Mechanisms of voltage- and ligand-activation in HCN channels

项目摘要 超极化激活和环核苷酸门控离子通道（HCN）在心脏中高度表达 和中枢神经系统，负责缓慢激活有助于起搏的电流 活动除了在神经元回路中产生节律性振荡的作用外，这些通道还发挥着 在工作记忆和运动学习中起着关键作用。它们是新药开发的重要药理学靶点 开发用于治疗疾病状况如癫痫和神经性疼痛。尽管在理解这些离子通道的结构和生理作用方面取得了进展，但在我们的研究中仍然存在重大差距。 了解HCN通道行为的生物物理机制。这些渠道是独一无二的， 电压门控离子通道超家族，并有可能提供新的见解向内整流和配体激活。例如，使用膜片钳荧光测定法的整体配体结合测量 最近提出了一种配体激活的显著模型，该模型涉及生理配体对通道活性的一系列正和负调节。尽管HCN通道的环核苷酸结合结构域（CNBD）的晶体结构众多，但这种不寻常的CNBD的机制仍然是未知的。 协同性的形式仍不清楚。该项目的中心目标是了解化学结构和所产生的力如何协调HCN通道中的配体激活。该提案利用了 在威斯康星大学麦迪逊分校的跨学科专业知识，结合配体结合的联合收割机单分子测量与 配体活化结构和功能分析。我们将检验HCN中的配体活化 通道可能涉及到二聚体构型的二聚体的破坏性转换。具体目标1： 使用零模式波导来测量单个配体与环状核苷酸结合结构域的结合。这将使我们能够直接测量每个配体结合步骤的能量，并跟踪与此过程相关的协同性。有了这个分析，我们将能够确定负责四个配体中每一个的关键分子决定因素。在具体目标2中，我们将使用X射线晶体学来确定HCN CNBD的未配位态的结构以及它们的新构象。 配体形式。在具体目标3中，我们将使用电生理学和生物化学结合研究进行配体激活的功能分析。这些研究结合诱变将确定分子 配体活化中同种型特异性差异的基础。拟议中的研究有望为我们提供新的视角， 在电压和配体激活的离子通道中的配体激活期间，分子力是构象变化的基础。

Project Summary Hyperpolarization-activated and cyclic nucleotide-gated ion channels (HCN) are highly expressed in the heart and central nervous system where they responsible for slowly activating currents that contribute to pacemaking activity. In addition to their role in generating rhythmic oscillations in neuronal circuits, these channels also play a crucial role in working memory and motor learning. They are important pharmacological targets for new drug development to treat disease conditions such as epilepsies and neuropathic pain. Despite the progress in understanding the structure and physiological role of these ion channels, there remains a significant gap in our knowledge of the biophysical mechanisms that underpin HCN channel behavior. These channels are unique in the voltage-gated ion channel superfamily and have the potential to provide new insights into inward rectification and ligand activation. For instance, ensemble ligand binding measurements using patch clamp fluorimetry have recently suggested a remarkable model of ligand activation that involves a sequence of positive and negative modulation of channel activity by physiological ligand. Although numerous crystal structures of cyclic nucleotide-binding domain (CNBD) from HCN channels are available, the mechanisms that underlie this unusual form of cooperativity remain unclear. The central goal of this project is to understand how the chemical structure and the resulting forces orchestrate ligand activation in HCN channels. This proposal takes advantage of the interdisciplinary expertise at UW-Madison to combine single molecule measurements of ligand binding with structural and functional analysis of ligand activation. We will test the hypothesis that ligand activation in HCN channels may involve a symmetry-breaking switch to a dimer of dimer configuration. In specific aim 1, we will use zero-mode waveguides to measure the binding of individual ligands to the cyclic-nucleotide binding domains. This will allow us to directly measure energetics of each ligand-binding step and to track the cooperativity associated with this process. With this analysis in hand, we will be able to identify the key molecular determinants responsible for each of the four ligands. In specific aim 2, we will use X-ray crystallography to determine the structures of the unliganded states of the HCN CNBDs as well as new conformations of their liganded forms. In specific aim 3, we will carry out functional analysis of ligand activation using electrophysiological and biochemical binding studies. These studies combined with mutagenesis will identify the molecular bases for isoform-specific differences in ligand activation. The proposed studies are expected to shed new light on the molecular forces that underlie conformational changes during the ligand activation in a voltage- and ligand-activated ion channel.