3D Structure and Function of CRAC Channels

 描述(由申请人提供)：本项目的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通道通过产生激活免疫反应基因所必需的钙离子持续流入淋巴细胞，在免疫系统中起着至关重要的作用。CRAC通道包含两个主要成分：Orai蛋白和STIM蛋白，前者在质膜上形成一个钙离子传导孔，后者是一种位于内质网(ER)中的膜蛋白。在一个被称为门控的过程中，CRAC通道随着内质网中存储的钙离子浓度的降低而从非导电(闭合)状态切换到导电(开放)状态。阻止门控的Orai突变会导致严重的联合免疫缺陷，这突显了这一过程在免疫功能中的重要性。Orai还与癌症进展有牵连。在几个方面，Orai与其他离子通道不同。我的实验室确定了Orai的三维结构，它代表了通道的封闭构象。这张通道的“蓝图”揭示了Orai的结构和它的离子孔。在这些研究中，我们将研究该通道史无前例的门控机制：内质网中存储的钙离子的耗竭使Orai和STIM跨越细胞质空间，分离它们不同的膜定位，这种分子‘握手’导致Orai中的离子孔打开。开放的通道对钙离子有极高的选择性，有效地只允许钙离子进入细胞。拟议的研究还旨在解决钙离子如何通过通道渗透的机制，以及通道如何实现如此高的离子选择性。这项研究使用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，包括在细胞和重组系统中测量CRAC通道功能的分析，将被用来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)确定开放构象中Orai的三维结构，ii)可视化Orai和STIM之间作为门控基础的分子“握手”，iii)研究高选择性钙渗透的机制，以及iv)研究CRAC通道的小分子抑制剂如何抑制该通道。这些研究将揭示CRAC通道的功能原理，从而为包括钙信号、离子通道和膜蛋白结构生物学在内的多个领域的研究做出重大贡献。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，这些抑制剂可能作为抗癌药物和免疫反应的调节剂。

 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.