Structural dynamics underlying voltage and pH gating of the human proton channel

人体质子通道电压和 pH 门控的结构动力学

• 批准号: 10610652
• 负责人: SHIZHEN WANG
• 金额: $ 6.27万
• 依托单位: UNIVERSITY OF MISSOURI KANSAS CITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-05-01 至 2023-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10610652
• 关键词: Administrative Supplement; Alkylation; Award; Biological; Cations; Cells; Charge; Electrophysiology (science); Fluorescence Resonance Energy Transfer; Funding; Human; Image; Ion Channel Gating; Liposomes; Membrane; Metabolic; Microscope; Molecular Conformation; NADPH Oxidase; Parents; Phagocytes; Physiological; Production; Proteins; Protons; Reactive Oxygen Species; Resolution; Respiratory Burst; Role; Semiconductors; Speed; Structure-Activity Relationship; Time; biophysical properties; cancer invasiveness; conformational conversion; fluorescence microscope; insight; kinetic model; metal oxide; millisecond; neuron loss; pH Homeostasis; pH gradient; patch clamp; pathogen; sensor; single molecule; single-molecule FRET; sperm cell; voltage; Administrative Supplement; Alkylation; Award; Biological; Cations; Cells; Charge; Electrophysiology (science); Fluorescence Resonance Energy Transfer; Funding; Human; Image; Ion Channel Gating; Liposomes; Membrane; Metabolic; Microscope; Molecular Conformation; NADPH Oxidase; Parents; Phagocytes; Physiological; Production; Proteins; Protons; Reactive Oxygen Species; Resolution; Respiratory Burst; Role; Semiconductors; Speed; Structure-Activity Relationship; Time; biophysical properties; cancer invasiveness; conformational conversion; fluorescence microscope; insight; kinetic model; metal oxide; millisecond; neuron loss; pH Homeostasis; pH gradient; patch clamp; pathogen; sensor; single molecule; single-molecule FRET; sperm cell; voltage

Project Summary/Abstract Voltage-gated proton (Hv) channels carry robust proton currents across membranes and are gated by both voltage and transmembrane pH gradient (∆pH). They normally serve as proton extruders to maintain the pH homeostasis of metabolically active cells. In phagocytes, human Hv1 (hHv1) channels compensate for charge and pH imbalance during the respiratory burst of NADPH oxidase to promote the production of reactive oxygen species (ROS) for pathogen defense. The sperm hHv1 channels trigger intracellular alkylation essential for capacitation. The hHv1 channel also highly correlates with cancer invasiveness and ischemic neuronal cell death. Voltage and ∆pH gating are two fundamental biophysical properties determining the dynamics of proton currents through Hv channels, which in turn underlie their physiological and pathophysiological roles in the cells mentioned above. Funded by the parent award, we examined the conformational dynamics of the purified hHv1 proteins in liposomes using single molecule FRET (Fluorescence Resonance Energy Transfer). We have provided the first glimpse of real-time conformational transitions in the hHv1 voltage sensor and showed that both voltage and pH gate the hHv1 channel by modifying the conformational landscapes of the voltage sensor. We also generated a kinetic model to explain how voltage pH interplay determines hHv1 channel gating. To maximize the impacts of the exciting findings that have been made, we need to obtain accurate rate constants of conformational transitions described by the kinetic model, which can provide key mechanistic insights into the voltage sensing and gating in other voltage-gated cation channels. The administrative supplement for the accessory will upgrade the existing TIRF (Total Internal Reflection Fluorescence) microscope for single molecule FRET imaging, which will provide the critical technical strength to maximize the scientific impacts of the parent award. The accessory contains the patch-clamp module for electrophysiological recording and the single molecule FRET imaging module containing a high-speed sCMOS (scientific Complementary Metal- Oxide-Semiconductor) camera reaching a time resolution close to 1 millisecond. With the accessory, the existing TIRF microscope will be upgraded to perform single molecule FRET imaging and electrophysiological recordings simultaneously. As a result, we will be able to control the voltage and pH applied to hHv1 channels more precisely to get accurate rate constants of the conformational transitions in the hHv1 channel induced by pH and voltage. In addition, we will be able to examine the channel gating dynamics by patch-clamp recordings and conformational dynamics using single molecule FRET imaging simultaneously, thus defining the structure and function relationship directly.

项目摘要/摘要 电压门控质子（HV）通道横跨膜携带强大的质子电流 电压和跨膜pH梯度（∆PH）。它们通常用作维持pH的质子挤出机 代谢活性细胞的稳态。在吞噬细胞中，人类HV1（HHV1）通道补偿了电荷 在氧化NADPH氧化物的呼吸爆发期间，pH不平衡以促进活性氧的产生 病原体防御的物种（ROS）。精子HHV1通道触发细胞内烷基化必不可少的 电容。 HHV1通道还高度与癌症的侵入性和缺血性神经元细胞相关 死亡。电压和∆PH门控是确定质子动力学的两个基本生物物理特性 通过HV通道的电流，这又是其身体和病理生理作用的基础 上面提到的单元格。由父母奖资助，我们检查了纯化的会议动态 脂质体中的HHV1蛋白使用单分子fret（荧光共振能传递）。我们有 提供了HHV1电压传感器中实时构象过渡的第一概述，并表明这一点 电压和pH门通过修改电压传感器的构象景观，均可通过HHV1通道。 我们还生成了一个动力学模型，以解释电压pH相互作用如何决定HHV1通道门控。到 最大化已经提出的令人兴奋的发现的影响，我们需要获得准确的速率常数 动力学模型所描述的构象转变，该转变可以提供关键的机械见解 其他电压门控阳离子通道中的电压敏感性和门控。的行政补充 附件将升级单个现有的TIRF（总内反射荧光）显微镜 分子FRET成像，它将提供关键的技术强度，以最大程度地提高科学影响 父母奖。配件包含用于电生理记录的贴片钳模块， 单分子FRET成像模块，该模块包含高速SCMO（科学互补金属） 氧化物 - 轴导剂）相机达到接近1毫秒的时间分辨率。使用配件， 现有的TIRF显微镜将升级以执行单分子FRET成像和电生理学 录音简单。结果，我们将能够控制电压并应用于HHV1通道 更精确地获得了由HHV1通道中构象转变的准确速率常数 pH和电压。此外，我们将能够通过贴片钳记录来检查通道门控动力学 并同时使用单分子FRET成像构象动力学，从而定义结构 和功能关系直接。