Ion Channel Characterization using Current Voltage Resonance Spectroscopy

描述（由申请人提供）：可兴奋膜的离子通道负责同步可兴奋细胞（如心肌细胞）的放电和恢复。众所周知，离子通道功能的异质性和丧失是致死性疾病的主要组成部分，例如心力衰竭中的心源性猝死和遗传性离子通道突变的家族形式。由于诸如膜片钳的技术，个别离子通道在相对隔离的条件下的行为被很好地定义，所述技术可以测量单个离子通道、在异源系统中过表达的离子通道或在所有其它离子通道被抑制的条件下心肌细胞中的离子通道的功能。然而，很少有人知道如何离子通道作为一个家庭在生理条件下，如心脏动作电位时，一个以上的离子通道积极通过电流。具体地，电流通过多个离子通道限定了电压形态，并有助于激活或进入/停用其他离子通道的反馈机制。本建议的目的是验证阻抗谱同时定量跨肌膜电流INa和IK 1的具体。我们选择这两个通道是基于我们小组获得的有趣的先前结果。简而言之，传导较快的右心室表达的Nav1.5相对于左心室显著较少。 我们证明IK 1比Nav1.5更大程度地调节正常心脏传导。 阻抗谱将用于证明同时定量INa和IK 1的可行性。为了解决每个离子通道由于结构差异而具有独特特征频率响应的一般假设，将测试以下特定目标。1.确定异源细胞中INa和IK 1的特征共振频率特征2.确定钠和钾通道中至少一个特征共振频率的机制3.证明IK 1和INa可以同时测量。在初步数据中，我们现在证明INa和IK 1表现出与其各自电流幅度相关的相似且独特的频率。此外，初步数据表明，电流的时间过程（INa或IK 1）占主导地位的特征频率响应。因此，为了同时定量INa或IK 1，必须通过“差频响应校正”去除主要信号。“阻抗谱并不新鲜。然而，应用阻抗谱校正占主导地位的信号是一种新的方法，同时量化跨肌膜离子通道。这是一个重要的工具，因为它将使研究人员能够最终量化当通道受到同时活跃的通道产生的电压影响时，其天然环境中的电流。成功完成这一提议将允许同时定量任何可兴奋细胞中的肌膜电流，而不仅仅是心肌细胞。公共卫生相关性：离子通道描述可兴奋心肌细胞的电压分布。当离子通道功能改变或在心脏区域之间变得异质时，个体对心源性猝死的易感性，心源性猝死是美国的主要死亡原因，显著增加。该提案旨在开发一种方法，以同时定量在生理动作电位期间心肌细胞中的多个功能离子通道，以确定离子通道功能异质性。

DESCRIPTION (provided by applicant): Ion channels of excitable membranes are responsible for synchronizing the firing and recovery of excitable cells such as cardiac myocytes. It is well established that heterogeneities and loss of ion channel function is a major component of lethal diseases such as sudden cardiac death in heart failure and familial forms of genetic ion channel mutations. The behavior of individual ion channels in relatively isolated conditions is well defined due to techniques such as patch clamping which can measure the function of a single ion channel, an ion channel over- expressed in a heterologous system, or an ion channel in a cardiac myocyte under conditions where all other ion channels are suppressed. However, little is known about how ion channels behave as a family under physiological conditions such as a cardiac action potential when more than one ion channel is actively passing current. Specifically, the passage of current by multiple ion channels defines the voltage morphology and contributes to feedback mechanisms activating or in/deactivating other ion channels. The purpose of this proposal is to validate impedance spectroscopy for simultaneously quantifying transsarcolemmal currents INa and IK1 in specific. We chose these two channels based on intriguing previous results our group obtained. In short, the faster conducting right ventricle expresses significantly less Nav1.5 relative to left. We demonstrated that IK1 modulates normal cardiac conduction to a greater extent than Nav1.5. Impedance spectroscopy will be used to demonstrate the feasibility of simultaneously quantifying INa and IK1. In order to address the general hypothesis that each ion channel has a unique characteristic frequency response due to structural differences, the following specific aims will be tested. 1. Determine the characteristic resonant frequency signatures of INa and IK1 in heterologous cells 2. Determine the mechanisms underlying at least one characteristic resonant frequency in sodium and potassium channels 3. Demonstrate that IK1 and INa can be measured simultaneously. In the preliminary data, we now demonstrate that INa and IK1 exhibit similar and unique frequencies that correlate to their respective current amplitudes. Additionally, the preliminary data demonstrates that the time course of the current (INa or IK1) predominates the characteristic frequency response. Therefore, in order to quantify INa or IK1 simultaneously, the predominant signal must be removed by a "difference frequency response correction." Impedance spectroscopy is not new. However, the application of impedance spectroscopy corrected for the predominating signal is a novel method to simultaneously quantify transsarcolemmal ion channels. This is an important tool, because it will allow researchers to finally quantify currents in their native environment when the channels are being affected by the voltage produced by concurrently active channels. Successful completion of this proposal would allow simultaneous quantification of sarcolemmal currents in any excitable cell, not just cardiomyocytes. PUBLIC HEALTH RELEVANCE: Ion channels describe the voltage profile of excitable cardiac cells. When ion channel function changes or becomes heterogeneous between regions of the heart, an individual's susceptibility to sudden cardiac death, the leading cause of death in the U.S., increases significantly. This proposal seeks to develop a method to simultaneously quantify multiple functioning ion channels in cardiac myocytes during a physiologic action potential in order to determine ion channel functional heterogeneity.