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

使用结构和功能工具深入了解真核环核苷酸调节离子通道门控的分子机制

• 批准号: 271275232
• 负责人: Dr. Philipp Schmidpeter
• 金额: --
• 依托单位: Department of Anesthesiology
• 依托单位国家: 德国
• 项目类别: Research Fellowships
• 财政年份: 2015
• 资助国家: 德国
• 起止时间: 2014-12-31 至 2015-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/271275232?language=en
• 关键词: Insights; into; molecular; mechanism; gating

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.

环核苷酸（cN）调节的离子通道如cN门控（CNG）、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器活动中发挥重要作用。它们是含有s4的电压门控离子通道家族的成员，具有额外的细胞质cN结合域，通过称为c -连接器的螺旋束连接到孔上。迄今为止，没有全长通道的高分辨率结构，也没有纯化通道蛋白的功能表征。我们的目标是通过在特定的膜环境中表征纯化通道的结构和功能，帮助理解配体结合如何导致CNG/HCN通道中的孔门控。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圆盘上，以提高蛋白质的稳定性。我们将使用电生理学技术表征纯化通道。cN与纯化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形成的理解。