The Electrophysiological Studies of Voltage Gated Channels

描述(申请人提供)：这项建议的长期目标是在分子水平上理解膜蛋白中的电压传感。电压传感在神经、肌肉和心脏等兴奋性组织中发挥着重要作用。我们建议通过研究电压变化如何引起电压门控钠、钾通道和电压敏感磷酸酶Ci-VSP的构象变化，方法是将门控电流与放置在特定位置的探针的同时重排和荧光变化相关联。最终，我们希望用从结构变化中获得的能量来重现蛋白质的功能。在这一时期，我们有三个具体目标。目的1：电压感受器区域与激活的能量图景的关联。以Shaker K通道为模型，我们将研究通过用Trp猝灭的精氨酸的荧光替代通过疏水塞的门控电荷的轨迹。现场定向电致变色荧光法将被用来测试不同状态下的局域场 门控过程中的传感器。在激活和失活过程中，将研究电荷与插头的相互作用作为时间和电压的函数。我们将寻找S2和S3可能的运动，尝试使用LRET定义激活状态和松弛状态之间的差异，并寻找稳定松弛状态的条件。目的2：构象和动力学与Ci-VSP结构的相关性。最近Ci-VSP在静止和激活/松弛状态下的晶体结构将与在过渡过程中检测到的功能和结构变化进行比较和关联，以解决每个分子有多少电荷移动，在门控过程中单个电荷的大小，TH疏水塞子残基对动力学和稳态的影响，S4在门控过程中可能的二级结构变化，以及通过纯化和重组蛋白的单分子荧光记录单个传感器的运动或在卵母细胞中表达。对于目标1和目标2，我们希望得到构象变化的一般规律，但也得到Shaker和Ci-VSP之间的具体差异。目的3：真核细胞钠通道的构象变化和动力学。在这一时期，我们的目标是两个总体目标。首先，通过使用带有新的荧光毒素和定点荧光的LRET，我们将测量Nav1.4每个单独结构域的构象变化，作为有和没有Beta1亚基的电压的函数。其次，我们将定义由β亚基诱导的钠通道快速动力学的分子基础，它对动作电位的产生至关重要。我们将检验β1亚基诱导正协作性的假设。我们将用LRET测量β1亚基和β亚基之间的距离，并用LRET和单分子荧光法测定每个α中β1‘S的数量。这项研究预计将影响我们对特定残基如何影响动力学和稳态特性的知识，这些特性在许多情况下可以追溯到导致癫痫、心律失常、肌强直和猝死的突变。

DESCRIPTION (provided by applicant): The long term objective of this proposal is the understanding of voltage sensing in membrane proteins at the molecular level. Voltage sensing plays a major role in excitable tissues such as nerve, muscle and heart. We propose to study how voltage changes induce conformational changes in voltage gated sodium and potassium channels and in the voltage sensitive phosphatase Ci-VSP by correlating the gating currents with simultaneous rearrangements followed by fluorescence changes of probes placed in specific sites. Ultimately we would like to reproduce the function of the proteins with the landscape of energy obtained from structural changes. In this period we have three specific aims. Aim 1: Correlation of voltage sensor regions with the energy landscape of activation. Using Shaker K channels as a model we will study the trajectory of the gating charges through the hydrophobic plug using a fluorescent replacement of arginine that is quenched by Trp. Site-directed electrochromic fluorometry will be used to test the local field in different states of the sensor during gating. The interactions of the charges with the plug will be studied during activation and deactivation as a function of time and voltage. We will look for possible movements of S2 and S3, try to define the differences between the activated and relaxed states using LRET, and search for conditions that stabilize the relaxed state. Aim 2: Correlation of conformations and kinetics with Ci-VSP structures. Recent crystal structures of Ci-VSP in putative resting and activated/relaxed states will be compared and correlated with the function and structural changes detected during transitions to address how many charges move per molecule, the size of the individual shot of charge during gating, the effect of the residues of th hydrophobic plug on kinetics and steady-state, a possible secondary structure change of S4 during gating, and recording of single sensor movements by single molecule fluorescence of purified and reconstituted proteins or expressed in oocytes. With aims 1 and 2 we expect to obtain general rules of conformational changes but also specific differences between Shaker and Ci-VSP. Aim 3: Conformational changes and kinetics of the eukaryotic sodium channels. In this period we aim at two general objectives. First, by using LRET with new fluorescent toxins and site- directed fluorescence we will measure conformational changes of each individual domain of Nav1.4 as a function of voltage with and without the beta1 subunit. Second, we will define the molecular basis of the fast kinetics of Na channels induced by the beta subunit, which is crucial for action potential generation. We will test the hypothesis that the beta 1 subunit induces positive cooperativity. We will measure the distances between the beta 1 subunit and beta subunit with LRET, and determine the number of beta 1's per alpha with LRET and single molecule fluorescence. This research is expected to impact our knowledge of how specific residues affect kinetics and steady-state properties that in many cases can be traced to mutations that cause epilepsy, arrhythmia, myotonias and sudden death.