3D Structure and Function of CRAC Channels

描述（申请人提供）：CRAC通道的三维结构和功能。本研究的长期目标是了解钙释放活化钙（CRAC）通道中离子渗透、离子选择性和门控的机制。CRAC通道通过产生持续的钙（Ca2+）内流在免疫反应中发挥核心作用，这是诱导T细胞激活基因所必需的。CRAC通道是位于质膜上的完整膜蛋白，在内质网（ER）内储存的Ca2+耗尽时打开。尽管经过了20多年的研究，这种储存操作钙进入（SOCE）过程的分子成分仍是未知的。最近，Orai蛋白被确定为孔亚基，STIM蛋白被确定为ER Ca2+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著差异。除了作为一个完整的膜蛋白外，Orai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Ca2+离子具有高度选择性，但这种离子选择性必须通过不同于其他Ca2+通道的结构来实现。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）：存储在内质网中的Ca2+的耗尽将Orai通道及其激活剂STIM结合在一起，尽管它们的膜定位不同。本研究提出使用x射线晶体学来确定CRAC通道的三维结构。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如何实现对Ca2+的高选择性，以及iii)研究如何通过与STIM的相互作用对通道进行门控。本研究将揭示CRAC通道功能的基本原理，为钙信号、离子通道、膜蛋白结构生物学等多个领域的研究做出重要贡献。

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. 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.