The Electrophysiological Studies of Voltage Gated Channels

描述(由申请人提供)：这个项目的长期目标是在分子水平上了解电压依赖的膜蛋白的电压依赖门控的动力学，重点是电压门控通道。在这项建议中，实验旨在将结构变化与电压依赖性钠、钾和磷酸酶(Ci-VSP)的功能联系起来。克隆和工程蛋白将在非洲爪哇卵母细胞中表达，或在脂双层中纯化和重组。电生理技术将用于跟踪功能，结构将通过荧光光谱和化学修饰的组合在整体和单分子水平上进行探测。具体目标是：1)描述S4段在电压检测过程中的轨迹。这一目标将使用许多技术，包括通道不同状态下的金属桥，传感器疏水插头中关键残基的替换，荧光偏振和各向异性，分子模拟和可用的晶体结构。通过将函数的修改与结构测量相关联，将几个残基的位置限制在静止、中间、活动和松弛状态，以提出传感器的动态轨迹，包括可能的二级结构变化。2)每个亚基在K+通道的激活和失活中的作用以及一个感受器的孤立工作。目的是了解一个传感器在虚拟隔离中的功能。这将通过在只有一个功能S4片段的串联结构和Ci-VSP中使用荧光来研究离子电流和构象变化来完成。3)电压门控性钠离子通道的结构和功能相关性。这将通过测量分子内距离和通道每个区域中基于镧系元素的共振能量转移来实现，以及它与电压的依赖关系和与功能的相关性，包括β亚基。4)用单分子荧光法在单分子水平上描述门控过程中的构象变化。用荧光在单分子水平上研究通道门的动力学，有望揭示隐藏在宏观测量中的结构变化，并需要它们来完成对门中分子事件的描述。与公共健康相关：本方案中的实验是使用电生理、荧光光谱和蛋白质和化学修饰同时对电压依赖蛋白质(如钠和钾通道)进行的功能和结构研究。重点是了解跨膜电压检测的基本机制背后的分子事件的动力学，以及这些事件如何影响它们在跨细胞膜的离子传导中的作用。由于电压依赖机制是神经冲动传导、心脏收缩和细胞稳态等基本生物学过程的基础，这些研究有望对健康和疾病产生影响。

DESCRIPTION (provided by applicant): This project has the long term objective of understanding at the molecular level the dynamics of voltage- dependent gating of voltage-dependent membrane proteins with emphasis in voltage-gated channels. In this proposal the experiments are designed to correlate structural changes with the function on voltage dependent Na, K and phospatase (Ci-VSP). Cloned and engineered proteins will be expressed in Xenopus oocytes or purified and reconstituted in lipid bilayers. Electrophysiological techniques will be used to follow the function and the structure will be probed with a combination of fluorescence spectroscopy and chemical modifications in the ensemble as well as at the single molecule level. The specific aims are: 1) Description of the trajectory of the S4 segment during voltage sensing. This aim will use many techniques including metal bridges in different states of the channel, replacement of critical residues in the hydrophobic plug of the sensor, fluorescence polarization and anisotropy, molecular modeling and the crystal structures available. By correlating the modification of the function with the structural measurements the position of several residues will be constrained in the resting, intermediate, active and relaxed states to propose the dynamic trajectory of the sensor including possible secondary structure changes. 2) Role of each subunit in the activation and inactivation of the K+ channel and the operation of one sensor in isolation. The objective is to understand the function of one sensor in virtual isolation. This will be done by studying ionic currents and conformational changes using fluorescence in tandem constructs with only one functional S4 segment and in Ci-VSP. 3) Structural and functional correlates of the voltage-gated Na+ channel. This will be approached by measuring intramolecular distances with lanthanide-based resonance energy transfer in each one of the domains of the channel and its voltage dependence and correlation with the function, including the beta subunit. 4) Description of conformational changes during gating at the single molecule level using single molecule fluorescence. The dynamics of channel gating studied at the single molecule level with fluorescence is expected to reveal structural changes that are hidden in macroscopic measurements and they are required to complete the description of molecular events in gating. PUBLIC HEALTH RELEVANCE: The experiments in this proposal are functional and structural studies using electrophysiological, fluorescence spectroscopy and protein and chemical modification done simultaneously in voltage dependent proteins such as Na and K channels. The emphasis is understanding the dynamics of the molecular events underlying the fundamental mechanism of voltage detection across the membrane and how those events can effect their action in the conduction of ions across the cell membrane. As voltage dependent mechanisms underlie basic biological processes such as the conduction of the nerve impulse, heart contraction, and cell homeostasis, these studies are expected to have impact in health and disease.