Three-dimensional structure of a CIC-type CI Channel

描述（由申请人提供）：C1通道在 生物膜研究了C1通道的C1 C族 在过去的20年里， 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。真核生物的分子量 具有特征性跨膜拓扑结构，包括 约10个跨膜片段。越来越多的证据表明C1 C孔隙 都是由一个亚单位组成的然而，它们的四级结构是 同源二聚体。这些独特的特征预示着一种全新的结构-功能 离子通道家族的典范。 我和我的同事成功地重组了细菌C1 C 类似物EriC转化成大的、高度有序的二维晶体， 用于高分辨率电子冷冻显微镜。我们建议确定 以3.7 A或更高分辨率显示EriC的三维结构 其二级结构和孔隙。为了帮助确定 多肽链在地图上不清楚的区域，例如在表面 环，将使用半胱氨酸诱变与重原子标记的组合。 半胱氨酸诱变和标记也将用于鉴定关键的功能性突变。 结构中的组。标签的位置将使用 差分傅立叶映射这项工作将产生第一个模型的C1 C型 离子通道，关键是更彻底地了解C1 C通道的功能。

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.