Three-dimensional structure of a CIC-type CI Channel

描述(申请人提供)：C1-渠道在以下方面扮演多种角色 生物膜。研究了C1C家族的C1型通道 在过去的20年里，在功能上，它只被从分子上鉴定出来 十年前。CIC渠道结构与许多其他渠道完全不同 例如电压门控阳离子通道。真核生物的成员有一个分子质量 大约100 kDa，具有特征的跨膜拓扑结构，包括 约10个跨膜节段。越来越多的证据表明，C1C毛孔 都是由一个亚基组成的。然而，它们的四元结构是 同二聚体。这些独特的特征预示着一种全新的结构--功能 这一系列离子通道的范例。 我和我的同事成功地重组了细菌C1C 同系物，埃里克，变成大的，高度有序的二维晶体适合 用于高分辨率电子冷冻显微镜。我们建议确定 在3.7A或更高分辨率下显示Eric的三维结构 它的二级结构和毛孔。以帮助确定 地图上不清楚区域的多肽链，例如在表面 循环、半胱氨酸诱变和重原子标记相结合的方法。 半胱氨酸诱变和标记也将被用来确定关键功能 结构中的组。标签的位置将通过以下方式确定 差分傅立叶映射。这项工作将生产出第一个C1C型模型 离子通道，是更深入了解C1C通道功能的关键。

DESCRIPTION (provided by applicant): C1- channels play a multitude of roles in biological membranes. The C1C family of C1- channels has been studied functionally for the past 20 years, and was identified molecularly only about a decade ago. CIC channels structure differs entirely from many other channels such as voltage-gated cation channels. Eukaryotic members have a molecular mass of approximately 100 kDa with a characteristic transmembrane topology including about 10 transmembrane segments. Accumulating evidence suggests that C1C pores are formed by a single subunit. However, their quaternary structure is homodimeric. These unique features predict an entirely new structure-function paradigm for this family of ion channels. My colleagues and I have succeeded in reconstituting the bacterial C1C homologue, EriC, into large, highly ordered two-dimensional crystals suitable for high-resolution electron cryo-microscopy. We propose to determine the three-dimensional structure of EriC at 3.7 A resolution or higher to visualize its secondary structure and the pore. To help determine the path of the polypeptide chain in unclear regions of the map, for example in the surface loops, cysteine mutagenesis combined with heavy atom labeling will be used. Cysteine mutagenesis and labeling will also be used to identify key functional groups in the structure. The location of the labels will be determined using difference Fourier maps. The work will produce the first model of a C1C-type ion channel, key to a more thorough understanding of C1C channel function.