3D Structure and Function of CRAC Channels

描述(申请人提供)：CRAC频道的3D结构和功能。这项建议的长期目标是了解钙释放激活钙通道中离子渗透、离子选择性和门控的机制。CRAC通道通过产生诱导T细胞激活基因所必需的钙离子持续内流，在免疫反应中发挥核心作用。CRAC通道是位于质膜上的完整的膜蛋白，在内质网(ER)内储存的钙离子耗尽时开放。尽管有20多年的研究，但这一储存操作钙进入(SOCE)过程背后的分子组成尚不清楚。最近，Orai蛋白被确定为孔亚基，STIM蛋白被确定为内质网钙离子感受器。这些期待已久的发现加速了关于CRAC通道功能的分子机制的研究，揭示了与其他离子通道的显著差异。Orai除了是一个完整的膜蛋白外，与其他已知的离子通道没有显著的氨基酸序列同源性。ORAI通道对钙离子具有很高的选择性，但这种离子选择性必须通过不同于其他钙离子通道的架构来实现。该通道还有一种前所未有的门控机制(打开和关闭通道的过程)：内质网中存储的钙离子的枯竭将Orai通道及其激活剂STIM结合在一起，尽管它们的膜定位不同。这项研究建议使用X射线结晶学来确定CRAC通道的三维结构。生化和生物物理技术，包括一种测定体外离子通道活性的方法，将被用于将通道功能与结构分析联系起来。通过这些方法，我们的目标是：i)确定Orai的三维结构，ii)研究Orai是如何实现对钙的高选择性的，以及iii)研究通道是如何通过与STIM的相互作用而被门控的。这项研究将揭示CRAC通道功能的基本原理，从而为钙信号、离子通道和膜蛋白结构生物学等多个领域的研究做出重大贡献。 与公共健康相关：这项提议寻求在原子水平上详细了解钙离子通道Orai的结构和分子机制。Orai在免疫系统中起着重要作用。Orai的突变是导致人类患者严重联合免疫缺陷的致命形式之一。这项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，Orai通道功能也是血小板激活和乳腺肿瘤细胞迁移所必需的，这表明通道抑制剂可能在预防动脉血栓形成、缺血性脑梗塞和乳腺癌转移方面具有治疗应用。

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.