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，温度传感和温度依赖门控的分子原理可能是最不了解的。在这里，我们试图了解温度敏感性的分子机制，通过系统地研究工程化的Shaker钾通道。由于我们对Shaker钾离子通道的结构和动力学有了充分的了解，它将被开发为温度依赖性门控的生物物理研究的模型系统。我们建议测试的假设，溶剂介导的相互作用的氨基酸侧链在网站上经历的溶剂可及性的变化可能是离子通道的温度敏感性响应的基础。我们的研究将结合联合收割机新开发的自由能测量通道门控与电生理学，荧光光谱学和分子模拟。我们将广泛地集中我们的调查对电压敏感域的振动钾通道。首先，我们将测试电压灵敏度和温度灵敏度之间的相关性。热力学分析的温度和电压敏感特性的专门的温度敏感的离子通道导致的想法，电压和温度的离子通道的敏感性是负相关的。将通过表征钾离子通道的突变体的温度依赖性响应来测试该假设，钾离子通道的电压依赖性通过负责其电压依赖性的电荷残基的中和而降低。其次，我们将测试非极性残基在Shaker通道的S4段中的重要性及其对温度灵敏度的影响。S4片段的疏水残基可能随着通道激活而经历环境极性的改变。我们将测试改变这些位点的极性是否会导致温度依赖性表型。我们还将利用重水作为探针，研究溶剂在这些网站的可及性。这些实验将与新的光谱方法相结合，以测试温度敏感的取代是否改变了蛋白质中发生的结构变化的性质。最后，我们将评估蛋白质裂缝内的水可及残留物的重要性。改变这些残基的极性预计会改变与其溶剂化/去溶剂化过程相关的能量。我们将在这些位点中的每一个引入极性和非极性取代，并测试这些突变体的功能性温度敏感性。这些取代的裂缝的几何形状的影响将通过测量的离子强度依赖的电荷易位过程进行评估。这些实验将与分子动力学模拟相结合，以评估这些扰动对裂缝内水动力学的作用。在这些研究的结论，我们将取得重大进展，在测试分子理论，可能是离子通道门控的温度依赖性的基础，开发了一个新的模型系统，并完善了我们的知识，水在离子通道门控的作用。