3D Structure and Function of CRAC Channels

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。

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.