Three-dimensional structure of a CIC-type CI Channel

描述（由申请人提供）：C1-通道在

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.