Structural mechanisms for gating of bacterial cyclic nucleotide-gated ion channels

细菌环核苷酸门控离子通道门控的结构机制

• 批准号: 10224689
• 负责人: William N Zagotta
• 金额: $ 39.64万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-09-20 至 2022-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10224689
• 关键词: Agonist; Architecture; Binding; Biochemical; Biological Models; Brain; Cations; Cryoelectron Microscopy; Cyclic AMP; Cyclic GMP; Cyclic Nucleotides; Data; Detergents; Electrons; Environment; Enzymes; Freezing; Functional disorder; Goals; Heart; Heterogeneity; Ion Channel; Kinetics; Ligands; Light; Lipids; Magnetic Resonance; Measurement; Measures; Membrane Lipids; Methodology; Methods; Microfluidics; Modeling; Molecular; Molecular Conformation; Molecular Machines; Nervous System Physiology; Neurons; Pacemakers; Photoreceptors; Physiological; Physiology; Property; Proteins; Publishing; Research Personnel; Resolution; Rest; Spectrum Analysis; Spin Labels; Structure; System; Testing; base; cyclic-nucleotide gated ion channels; molecular modeling; nanodisk; novel; olfactory receptor; reconstitution; response

Cyclic nucleotide-regulated ion channels are exquisite molecular machines that underlie important physiological functions. Cyclic nucleotide-gated (CNG) channels generate the primary electrical response to light in photoreceptors and to odorant in olfactory receptors. The related hyperpolarization-activated cyclic nucleotide-gated (HCN) channels underlie the pacemaker activity of the heart and many neurons in the brain. These cation selective channels are opened by the direct binding of cyclic nucleotides (cAMP and cGMP) to an intracellular domain of the channel. Our goal is to reveal the molecular mechanism for this allostery in CNG channels. Our approach will be to study bacterial CNG channels as a model system for the eukaryotic channels because of the huge advantages they provide for our biochemical methods . We will leverage the power of four different methodologies to determine the structure, conformational heterogeneity, and dynamics of these channels: 1) cryoelectron microscopy (cryo-EM), 2) double electron-electron resonance (DEER), 3) microfluidic rapid freeze quench (µRFQ) in combination with DEER, and 4) Rosetta-based molecular modeling. The proposal includes four investigators who are pioneers in each of these methods. The use of all four methods on the same ion channel under the same conditions is synergistic and ultimately will lead to a comprehensive structural and energetic model for the allostery of this channel. Ultimately a molecular understanding of these channels would inform not only the physiology and pathophysiology of the heart and brain, but also the general mechanisms for allosteric control of many enzymes.

环状核调节的离子通道是独家分子机器 重要的生理功能是基础。环状核苷酸门控（CNG）通道 产生对光感受器的光的主要电响应，并在 嗅觉受体。相关的超极化激活的循环核苷酸门控 （HCN）通道是心脏的起搏器活动和许多神经元的基础 脑。这些阳离子选择性通道通过循环的直接结合打开 核苷酸（CAMP和CGMP）到通道的细胞内结构域。我们的目标是 揭示了CNG通道中这种变构的分子机制。我们的做法意愿 研究细菌CNG通道作为真核通道的模型系统 由于它们为我们的生化方法提供了巨大的优势。我们将 利用四种不同方法的功率来确定结构， 构象异质性和这些通道的动力学：1）冷冻电子 显微镜（冷冻EM），2）双电子电子共振（鹿），3）微流体 快速冷冻淬火（µRFQ）与鹿相结合，4）基于Rosetta的分子 造型。该提议包括四名调查人员 方法。在同一离子通道上使用所有四种方法 条件是协同作用的，最终将导致全面的结构和 该通道变构的能量模型。最终一种分子理解 这些渠道不仅会告知心脏的生理和病理生理学 和大脑，也是许多酶的变构控制的一般机制。