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脑的起搏器活动中起着重要作用。它们是含有S4的电压门控离子通道家族的成员，具有一个额外的细胞质CN结合域，通过称为C-接头的螺旋束连接到孔上。到目前为止，还没有全长通道的高分辨率结构，也没有纯化的通道蛋白的功能表征。我们的目的是通过表征纯化的通道在特定的膜环境中的结构和功能，帮助理解配体结合如何导致CNG/HCN通道的孔门。细菌、体外翻译、酵母和昆虫细胞将被用来优化这些通道的表达。对于功能分析，纯化的通道将被转移到脂质体或纳米盘中，以提高蛋白质的稳定性。我们将使用电生理学技术对纯化的通道进行表征。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特性。这将加强我们对这些通道如何在分子水平上进行门的理解。

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.