Thermodynamics and energetics of voltage-gated ion channels

描述(申请人提供)：离子通道直接感应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被理解的。在这里，我们试图通过对工程振荡钾通道的系统研究来理解温度敏感性的分子机制。由于我们对振荡钾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门控的生物物理研究的模型系统。我们建议检验这一假设，即溶剂介导的氨基酸侧链相互作用在经历溶剂可及性改变的位置可能是离子通道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自由能测量与电生理学、荧光光谱和分子模拟。我们将广泛地将我们的研究集中在Shaker钾通道的电压敏感区域。首先，我们将测试电压和温度敏感度之间的相关性。对特殊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导致了离子通道的电压和温度敏感性是反向相关的想法。这一假说将通过表征钾离子通道突变体的温度依赖反应来验证，其电压依赖性通过中和导致其电压依赖性的电荷残基来降低。其次，我们将测试振荡器通道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相关的表型。我们还将利用重水作为探针，研究这些地点的溶剂可获得性。这些实验将与新的光谱方法相结合，以测试对温度敏感的取代是否会改变蛋白质结构变化的性质。最后，我们将评估蛋白质缝隙内水可及残留物的重要性。改变这些残基的极性有望改变与它们的溶剂化/去溶化过程相关的能量。我们将在每个位置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转移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，我们将在测试离子通道门控的温度依赖关系的分子理论方面取得重大进展，开发一个新的模型系统，并完善我们对水在离子通道门控中的作用的认识。

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.