ELECTROPHYSIOLOGICAL STUDIES OF VOLTAGE GATED CHANNELS

这个项目的长期目标是在分子水平上理解离子通道的电压依赖门控。 水平。在这个方案中，实验被设计来描述振荡器和鱿鱼钾通道的结构方面 人骨骼肌钠离子通道。克隆的、工程化的通道将在非洲爪哇卵母细胞中表达， 功能将通过电生理技术进行评估，而结构将通过光学和 化学修饰技术。有四个具体目标。1)结构变化与功能的相关性 电压传感器。这将使用组氨酸扫描突变技术对 S4和S2段。这项技术利用质子来探测工程组氨酸残基的可及性，通常 取代蛋白质的碱性残基。此外，连接到通道特定位置的荧光探针(突变) 半胱氨酸)将被用来评估环境变化，并将它们与门控电流相关联。2)测量 通道分子中的距离。这个目的将使用荧光共振能量转移及其变体， 基于镧系元素的共振能量转移，以测量跨亚基的特定位置之间的距离或 通道中的特定位置和位于通道毛孔上的特定毒素。的附着点 荧光团和稀土元素是通道分子和毒气分子中的半胱氨酸工程物质。II.距离测量 将使用新开发的光学设置在S2、S3和S4网段的站点上完成，该光学设置允许同时 电压钳位和门控电流的精确测量。距离测量将在不同的位置执行 膜电位，以评估在电导激活期间可能的距离。3)激活剂的研究 失活途径。为了达到这个目的，对门控的初始快速事件进行了研究，并对这些事件进行了详细的描述 将通过对振动器K中门控电流的噪声分析来研究导致通道打开和缓慢失活的原因。 与钠通道中的门控电流相关联，以将它们与在AIMS中获得的结构信息相关联 1和2。4)建模。将进行动力学建模以解释电生理学和光学方面的结果 实验。对电压斜坡产生的波动的模拟将与噪声分析实验进行比较 目标3测试激活和失活模型。此外，还将根据以下结果进行分子建模 距离测量，包括激活期间可能发生的距离变化。这些实验是可以预料到的 让我们深入了解与电压相关的门控伴随的分子重排，这是一种基本特性 在许多膜机制中，它在兴奋性和细胞内稳态中具有至关重要的作用。

The long term objective of this project is the understanding of voltage-dependent gating of ion channels at the molecular level. In this proposal experiments are designed to describe structural aspects of Shaker and squid potassium channelsand human skeletal muscle sodium channels. Cloned, engineered channels will be expressed in Xenopus oocytes and the function will be assessed with electrophysiological techniques while the structure will be probed with optical and chemical modification techniques. There are four specific aims. 1) Correlation of structural changes with the function of the voltage sensor. This will be approached using the technique of histidine scanning mutagenesis on the charges of the S4 and S2 segments. This technique utilizes protons to probe the accessibility of engineered histidine residues, usually replacing basic residues of the protein. In addition, fluorescent probes attached to specific sites of the channels (mutated to cysteine) will be used to assess changes in environment and correlate them with gating currents. 2) Measurements of distances in the channel molecule. This aim will use the fluorescence resonance energy transfer and its variant, lanthanide-based resonance energy transfer, to measure distances between specific sites across subunits or between an specific site in the channel and an specific toxin sitting on the pore of the channel. The sites of attachment of the fluorophores and lanthanides are engineered cysteines in the channel molecule and Agitoxin II. Distance measurements will be done on sites in the S2, S3 and S4 segments using a newly developed optical setup that allows simultaneous voltage clamp and accurate measurements of gating currents. Distance measurements will be performed at different membrane potentials to assess possible distance during activation of the conductance. 3) Study of the activation and inactivation pathways. In this aim a study of the initial fast event of gating and a detailed characterization of the events leading to channel opening and slow inactivation will be studied with noise analysis of gating currents in the Shaker K. channel and with gating currents in the Sodium channel to correlate them with the structural information obtained in aims 1 and 2. 4) Modeling. Kinetic modeling will be done to account for the results in electrophysiologyical and optical experiments. Simulations of fluctuations produced by voltage ramps will be compared to the noise analysisexperiments of aim 3 to test models of activation and inactivation. In addition, molecular modeling will be done based on the results of distance measurements, including possible distance changes occurring during activation. These experiments are expected to give us insight on the molecular rearrangements concomitant with voltage-dependent gating, which is a basic property of many membranechannelsand it has critical importance in excitability and cell homeostasis.