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。除了它们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至关重要的作用。它们是新药的重要药理靶点。 开发用于治疗癫痫和神经性疼痛等疾病的药物。尽管在了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 了解支持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，有可能为内向整流和配体激活提供新的见解。例如，使用膜片钳荧光法测量整体配体结合 最近提出了一种值得注意的配体激活模型，该模型涉及生理配体对通道活性的一系列正负调节。尽管来自HCN通道的环核苷酸结合域(CNBD)的晶体结构很多，但这种不寻常的机制 合作的形式仍然不清楚。这个项目的中心目标是了解化学结构和由此产生的作用力如何协调HCN通道中的配体激活。这项建议充分利用了 威斯康星大学麦迪逊分校的跨学科专业知识将配体结合的单分子测量与 配基活化的结构和功能分析。我们将检验一个假设，即HCN中的配体激活 通道可能涉及到对称破坏切换到二聚体配置的二聚体。在具体目标1中，我们将 使用零模波导来测量单个配体与环核苷酸结合域的结合。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作性。有了这份分析，我们将能够确定对四个配体中的每一个负责的关键分子决定因素。在具体目标2中，我们将使用X射线结晶学来确定HCN CNBD的未连接状态的结构以及它们的新构象 已连接的表格。在具体目标3中，我们将通过电生理和生化结合研究来进行配体激活的功能分析。这些研究与突变相结合将识别出分子 异构体特异性配体激活差异的基础。拟议的研究有望带来新的曙光。 关于电压和配体激活离子通道中配体激活过程中的构象变化背后的分子作用力。

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.