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。在这项提议中，实验被设计来描述摇床和鱿鱼钾通道以及人骨骼肌钠通道的结构方面。克隆的工程化通道将在非洲爪哇卵母细胞中表达，其功能将通过电生理技术进行评估，结构将通过光学和化学修饰技术进行探测。有四个具体目标。1)结构变化与电压传感器功能的相关性。这将使用组氨酸扫描突变技术对S4和S2片段的电荷进行研究。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(突变为半胱氨酸)将被用来评估环境的变化，并将它们与门控电流相关联。2)测量通道分子中的距离。这个目标将使用荧光共振能量转移及其变种，基于镧系元素的共振能量转移，来测量跨亚单位的特定位置之间的距离，或者通道中的特定位置与位于通道孔上的特定毒素之间的距离。荧光团和稀土元素的附着位置是通道分子和Agitoxin II中的工程半胱氨酸。将使用新开发的光学装置在S2、S3和S4段的位置进行距离测量，该装置允许同时进行电压钳制和准确测量门控电流。将在不同的膜电位下进行距离测量，以评估在电导激活期间可能的距离。3)激活和失活途径的研究。为此，将通过Shaker K通道中的门控电流和钠通道中的门控电流的噪声分析来研究门控的初始快速事件以及导致通道打开和缓慢失活的事件的详细特征，以将它们与在AIMS 1和2)建模中获得的结构信息相关联。将对电生理学和光学实验的结果进行动力学建模。对电压斜坡产生的波动的模拟将与Aim 3的噪声分析实验进行比较，以测试激活和失活模型。此外，分子建模将基于距离测量的结果，包括激活期间可能发生的距离变化。这些实验有望让我们深入了解伴随着电压依赖门控的分子重排，这是许多膜通道的基本特性，在兴奋性和细胞动态平衡方面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 channels and 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 analysis experiments 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 membrane channels and it has critical importance in excitability and cell homeostasis.