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: 10609452
• 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 2028-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10609452
• 关键词: Address; Algorithmic Analysis; Architecture; Arrhythmia; Binding; Biological Models; Biophysical Process; Brain; Chemicals; Cyclic Nucleotides; Development; Disease; Elements; Epilepsy; Exhibits; Fluorometry; Functional disorder; Heart; High temperature of physical object; Ion Channel; Ion Channel Gating; Length; Life; Ligand Binding; Ligands; Measures; Membrane; Modeling; Molecular; Molecular Probes; Muscle; Property; Protein Dynamics; Proteins; Regulation; Second Messenger Systems; Signal Transduction; Spectrum Analysis; Stimulus; Structure; Temperature; Temperature Sense; Testing; Thermodynamics; 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; waveguide

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.

项目概要 电压门控离子通道 (VGIC) 的成员对于整个过程中的电和化学信号传导至关重要 生命的三个王国。离子通道功能障碍是多种病理生理学的基础，它们是 新药开发的首要目标之一。尽管它们具有共同的膜结构， 这个超家族中的通道表现出令人惊讶的功能多样性。对膜的反应最开放 去极化，但有些对超极化持开放态度。其中许多也是多模式的——它们的活动受到监管 通过第二信使（例如环核苷酸）或物理刺激（例如温度）。主要目标 该提案的目的是探讨分子驱动力，以了解其基本机制 电压门控及其通过温度和配体的调节。当前的机械方法倾向于结构 重点关注蛋白质动力学要么被忽略，要么被视为次要的。虽然结构 现在有许多对温度高度敏感的离子通道可用，我们对温度机制的理解 热敏感性在很大程度上仍然受到限制，因为我们无法直接探测分子力。到 为了解决这个问题，我们正在采用多管齐下的方法，结合新的和现有的工具来系统地 表征决定电压门控极性的分子相互作用，精致的温度敏感 VGIC 中的性和不寻常的变构。我们使用 HCN 通道作为模型系统来研究选通极性 和配体激活。使用零模型波导和新开发的高通量分析算法 我们能够探测模型系统中配体结合的协同性。我们现在准备扩展这些 对全长通道和受体的研究。关于门控极性的机制，我们做了一个 令人惊讶的发现是二分开关调节 HCN 通道中的门控极性。微秒级模拟 安东超级计算机中的关系提出了一个门控模型，我们将对其进行进一步测试。我们将进行结构性 研究并将其与电压钳荧光测定法结合起来，以注释这些结构。接下来我们还将 使用祖先蛋白质重建方法，识别调节门控的深层变构网络 这些渠道的稀有性。我们对温度相关门控的研究基于两个模型系统：a) Tem- 温度敏感的 Shaker 突变体和 b) 古菌 MthK 通道。为了确定基本要素 由于负责“感知”温度，我们必须测量热力学性质，例如热量 容量。我们建议开发一种涉及单分子力谱的新方法来提取这些 能量参数。总体而言，我们以“分子力”为中心的方法有潜力提供无与伦比的 深入了解 VGIC 中的电压门控机制及其温度调节。