Biophysical mechanisms of gating and modulation in voltage-gated ion channel superfamily

电压门控离子通道超家族的门控和调节的生物物理机制

• 批准号: 10266191
• 负责人: Baron Chanda
• 金额: $ 98.55万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-05-03 至 2029-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10266191
• 关键词: Address; Algorithmic Analysis; Architecture; Arrhythmia; Binding; Biological Models; Biophysical Process; Brain; Chemicals; Cyclic Nucleotides; Disease; Elements; Epilepsy; Exhibits; Fluorometry; Functional disorder; Ion Channel; Ion Channel Gating; Length; Life; Ligand Binding; Ligands; Measures; Membrane; Modeling; Molecular; Molecular Probes; Myocardium; Property; Protein Dynamics; Proteins; Regulation; Second Messenger Systems; Signal Transduction; Spectrum Analysis; Stimulus; Structure; Temperature; Temperature Sense; Testing; Thermodynamics; base; driving force; drug development; high throughput analysis; insight; member; mutant; new therapeutic target; novel strategies; novel therapeutics; receptor; reconstruction; response; simulation; single molecule; supercomputer; tool; voltage; voltage clamp

Project Summary Members of the voltage-gated ion channels (VGICs) are critical for electrical and chemical signaling throughout the three kingdoms of life. Dysfunction of ion channels underlie a wide range of pathophysiology and they are one of the primary targets for new drug development. Although they share a common membrane architecture, the channels in this superfamily exhibit surprising diversity of function. Most open in response to a membrane depolarization but some open on hyperpolarization. Many of them are also polymodal- their activity is regulated by second messengers such as cyclic nucleotide or a physical stimulus such as temperature. The main objective of this proposal is to probe the molecular driving forces in order to understand the fundamental mechanisms of voltage-gating and its modulation by temperature and ligand. Current mechanistic approach tends to be structure focused to the extent that protein dynamics is either ignored or treated as secondary. Although the structures of many highly temperature-sensitive ion channels are now available, our understanding of the mechanism of tem- perature-sensitivity remains limited, in large part, due to our inability to directly probe the molecular forces. To address this issue, we are using a multi-pronged approach that combines new and existing tools to systematically characterize the molecular interactions that determine polarity of voltage-gating, exquisite temperature-sensitiv- ity and unusual allostery in VGICs. We are using the HCN channel as a model system to study gating polarity and ligand activation. Using zero model waveguides and newly developed high-throughput analysis algorithms we were able to probe the cooperativity of ligand binding in a model system. We are now poised to extend these studies to full-length channels and receptors. With regards to mechanisms of gating polarity, we have made a surprising discovery that a bipartite switch regulates gating polarity in HCN channels. Microsecond scale simu- lations in Anton supercomputer suggest a gating model which we will be tested further. We will carry out structural studies and combine it with voltage clamp fluorometry in order to annotate these structures. Next, we will also use ancestral protein reconstruction approach, to identify the deep allosteric networks that regulate gating po- larity in these channels. Our studies on temperature-dependent gating is based on two model systems: a) Tem- perature-sensitive Shaker mutant and, b) archaeal MthK channel. In order to determine the essential elements that are responsible for “sensing” temperature, we have to measure the thermodynamic properties such as heat capacity. We propose to develop a new approach involving single molecule force spectroscopy to extract these energetic parameters. Overall, our “molecular forces” focused approach has the potential to provide unparalleled insights into the mechanisms of voltage gating and its regulation by temperature in VGICs.

项目摘要 电压门控离子通道(VGIC)的成员对于贯穿整个过程的电和化学信号是至关重要的 生命的三大王国。离子通道功能障碍是广泛的病理生理学基础，它们是 新药开发的主要目标之一。尽管它们共享共同的膜体系结构， 这个超级家族中的通道展示了令人惊讶的功能多样性。最开放的是对膜的响应 去极化，但有些是开放的超极化。它们中的许多也是多式联运的--它们的活动是受监管的 通过第二信使，如环核苷酸或物理刺激，如温度。主要目标 这一建议的目的是探索分子驱动力，以了解其基本机制 电压门控及其温度和配基的调制。当前的机械论方法倾向于结构化 聚焦到蛋白质动力学要么被忽视，要么被视为次要的程度。尽管它的结构 目前已有许多对温度高度敏感的离子通道可供选择，我们对温度敏感离子通道的作用机理的了解将有助于我们对温度敏感离子通道的研究。 温度敏感性仍然有限，这在很大程度上是因为我们无法直接探测分子作用力。至 为了解决这个问题，我们正在使用一种多管齐下的方法，将新的和现有的工具结合起来，系统地 表征决定电压门控的极性的分子相互作用，精致的温度敏感性- VGIC中的丰度和异常变构。我们正在使用HCN通道作为模型系统来研究门极极性 和配基激活。使用零模波导和新开发的高通量分析算法 我们能够在一个模型系统中探索配体结合的协作性。我们现在准备延长这些 对全长通道和受体的研究。关于门控极性的机制，我们已经做了一个 一项令人惊讶的发现：两部分开关调节着HCN通道中的门控极性。微秒级模拟- Anton超级计算机上的数据提出了一个门控模型，我们将对其进行进一步的测试。我们将开展结构性改革。 研究它并将其与电压钳荧光法相结合，以注释这些结构。接下来，我们还将 利用祖先蛋白重构的方法，识别调节门控位置的深层变构网络。 在这些渠道中出现了严重问题。我们对温度相关浇注的研究是基于两个模型系统的：a)瞬变浇注； 温度敏感的摇动器突变体和，b)古生菌MthK通道。为了确定基本要素 负责“感应”温度，我们必须测量热力学性质，如热 容量。我们建议开发一种新的方法，涉及单分子力谱来提取这些 能量参数。总体而言，我们专注于“分子力”的方法有可能提供无与伦比的 对VGIC中电压门控及其温度调节机制的认识。