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.

项目摘要该项目的目的是了解HCN通道的门控如何受到位于蛋白质的细胞质，细胞内部分。 HCN通道调节心脏中的起搏，并大脑中的神经元兴奋性。它们具有电压门控的K+通道的一般结构，但在膜超极化上激活并进行内向，去极化电流。重要的是，他们的 cAMP与细胞内环形核苷酸结合结构域（CNBD）的直接结合来调节门控位于蛋白质的C末端部分。最近的结构数据也揭示了存在其次，在蛋白质的细胞内N末端部分中高度结构化结构域，紧接第一个跨膜结构域（HCN域）。在这项研究中，我们将结合结构和确定HCN结构域在通道门控的调制中的潜在作用的功能方法，以及C末端CNBD和N端HCN结构域之间相互作用的性质。与众不同 HCN同工型（HCN1-4）具有明显不同的生物物理特性，我们将这些差异利用确定可能有助于调节HCN通道门控的关键接触和残基。在AIM 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营地，并将其特征与HCN1的冷冻结构进行比较。在AIM 2中，我们将在存在干扰营地介导的促进的配体的情况下解决HCN4的结构通道开口（辅助亚基Tir8b，biphenylurea bpu），并使用结构数据通过目标1和2获取，以告知旨在测试和解释任何推断的功能实验结构/功能关系的模型。最后，在AIM 3中，我们将使用类似的冷冻和在遗传癫痫患者和心律不齐。