Structural dynamics in cyclic nucleotide-modulated channels

环核苷酸调节通道的结构动力学

• 批准号: 10458032
• 负责人: Crina M Nimigean
• 金额: $ 39.97万
• 依托单位: WEILL MEDICAL COLL OF CORNELL UNIV
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-09-01 至 2025-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10458032
• 关键词: Agonist; Arrhythmia; Atomic Force Microscopy; Behavior; Binding; Biological Assay; Brain; Cell physiology; Cells; Color blindness; Cryoelectron Microscopy; Cyclic AMP; Cyclic GMP; Cyclic Nucleotides; Defect; Disease; Electrophysiology (science); Enzymes; Epilepsy; Equilibrium; Goals; Grant; HCN1 gene; Heart; Heterogeneity; Image; Investigation; Ion Channel; Kinetics; Lead; Length; Ligands; Light; Lipid Bilayers; Lipids; Liposomes; Mass Spectrum Analysis; Measures; Modality; Modeling; Molecular; Molecular Conformation; Monitor; Mutate; Mutation; Nervous system structure; Output; Peptidylprolyl Isomerase; Phase; Physiological; Physiological Processes; Play; Population; Probability; Process; Proline; Property; Proteins; Regulation; Reporting; Research; Resolution; Role; Sampling; Signal Transduction; Spectrum Analysis; Structure; Surface; Techniques; Testing; Time; Visual; Visual Signal Transduction Pathway; antagonist; base; cis trans isomerization; combinatorial; cyclic-nucleotide gated ion channels; design; experimental study; improved; insight; mutant; novel; novel therapeutics; particle; patch clamp; reconstitution; response; sensor; single molecule; stopped-flow fluorescence; voltage

ABSTRACT Cyclic nucleotide-modulated channels play major roles in pacemaking activity in heart and brain as well as in olfactory and visual signal transduction in the nervous system. Defects in the functioning of these channels lead to diseases such as epilepsy, cardiac arrhythmia, and color blindness. The overall objective of this grant is to understand how binding of cyclic nucleotides gates (opens/closes) the channels and how other factors such as lipids and proline isomerization modulate this gating. We will accomplish this by combining state-of-the-art techniques: single-particle cryo electron microscopy (cryo-EM) with atomic force microscopy force spectroscopy (AFM-FS), native mass spectrometry (MS), and functional assays like single-channel electrophysiology and stopped flow fluorescence assays of channels incorporated in liposomes. We will employ SthK, a model prokaryotic cyclic nucleotide-modulated channel, and also eukaryotic HCN1 and HCN2 for select sub-aims. Our first aim is to determine the molecular mechanisms for partial agonism and ligand selectivity in SthK. We will determine the structures of specific voltage-sensor SthK mutants that display increased open probability and correlate class averages with the single-channel electrophysiology. To determine the molecular mechanism for ligand selectivity we will use AFM-FS to determine at the single-molecule level the binding kinetics of cAMP and cGMP to either the SthK cyclic nucleotide binding domain alone or in the context of the full-length channel. This will yield the energetics of binding of both cyclic nucleotides and will isolate the contribution of the pore to the binding. This aim will shed light on why cAMP binding does not fully open the SthK channel and why cGMP is an antagonist, although its binding modality to the binding pocket is similar to that of cAMP. Our second aim is to understand how lipids modulate channel activity. We will systematically test the effect of lipids on SthK activity using stopped-flow fluorescence assays and single-channel electrophysiology where channels are in liposomes of controlled composition. We will determine the lipids tightly bound to the channels (both SthK and HCN1) using native MS and determine the mechanism of how they increase activity by perturbing the residues that appear to coordinate these lipid-protein interactions with functional assays. The third aim is to characterize functionally and structurally the regulation of SthK as well as potentially HCN channels by a newly discovered modality: prolyl isomerization of a conserved proline in the cyclic nucleotide binding domain, which appears to be responsible for SthK’s biphasic activation with cAMP. This can be highly impactful, as proline isomerization may turn out to be yet another means to regulate pacemaking activity in the heart and brain. All aims are geared towards unravelling the molecular mechanisms of cyclic nucleotide-modulated channels’ synergistic regulation by ligands, lipids and enzymes, which integrate to yield the channel activation levels required by the physiology of the cell.

抽象的 环状核苷酸调节的通道在心脏和大脑以及在 神经系统中的嗅觉和视觉信号转导。这些通道的功能缺陷。 癫痫，心律不齐和色盲等疾病。这笔赠款的总体目标是 了解环状核苷酸门的结合（打开/关闭）如何以及其他因素等因素如何 脂质和脯氨酸异构化调节该门控。我们将通过结合最先进的 技术：具有原子力显微镜光谱的单粒子冷冻电子显微镜（冷冻EM） （AFM-FS），天然质谱法（MS）以及单通道电生理学等功能测定法 脂质体中掺入的通道的停止流量荧光测定。我们将采用模型STHK 原核环核苷酸调节的通道，以及针对精选子IAM的真核HCN1和HCN2。我们的 第一个目的是确定STHK中部分激动和配体选择性的分子机制。我们将 确定显示出显示开放概率和 将类平均值与单通道电生理学相关。确定分子机制 配体选择性我们将使用AFM-FS在单分子水平上确定CAMP和 CGMP单独或在全长通道的背景下单独或单独使用STHK循环核苷酸结合结构域。这 将产生两种环核苷酸的结合能量，并将隔离孔对孔的贡献 结合。这个目标将阐明为什么营地绑定不会完全打开STHK频道以及CGMP为什么 拮抗剂，尽管它与结合口袋的结合方式类似于营地。我们的第二个目标是 了解脂质如何调节通道活动。我们将系统地测试脂质对STHK活性的影响 使用停止流量荧光测定和单通道电生理学，其中通道在脂质体中 受控组成。我们将使用紧密结合的脂质使用通道（STHK和HCN1）。 天然MS并确定它们如何通过扰动看起来的残差来增加活动的机制 将这些脂质 - 蛋白质相互作用与功能测定协调。第三个目的是表征功能 从结构上讲，通过新发现的模态对STHK以及潜在的HCN通道的调节：prolyl 在环状核苷酸结合结构域中保守脯氨酸的异构化，这似乎是负责的 用于STHK的双相激活。这可能具有很高的影响，因为脯氨酸异构化可能结果可能 是调节心脏和大脑起搏活动的另一种手段。所有目标都适合 通过通过循环核调节通道的协同调节的分子机制来阐明 配体，脂质和酶，它们整合以产生由生理学的通道激活水平 细胞。