The Electrophysiological Studies of Voltage Gated Channels

描述（由申请人提供）：本项目的长期目标是在分子水平上理解电压依赖性膜蛋白的电压依赖性门控动力学，重点是电压门控通道。本实验旨在将结构变化与电压依赖性Na、K和磷酸酶（Ci-VSP）的功能联系起来。克隆和工程蛋白将在爪蟾卵母细胞中表达或纯化并在脂质双层中重组。电生理技术将用于跟踪其功能，并结合荧光光谱和化学修饰在整体和单分子水平上对其结构进行探测。具体目的是：1)描述S4段在电压传感过程中的运动轨迹。这一目标将使用许多技术，包括不同状态的金属桥，传感器疏水插头中关键残留物的替换，荧光偏振和各向异性，分子建模和晶体结构。通过将函数的修改与结构测量相关联，将若干残差在静息、中间、主动和放松状态下的位置进行约束，从而提出包含可能的二次结构变化的传感器动态轨迹。2)每个亚单元在K+通道激活和失活以及一个传感器的隔离操作中的作用。目的是了解一个传感器在虚拟隔离中的功能。这将通过研究离子电流和构象变化使用荧光串联结构只有一个功能的S4段和Ci-VSP来完成。3)电压门控Na+通道的结构和功能相关性。这将通过测量分子内距离与基于镧系元素的共振能量转移在通道的每个域及其电压依赖性和与函数的相关性来实现，包括β亚基。4)利用单分子荧光在单分子水平上描述门控过程中的构象变化。利用荧光在单分子水平上研究通道门控的动力学，有望揭示隐藏在宏观测量中的结构变化，并完成对门控分子事件的描述。公共卫生相关性：本提案中的实验是使用电生理学、荧光光谱和蛋白质和化学修饰同时对电压依赖性蛋白质（如Na和K通道）进行的功能和结构研究。重点是理解细胞膜上电压检测基本机制的分子事件的动力学，以及这些事件如何影响它们在细胞膜上离子传导中的作用。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过程的基础，因此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.