Thermodynamics and energetics of voltage-gated ion channels

电压门控离子通道的热力学和能量学

• 批准号: 8690188
• 负责人: Baron Chanda
• 金额: $ 31.89万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-09-15 至 2017-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8690188
• 关键词: Address; Amino Acids; Biological Models; Biophysical Process; Characteristics; Charge; Chemicals; Dependence; Dependency; Deuterium Oxide; Disease; Electrophysiology (science); Electrostatics; Engineering; Environment; Erythromelalgia; Esthesia; Family; Fluorescence Spectroscopy; Free Energy; Gated Ion Channel; Geometry; Heating; Inherited; Investigation; Ion Channel; Ion Channel Gating; Ionic Strengths; Kinetics; Knowledge; Measurement; Measures; Mediating; Methods; Modeling; Molecular; Movement; Multiple Sclerosis; Mutagenesis; Mutate; Mutation; Nature; Organism; Phenotype; Physiological; Positioning Attribute; Potassium Channel; Process; Proteins; Role; Shaker potassium channel; Side; Signal Transduction; Site; Solvents; Stimulus; Structure; TRPV1 gene; Temperature; Temperature Sense; Testing; Thermodynamics; Water; analytical tool; base; insight; member; molecular dynamics; mutant; novel; research study; response; sensor; simulation; success; theories; voltage; voltage clamp

DESCRIPTION (provided by applicant): Ion channels directly sense a wide variety of physical and chemical stimuli. Of these, the molecular principles of temperature-sensing and temperature-dependent gating are perhaps the least understood. Here we seek to understand the molecular mechanism of temperature-sensitivity by systematically studying the engineered Shaker potassium channel. The Shaker potassium channel will be developed as a model system for biophysical studies of temperature-dependent gating because of our substantial understanding of its structure and dynamics. We propose to test the hypothesis that solvent mediated interactions of amino acid side-chains at sites undergoing a change in solvent accessibility may underlie temperature-sensitive response of ion channels. Our studies will combine newly developed free-energy measurements of channel gating with electrophysiology, fluorescence spectroscopy and molecular simulations. We will broadly focus our investigations on the voltage-sensing domain of the Shaker potassium channel. First, we will test the correlation between voltage- and temperature-sensitivity. Thermodynamic analysis of the temperature- and voltage-sensitive characteristics of the specialized temperature-sensitive ion channels led to the idea that the voltage- and temperature-sensitivities of ion channels are inversely correlated. This hypothesis will be tested by characterizing the temperature dependent response of mutants of the potassium ion channels, whose voltage-dependencies are reduced by neutralization of charge residues responsible for their voltage-dependence. Second, we will test the importance of the non-polar residues in the S4 segment of the Shaker channel and its influence on temperature sensitivity. The hydrophobic residues of S4 segment are likely to undergo a change in environment polarity as the channel activates. We will test whether altering the polarity of these sites leads to temperature-dependent phenotypes. We will also utilize heavy water as a probe for studying solvent accessibility at these sites. These experiments will be combined with novel spectroscopic approach to test whether the temperature sensitive substitutions alter the nature of structural changes occurring in the proteins. Finally, we will evaluate the importance of water-accessible residues within protein crevices. Altering the polarity of these residues is expected to change the energies associated with their solvation/desolvation process. We will introduce polar and non-polar substitutions at each of these sites and test the functional temperature sensitivity of these mutants. The effects of these substitutions on the geometry of the crevices will be assessed by measuring the ionic strength dependence of charge translocation process. These experiments will be combined with molecular dynamics simulations to evaluate the role of these perturbations on water dynamics within the crevices. At the conclusion of these studies, we would have made significant headway in testing molecular theories that may underlie the temperature-dependence of ion channel gating, developed a new model system and refined our knowledge of the role of water in ion channel gating.

描述（由申请人提供）：离子通道直接感知各种物理和化学刺激。其中，温度敏感性和温度依赖性门的分子原理也许是最不了解的。在这里，我们试图通过系统地研究工程振荡钾通道来理解温度敏感性的分子机制。由于我们对其结构和动力学的深刻理解，将开发出振荡器钾通道作为温度依赖门控的生物物理研究的模型系统。我们建议测试以下假设：溶剂介导的氨基酸侧链在经历溶剂可及性发生变化的位点可能是离子通道对温度敏感反应的基础。我们的研究将结合新开发的通道门控的自由化测量，以及电生理学，荧光光谱和分子模拟。我们将大致将调查集中在振荡器钾通道的电压感应域上。首先，我们将测试电压和温度敏感性之间的相关性。对特殊温度敏感离子通道的温度和电压敏感性特性的热力学分析导致​​了这样的想法，即离子通道的电压和温度敏感性成反比。该假设将通过表征钾离子通道突变体的温度依赖性响应来检验，钾离子通道的温度依赖性响应，其电压依赖性通过中和电荷残基负责其电压依赖性而降低。其次，我们将测试非极性残基在振荡器通道的S4段中的重要性及其对温度灵敏度的影响。随着通道激活，S4段的疏水残基可能会发生环境极性的变化。我们将测试改变这些位点的极性是否导致温度依赖性表型。我们还将利用重水作为研究这些地点溶剂可及性的探测。这些实验将与新型的光谱方法结合使用，以测试温度敏感的取代是否会改变蛋白质中发生的结构变化的性质。最后，我们将评估蛋白质缝隙中可接收残基的重要性。预计改变这些残基的极性会改变与它们的溶剂化/脱染过程相关的能量。我们将在这些位点引入极性和非极性取代，并测试这些突变体的功能温度敏感性。这些取代对缝隙几何形状的影响将通过测量电荷易位过程的离子强度依赖性来评估。这些实验将与分子动力学模拟相结合，以评估这些扰动对缝隙内水动力学的作用。在这些研究的结论中，我们将在测试分子理论方面取得了重大进展，这些理论可能是离子通道门控的温度依赖性，开发了一个新的模型系统，并完善了我们对水在离子通道门口中的作用的了解。