ELECTROPHYSIOLOGICAL STUDIES OF VOLTAGE GATED CHANNELS

该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性门控。在这个提议的实验旨在描述结构方面的震动和鱿鱼钾通道和人类骨骼肌钠通道。克隆的工程通道将在非洲爪蟾卵母细胞中表达，并将利用电生理技术评估其功能，同时利用光学和化学修饰技术探索其结构。有四个具体目标。1)结构变化与电压传感器功能的相关性。这将采用组氨酸扫描诱变技术对S4和S2片段的电荷进行处理。该技术利用质子探测工程组氨酸残基的可及性，通常取代蛋白质的基本残基。此外，荧光探针附着在通道的特定位置（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联系起来。2)通道分子内距离的测量。该目标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，来测量跨亚基的特定位点之间的距离，或通道中特定位点与通道孔上特定毒素之间的距离。荧光团和镧系元素的附着位点是通道分子中的工程半胱氨酸和Agitoxin II。距离测量将在S2、S3和S4段进行，使用新开发的光学装置，可以同时进行电压箝位和门控电流的精确测量。距离测量将在不同的膜电位下进行，以评估电导激活期间可能的距离。3)激活和失活途径的研究。在这个目标中，将研究门控的初始快速事件和导致通道打开和缓慢失活的事件的详细特征，并对激振器K通道中的门控电流和钠通道中的门控电流进行噪声分析，以将它们与目标1和目标2中获得的结构信息相关联。4)建模。动力学建模将用于解释电生理和光学实验的结果。由电压斜坡产生的波动模拟将与目标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，包括激活过程中可能发生的距离变化。这些实验有望让我们了解伴随电压依赖性门控的分子重排，这是许多膜通道的基本特性，在兴奋性和细胞稳态中具有至关重要的意义。

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.