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。本提案的长期目标是了解钙释放激活钙（CRAC）通道的离子渗透，离子选择性和门控机制。CRAC通道通过产生诱导T细胞活化基因所必需的钙（Ca2+）的持续内流在免疫应答中发挥核心作用。CRAC通道是位于质膜中的整合膜蛋白，响应于内质网（ER）内储存的Ca2+的耗尽而打开。尽管经过20多年的研究，这种钙库操纵的钙进入（SOCE）过程的分子组成仍然未知。最近，奥赖蛋白被鉴定为孔亚基，STIM蛋白被确定为ER Ca 2+传感器。这些期待已久的发现加速了有关CRAC通道功能分子机制的研究，揭示了与其他离子通道的显着差异。除了是一种完整的膜蛋白外，奥赖与其他已知的离子通道没有显著的氨基酸序列同源性。奥赖通道对Ca2+离子具有高度选择性，但这种离子选择性必须通过与其他Ca2+通道不同的结构来实现。该通道还具有前所未有的门控机制（打开和关闭通道的过程）：储存在ER中的Ca 2+的耗尽将奥赖通道及其激活剂STIM结合在一起，尽管它们具有不同的膜定位。本研究建议使用X射线晶体学来确定CRAC通道的三维结构。生物化学和生物物理技术，包括体外离子通道活性测定，将用于将通道功能与结构分析相关联。通过这些方法，我们的目标是：i）确定奥赖的三维结构，ii）研究奥赖如何实现对Ca 2+的高选择性，以及iii）研究通道如何通过与STIM的相互作用被门控。这项研究将揭示CRAC通道功能的基本原理，从而为包括钙信号传导、离子通道和膜蛋白结构生物学在内的多个研究领域做出重大贡献。 公共卫生相关性：该提案旨在了解奥赖（一种钙离子通道）在原子水平上的结构和分子机制。奥赖在免疫系统中起着重要作用。奥赖的突变是人类患者中致命形式的严重联合免疫缺陷的原因。拟议的研究将为开发可用于调节免疫反应的通道抑制剂提供基础。最近的数据表明，奥赖通道功能也是血小板活化和乳腺肿瘤细胞迁移所必需的，这表明通道抑制剂可能具有预防动脉血栓形成、缺血性脑梗死和乳腺癌转移的治疗应用。