3D Structure and Function of CRAC Channels

 描述（由申请人提供）：本项目的目的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通道通过产生持续的钙（Ca 2+）流入淋巴细胞而在免疫系统中起着至关重要的作用，这是激活免疫应答基因所必需的。CRAC通道含有两种主要组分：蛋白质奥赖，其在质膜中形成Ca 2+传导孔;和调节蛋白STIM，其是位于内质网（ER）中的膜蛋白。在称为门控的过程中，CRAC通道响应于ER内储存的Ca 2+浓度的降低而从非导电（闭合）状态切换到导电（打开）状态。阻止门控的奥赖突变导致严重的联合免疫缺陷，这强调了这一过程在免疫功能中的重要性。奥赖也与癌症进展有关。在几个方面，奥赖与其他离子通道不同。我的实验室确定了奥赖的三维结构，它代表了通道的封闭构象。这个通道的“蓝图”揭示了奥赖及其离子孔的结构。与拟议的研究，我们将调查通道的前所未有的门控机制：消耗钙储存在ER团结奥赖和STIM跨细胞溶质空间分离其不同的膜定位，这种分子“握手”导致离子孔在奥赖打开。开放的通道对Ca 2+离子具有极高的选择性，有效地只允许Ca 2+进入细胞。拟议的研究还旨在解决Ca 2+离子如何渗透通过通道的机制，以及通道如何实现这种卓越的离子选择性。本研究使用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，包括测定细胞和重建系统中CRAC通道功能的试验，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）确定开放构象中奥赖的三维结构，ii）可视化作为门控基础的奥赖和STIM之间的分子“握手”，iii）研究高选择性Ca 2+渗透的机制，以及iv）研究CRAC通道的小分子抑制剂如何抑制通道。这些研究将揭示CRAC通道功能的原理，从而为包括Ca 2+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。这些研究将为CRAC通道抑制剂的设计和优化提供有用的信息，这些CRAC通道抑制剂可用作抗癌剂和免疫应答调节剂。

 DESCRIPTION (provided by applicant): The objective of this project is to understand the mechanisms of ion permeation, ion selectivity, and gating in calcium release-activated calcium (CRAC) channels. CRAC channels play a vital role in the immune system by generating the sustained influx of calcium (Ca2+) into lymphocytes that is necessary for activation of immune-response genes. CRAC channels contain two major components: the protein Orai, which forms a Ca2+-conducting pore in the plasma membrane, and the regulatory protein STIM, which is a membrane protein located in the endoplasmic reticulum (ER). In a process known as gating, CRAC channels switch from a non- conductive (closed) state to a conductive (open) state in response to a reduction of the concentration of Ca2+ stored within the ER. Mutations in Orai that prevent gating cause severe combined immune deficiency, which underscores the importance of this process in immune function. Orai has also been implicated in cancer progression. In several respects, Orai is unlike other ion channels. My laboratory determined a 3-dimensional structure of Orai that represents a closed conformation of the channel. This `blueprint' of the channel revealed the architecture of Orai and its ion pore. With the proposed studies, we will investigate the channel's unprecedented mechanism of gating: the depletion of Ca2+ stored in the ER unites Orai and STIM across the cytosolic space separating their distinct membrane localizations, and this molecular `handshake' causes the ion pore in Orai to open. The opened channel is exquisitely selective for Ca2+ ions, effectively allowing only Ca2+ to enter the cell. The proposed studies also aim to address the mechanisms for how Ca2+ ions permeate through the channel and how the channel achieves such superb ion selectivity. This study uses X-ray crystallography to determine 3-dimensional structures of CRAC channels. Biochemical and biophysical techniques, including assays to measure CRAC channel function in cells and in reconstituted systems, will be used to correlate channel function with structural analysis. With these approaches we aim to: i) determine the 3-dimensional architecture of Orai in an open conformation, ii) visualize the molecular `handshake' between Orai and STIM that underlies gating, iii) investigate the mechanisms of highly selective Ca2+ permeation, and iv) investigate how small molecule inhibitors of CRAC channels inhibit the channel. The proposed studies will reveal principles of CRAC channel function, thereby making significant contributions to multiple fields of research including Ca2+ signaling, ion channels, and membrane protein structural biology. The studies will provide useful information for the design and optimization of CRAC channel inhibitors that could have utility as anti-cancer agents and as modulators of immune responses.