Structural studies of HCN channels in health and disease

HCN通道在健康和疾病中的结构研究

基本信息

批准号：
10438777
负责人：
WAYNE A. HENDRICKSON
金额：
$ 42.69万
依托单位：
COLUMBIA UNIVERSITY HEALTH SCIENCES
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-08-15 至 2024-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10438777
关键词：
Address Amino Acids Arrhythmia Binding Brain Brain region C-terminal Cardiac Cardiac pacemaker Cardiovascular Diseases Cations Cryoelectron Microscopy Cyclic AMP Cyclic Nucleotides Cytoplasmic Tail Data Detergents Disease Drug Targeting Drug or chemical Tissue Distribution Electrons Electrophysiology (science)Epilepsy Exhibits Generations Genes Genetic Genetic Diseases Goals HCN1 gene HCN4 gene Health Heart Heart Diseases Hormones Human Individual Kinetics Laboratories Ligands Mediating Membrane Microscopic Modeling Mutation N-terminal Nature Neurons Pathogenicity Patients Phenotype Physiological Play Property Protein Isoforms Proteins Publishing Resolution Role Specificity Structure Tertiary Protein Structure Testing Transmembrane Domain Urea Voltage-Gated Potassium Channel base biophysical properties childhood epilepsy design diphenyl disease phenotype disease-causing mutation experimental study gain of function heart electrical activity infancy insight mutant nervous system disorder neuronal excitability novel novel therapeutic intervention particle protein expression protein function response sensor small molecule voltage voltage gated channel

项目摘要

Project Summary The goal of this project is to understand how the gating of HCN channels is regulated by domains located in the cytoplasmic, intracellular portion of the protein. HCN channels regulate pacemaking in the heart, and neuronal excitability in the brain. They have the general structure of voltage-gated K+ channels, but are activated upon membrane hyperpolarization and conduct an inward, depolarizing current. Importantly, their gating is modulated by the direct binding of cAMP to an intracellular cyclic-nucleotide binding domain (CNBD) located in the C-terminal portion of the protein. Recent structural data have also revealed the presence of a second, highly structured domain in the intracellular N-terminal portion of the protein, immediately preceding the first transmembrane domain (HCN domain). In this study, we will use a combination of structural and functional approaches to determine the potential role of the HCN domain in the modulation of channel gating, and the nature of the interactions between the C-terminal CNBD and N-terminal HCN domain. As different HCN isoforms (HCN1-4) exhibit markedly distinct biophysical properties, we will exploit these differences to identify key contacts and residues that may contribute to modulate the gating of HCN channels. In Aim 1, we will use cryo-electron microscopy (cryoEM) to determine the structure of the HCN4 isoform, in the presence and absence of cAMP, and compare its features to the available cryoEM structure of HCN1. In Aim 2, we will resolve the structure of HCN4 in the presence of ligands that interfere with the cAMP-mediated facilitation of channel opening (auxiliary subunit TRIP8b, biphenylurea compound BPU), and use the structural data acquired through Aims 1 and 2 to inform functional experiments designed to test and interpret any inferred model of structure/function relation. Finally, in Aim 3, we will use a similar combination of cryoEM and functional studies to study select HCN1 and HCN4 mutations found in patients with genetic epilepsy and cardiac arrhythmias.

项目概要该项目的目标是了解 HCN 通道的门控是如何由位于蛋白质的细胞质、细胞内部分。 HCN 通道调节心脏起搏，并且大脑中神经元的兴奋性。它们具有电压门控 K+ 通道的一般结构，但在膜超极化时被激活并传导向内的去极化电流。重要的是，他们的门控通过 cAMP 与细胞内环核苷酸结合域 (CNBD) 的直接结合来调节位于蛋白质的C末端部分。最近的结构数据还表明存在第二个高度结构化的结构域位于蛋白质的细胞内 N 端部分，紧接在第一个跨膜结构域（HCN 结构域）。在本研究中，我们将结合使用结构和确定 HCN 结构域在通道门控调制中的潜在作用的功能方法，以及 C 端 CNBD 和 N 端 HCN 结构域之间相互作用的性质。一样不同 HCN 异构体 (HCN1-4) 表现出明显不同的生物物理特性，我们将利用这些差异来识别可能有助于调节 HCN 通道门控的关键接触点和残基。在目标 1 中，我们将使用冷冻电子显微镜 (cryoEM) 确定 HCN4 同工型的结构，和缺乏 cAMP，并将其特征与 HCN1 的可用冷冻电镜结构进行比较。在目标 2 中，我们将在干扰 cAMP 介导的配体存在的情况下解析 HCN4 的结构通道开放（辅助亚基TRIP8b，联苯脲化合物BPU），并使用结构数据通过目标 1 和 2 获得的信息，为旨在测试和解释任何推断的功能实验提供信息结构/功能关系模型。最后，在目标 3 中，我们将使用冷冻电镜和功能研究，研究遗传性癫痫患者中发现的选定 HCN1 和 HCN4 突变心律失常。