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）：离子通道直接感知多种物理和化学刺激。其中，温度传感和温度依赖性门控的分子原理可能是人们了解最少的。在这里，我们试图通过系统地研究工程化的 Shaker 钾通道来了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入了解，Shaker 钾通道将被开发为温度依赖性门控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溶剂可及性发生变化的位点的相互作用可能是离子通道的温度敏感响应的基础。我们的研究将把新开发的通道门控自由能测量与电生理学、荧光光谱和分子模拟结合起来。我们将广泛关注 Shaker 钾通道的电压传感域的研究。首先，我们将测试电压敏感性和温度敏感性之间的相关性。对专门的温度敏感离子通道的温度和电压敏感特性的热力学分析得出了离子通道的电压和温度敏感度成反比的想法。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，其电压依赖性通过中和负责其电压依赖性的电荷残基而降低。其次，我们将测试 Shaker 通道 S4 段中非极性残基的重要性及其对温度敏感性的影响。当通道激活时，S4 片段的疏水残基可能会经历环境极性的变化。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来研究这些地点的溶剂可及性。这些实验将与新颖的光谱方法相结合，以测试温度敏感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内可水残留物的重要性。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。我们将在每个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，以评估这些扰动对裂缝内水动力学的作用。在这些研究结束时，我们将在测试可能成为离子通道门控温度依赖性基础的分子理论方面取得重大进展，开发出新的模型系统并完善我们对水在离子通道门控中的作用的认识。