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了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。在该提议中，实验被设计为将结构变化与电压依赖性Na、K和磷酸酶（Ci-VSP）的功能相关联。克隆和工程化的蛋白质将在非洲爪蟾卵母细胞中表达或在脂质双层中纯化和重构。电生理学技术将用于跟踪功能，并将结合荧光光谱和化学修饰在合奏以及在单分子水平上探测结构。具体目标是：1）描述电压感测期间S4段的轨迹。这一目标将使用许多技术，包括在不同状态的通道中的金属桥，在传感器的疏水塞中的关键残基的替换，荧光偏振和各向异性，分子建模和可用的晶体结构。通过将函数的修改与结构测量相关联，几个残基的位置将被约束在静止、中间、活性和松弛状态，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传感器的独立操作。目的是了解虚拟隔离中一个传感器的功能。这将通过在仅具有一个功能性S4片段的串联构建体和Ci-VSP中使用荧光研究离子电流和构象变化来完成。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镧系元素为基础的共振能量转移在每个域的通道和其电压依赖性和相关性的功能，包括β亚基。4)使用单分子荧光在单分子水平上描述门控期间的构象变化。在单分子水平上用荧光研究通道门控的动力学，预计将揭示隐藏在宏观测量中的结构变化，并且需要它们来完成门控中分子事件的描述。公共卫生相关性：该提案中的实验是使用电生理学、荧光光谱学以及在电压依赖性蛋白质（如Na和K通道）中同时进行的蛋白质和化学修饰的功能和结构研究。重点是了解分子事件的动力学基础的电压检测跨膜的基本机制，以及这些事件如何可以影响他们的行动在跨细胞膜的离子传导。由于电压依赖性机制是基本生物过程的基础，例如神经冲动的传导、心脏收缩和细胞稳态，这些研究预计将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.