The Electrophysiological Studies of Voltage Gated Channels

描述（由申请人提供）：本提案的长期目标是在分子水平上理解膜蛋白的电压传感。电压传感在神经、肌肉和心脏等易兴奋组织中起着重要作用。我们建议研究电压变化如何诱导电压门控钠和钾通道的构象变化，并在电压敏感的磷酸酶Ci-VSP相关的门控电流与同时重排，然后荧光变化的探针放置在特定的网站。最终，我们希望通过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：电压传感器区域与激活能量景观的相关性。使用Shaker K通道作为模型，我们将研究通过疏水塞的门控电荷的轨迹，使用被色氨酸淬灭的精氨酸的荧光替换。现场定向电致变色荧光法将用于测试在不同状态下的局部场， 门控期间的传感器。在激活和失活期间，将研究电荷与插头的相互作用随时间和电压的变化。我们将寻找S2和S3的可能运动，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。将比较Ci-VSP在推定的静息和活化/松弛状态下的最新晶体结构，并将其与在转变期间检测到的功能和结构变化相关联，以解决每个分子移动多少电荷、门控期间单个电荷发射的大小、疏水塞的残基对动力学和稳态的影响、门控期间S4的可能二级结构变化，以及通过纯化的和重构的蛋白质或在卵母细胞中表达的蛋白质的单分子荧光来记录单传感器运动。通过目标1和2，我们期望获得构象变化的一般规则，以及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，我们有两个总的目标。首先，通过使用具有新的荧光毒素和定点荧光的LRET，我们将测量Nav1.4的每个单独结构域的构象变化，其作为具有和不具有β 1亚基的电压的函数。其次，我们将定义由β亚基诱导的Na通道快速动力学的分子基础，这对动作电位的产生至关重要。我们将检验β 1亚基诱导正协同性的假设。我们将用LRET测量β 1亚基和β亚基之间的距离，并用LRET和单分子荧光确定每个α的β 1数量。这项研究预计将影响我们对特定残留物如何影响动力学和稳态特性的认识，在许多情况下，这些特性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.