Ion Channel Characterization using Current Voltage Resonance Spectroscopy

描述（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命疾病的主要组成部分，如心力衰竭引起的心源性猝死和家族形式的基因离子通道突变。由于膜片箝位等技术可以测量单个离子通道、异种系统中过表达的离子通道或心肌细胞中所有其他离子通道被抑制的条件下的离子通道的功能，因此可以很好地定义相对孤立条件下单个离子通道的行为。然而，对于离子通道作为一个家族在生理条件下的行为知之甚少，例如当多个离子通道积极通过电流时，心脏动作电位。具体来说，多个离子通道的电流通过定义了电压形态，并有助于激活或激活/灭活其他离子通道的反馈机制。本提案的目的是验证阻抗谱同时定量跨细胞电流INa和IK1的具体方法。我们选择这两个渠道是基于我们小组之前获得的有趣的结果。简而言之，传导较快的右心室相对于左心室表达的Nav1.5明显较少。我们证明IK1比Nav1.5更能调节正常的心脏传导。阻抗谱将用于证明同时量化INa和IK1的可行性。为了解决一般假设，即每个离子通道由于结构差异而具有独特的特征频率响应，将测试以下具体目标。1. 测定异种细胞中INa和IK1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。证明了IK1和INa可以同时测量。在初步数据中，我们现在证明了INa和IK1表现出与其各自的电流幅值相关的相似且独特的频率。此外，初步数据表明，电流（INa或IK1）的时间过程主导了特征频率响应。因此，为了同时量化INa或IK1，必须通过“差频响应校正”去除主要信号。阻抗谱学并不是新事物。然而，利用阻抗谱对主导信号进行校正是一种同时定量跨细胞离子通道的新方法。这是一个重要的工具，因为当通道受到同时有源通道产生的电压影响时，它将使研究人员最终量化其原生环境中的电流。这一建议的成功完成将允许同时定量任何可兴奋细胞的肌层电流，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各区域之间变得不均匀时，个体对心脏性猝死的易感性显著增加，心脏性猝死是美国死亡的主要原因。本研究旨在开发一种在生理动作电位期间同时量化心肌细胞中多个功能离子通道的方法，以确定离子通道功能的异质性。

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.