Insights into the molecular mechanism of gating of eukaryotic cyclic nucleotide-modulated ion channels using structural and functional tools

环核苷酸（cN）调节的离子通道如cN门控（CNG）和超极化激活和cN门控（HCN）离子通道在嗅觉和视觉信号级联以及心脏和大脑的起搏活动中起重要作用。它们是含有S4的电压门控离子通道家族的成员，具有通过称为C-接头的螺旋束连接到孔的额外的细胞质cN结合结构域。到目前为止，有没有高分辨率的全长通道的结构和纯化的通道protein. We的目的是有助于了解如何配体结合导致孔门控在CNG/HCN通道的结构和功能的纯化通道在一个确定的膜环境的特征。细菌、体外翻译、酵母和昆虫细胞将用于优化这些通道的表达。对于功能测定，纯化的通道将被转移到脂质体或纳米盘以改善蛋白质稳定性。我们将使用电生理学技术表征纯化的通道。cN与纯化的CNG/HCN全长通道结合的能量学和结构数据将补充功能和生物物理表征。这将增强我们对这些通道如何在分子水平上进行门控的理解。

Cyclic nucleotide (cN) modulated ion channels such as cN gated (CNG), and hyperpolarization activated and cN gated (HCN) ion channels play important roles in olfactory and visual signaling cascades as well as the pacemaker activity in the heart and brain. They are members of the S4-containing voltage gated ion channel family with an additional cytoplasmic cN binding domain attached to the pore via a helical bundle called theC-linker. To date there is no high-resolution structure of the full-length channel and no functional characterization of the purified channel protein.Our aim is to contribute to the understanding how ligand binding leads to pore gating in CNG/HCN channels by characterizing the structure and function of purified channels in a defined membrane environment. Bacteria, in vitro translation, yeast, and insect cells will be employed to optimize the expression of these channels. For functional assays, the purified channels will be transfer to liposomes or nanodiscs to improve protein stability. We will characterize the purified channels using electrophysiology techniques. The energetics of cN binding to purified CNG/HCN full-length channels and structural data will complement the functional and biophysical characterization. This will enhance our understanding of how these channels gate at the molecular level.