Structural dynamics in cyclic nucleotide-modulated channels

环核苷酸调节通道的结构动力学

• 批准号: 10684676
• 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/10684676
• 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; Isomerism; Kinetics; Lead; Length; Ligands; Lipid Bilayers; Lipids; Liposomes; Mass Spectrum Analysis; Measures; Modality; Modeling; Molecular; Molecular Conformation; Monitor; Mutate; Mutation; Nervous System; Output; Peptidylprolyl Isomerase; Phase; Physiological; Physiological Processes; Play; Population; Probability; Process; Proline; Property; Proteins; Regulation; Reporting; Research; Resolution; Role; S phase; Sampling; Signal Transduction; Spectrum Analysis; Structure; Surface; Techniques; Testing; Time; Visual; Visual Signal Transduction Pathway; antagonist; 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 环核苷酸调节通道在心脏和大脑的起搏活动以及 神经系统中的嗅觉和视觉信号转导。这些渠道的功能缺陷会导致 癫痫、心律失常和色盲等疾病。这笔赠款的总体目标是 了解环核苷酸的结合如何门控（打开/关闭）通道以及其他因素如何 脂质和脯氨酸异构化调节这种门控。我们将通过结合最先进的技术来实现这一目标 技术：单粒子冷冻电子显微镜 (cryo-EM) 与原子力显微镜力谱 (AFM-FS)、天然质谱 (MS) 以及单通道电生理学和功能测定等 对脂质体中掺入的通道进行停流荧光测定。我们将采用 SthK，一个模型 原核环核苷酸调节通道，以及用于选择子目标的真核 HCN1 和 HCN2。我们的 第一个目标是确定 SthK 中部分激动和配体选择性的分子机制。我们将 确定特定电压传感器 SthK 突变体的结构，这些突变体表现出增加的开放概率和 将班级平均值与单通道电生理学相关联。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与 SthK 环核苷酸结合域结合。这 将产生两种环核苷酸结合的能量，并将分离孔对 绑定。这一目标将阐明为什么 cAMP 结合不能完全打开 SthK 通道以及为什么 cGMP 不能完全打开 SthK 通道。 拮抗剂，尽管其与结合袋的结合方式与 cAMP 相似。我们的第二个目标是 了解脂质如何调节通道活性。我们将系统地测试脂质对SthK活性的影响 使用停流荧光测定和单通道电生理学，其中通道位于脂质体中 的受控成分。我们将使用以下方法确定与通道（SthK 和 HCN1）紧密结合的脂质 天然 MS 并确定它们如何通过扰动似乎具有活性的残基来增加活性的机制 协调这些脂质-蛋白质相互作用与功能测定。第三个目标是从功能上表征 以及通过新发现的方式对 SthK 以及潜在的 HCN 通道进行结构性调节：脯氨酰 环核苷酸结合域中保守脯氨酸的异构化，这似乎是造成这种情况的原因 用于 SthK 与 cAMP 的双相激活。这可能会产生很大的影响，因为脯氨酸异构化可能会导致 是调节心脏和大脑起搏活动的另一种方法。所有的目标都是为了 揭示环核苷酸调节通道协同调节的分子机制 配体、脂质和酶，它们整合以产生生理学所需的通道激活水平 细胞。

项目成果

Discrimination between cyclic nucleotides in a cyclic nucleotide-gated ion channel.

• DOI: 10.1038/s41594-023-00955-3
• 发表时间: 2023-04
• 期刊: NATURE STRUCTURAL & MOLECULAR BIOLOGY
• 影响因子: 16.8
• 作者: Pan, Yangang;Pohjolainen, Emmi;Schmidpeter, Philipp A. M.;Vaiana, Andrea C.;Nimigean, Crina M.;Grubmueller, Helmut;Scheuring, Simon
• 通讯作者: Scheuring, Simon

Correlation of membrane protein conformational and functional dynamics.

• DOI: 10.1038/s41467-021-24660-1
• 发表时间: 2021-07-16
• 期刊: Nature communications
• 影响因子: 16.6
• 作者: Sanganna Gari RR;Montalvo-Acosta JJ;Heath GR;Jiang Y;Gao X;Nimigean CM;Chipot C;Scheuring S
• 通讯作者: Scheuring S

An iris diaphragm mechanism to gate a cyclic nucleotide-gated ion channel.

虹膜膜片机制，用于栅极循环核苷酸门控离子通道。

• DOI: 10.1038/s41467-018-06414-8
• 发表时间: 2018-09-28
• 期刊: Nature communications
• 影响因子: 16.6
• 作者: Marchesi A;Gao X;Adaixo R;Rheinberger J;Stahlberg H;Nimigean C;Scheuring S
• 通讯作者: Scheuring S