonist; malignant breast neoplasm; prevent; public health relevance; reconstitution; response; second messenger; sensor; structural biology; three dimensional structure

DESCRIPTION (provided by applicant): 3D Structure and Function of CRAC Channels. The long-term objective of this proposal is to understand the mechanisms of ion permeation, ion selectivity, and gating in calcium release-activated calcium (CRAC) channels. CRAC channels play a central role in the immune response by generating the sustained influx of calcium (Ca2+) that is necessary for induction of T cell activation genes. CRAC channels, which are integral membrane proteins located in the plasma membrane, open in response to depletion of the Ca2+ stored within the endoplasmic reticulum (ER). Despite more than 20 years of research, the molecular components underlying this process of store-operated calcium entry (SOCE) were unknown. Recently, Orai protein was identified as the pore subunit and the STIM protein was determined to be the ER Ca2+ sensor. These long-awaited findings have accelerated research pertaining to the molecular mechanisms of CRAC channel function, revealing significant differences from other ion channels. Aside from being an integral membrane protein, Orai has no significant amino acid sequence homology to other known ions channels. The Orai channel is highly selective for Ca2+ ions, but this ion selectivity must be achieved by an architecture that is different from other Ca2+ channels. The channel also has an unprecedented mechanism of gating (the process that opens and closes the channel): the depletion of Ca2+ stored in the ER unites the Orai channel and its activator, STIM, despite their distinct membrane localizations. This study proposes to use X-ray crystallography to determine 3- dimensional structures of CRAC channels. Biochemical and biophysical techniques, including an assay to measure ion channel activity in vitro, will be used to correlate channel function with structural analysis. With these approaches we aim to: i) determine the 3-dimensional architecture of Orai, ii) investigate how Orai achieves high selectivity for Ca2+, and iii) study how the channel is gated by interactions with STIM. The proposed study will reveal basic principles of CRAC channel function, thereby making significant contributions to multiple fields of research including calcium signaling, ion channels, and membrane protein structural biology. PUBLIC HEALTH RELEVANCE: This proposal seeks to understand the architecture and molecular mechanisms of Orai, a calcium ion channel, at an atomic level of detail. Orai plays an important role in the immune system. A mutation of Orai is a cause of a lethal form of severe combined immunodeficiency in human patients. The proposed studies will provide a basis for the development of channel inhibitors that could be used to modulate immune responses. Recent data indicates that Orai channel function is also required for platelet activation and breast tumor cell migration, suggesting that channel inhibitors may have therapeutic applications for preventing arterial thrombosis, ischemic brain infarction, and breast cancer metastasis.

描述（由申请人提供）：CRAC通道的3D结构和功能。该提案的长期目标是了解离子渗透，离子选择性和门控的机制，钙释放激活的钙（CRAC）通道。 CRAC通道通过产生诱导T细胞活化基因所必需的钙（Ca2+）的持续涌入（CA2+）在免疫反应中起着核心作用。 CRAC通道是位于质膜中的整体膜蛋白，其响应于内质网中储存的Ca2+的耗竭而开放。尽管进行了20多年的研究，但尚不清楚这一储存钙进入过程（SOCE）过程的分子成分。最近，Orai蛋白被确定为孔亚基，并确定刺激蛋白为ER Ca2+传感器。这些期待已久的发现加速了与CRAC通道功能的分子机制有关的研究，从而揭示了与其他离子通道的显着差异。除了作为整体膜蛋白外，Orai还没有明显的氨基酸序列与其他已知离子通道同源。 ORAI通道对Ca2+离子具有很高的选择性，但是必须通过与其他CA2+通道不同的体系结构来实现该离子选择性。该通道还具有通道的前所未有的门控机制（打开通道并关闭通道的过程）：尽管它们的膜定位明显，但在ER中储存的Ca2+耗尽。这项研究建议使用X射线晶体学来确定CRAC通道的3维结构。生化和生物物理技术，包括在体外测量离子通道活性的测定，将用于将通道功能与结构分析相关联。通过这些方法，我们的目的是：i）确定Orai的3维结构，ii）研究Orai如何对Ca2+的选择性高度选择性，iii）研究如何通过与Stim的相互作用来控制通道。拟议的研究将揭示CRAC通道功能的基本原理，从而为包括钙信号，离子通道和膜蛋白结构生物学（包括钙信号传导，离子通道和膜蛋白结构生物学）做出重大贡献。 公共卫生相关性：该提案旨在以原子的细节层面了解Orai（钙离子渠道）Orai的结构和分子机制。 Orai在免疫系统中起重要作用。 ORAI突变是人类患者严重合并免疫缺陷的致命形式的原因。拟议的研究将为开发通道抑制剂的开发提供基础，该抑制剂可用于调节免疫反应。最近的数据表明，血小板激活和乳腺肿瘤细胞迁移也需要ORAI通道功能，这表明通道抑制剂可能具有预防动脉血栓形成，缺血性脑梗塞和乳腺癌转移的治疗应用。