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使可兴奋细胞(如心肌细胞)的激发和恢复同步。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，如心力衰竭引起的心源性猝死和家族性遗传离子通道突变。在相对孤立的条件下，单个离子通道的行为被很好地定义，这是由于诸如膜片钳技术，其可以测量单个离子通道、在异种系统中过度表达的离子通道或在所有其他离子通道被抑制的条件下的心肌细胞中的离子通道的功能。然而，关于离子通道在生理条件下如何表现为一个家族，如当不止一个离子通道活跃地传递电流时，心脏动作电位，人们知之甚少。具体地说，多个离子通道的电流通道定义了电压形态，并有助于反馈机制激活或进入/停用其他离子通道。这项建议的目的是验证阻抗谱同时定量跨肌膜电流INA和Ik1。我们根据我们小组之前获得的有趣结果选择了这两个渠道。简而言之，传导速度越快的右心室表达的NaV1.5比左心室明显减少。我们证明了IK1对正常心脏传导的调节作用比NaV1.5更大。交流阻抗谱将被用来证明同时量化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唯一的特征频率响应，将测试以下特定目标。1.确定异种细胞中INA和INA的特征共振频率特征2.确定钠通道和钾通道中至少一个特征共振频率的机制3.证明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表现出与它们各自的电流幅度相关的相似和独特的频率。此外，初步数据表明，电流的时间过程(Ina或Ik1)在特征频率响应中占主导地位。因此，为了同时量化INA或Ik1，必须通过“差频响应校正”来去除主要信号。阻抗谱不是什么新鲜事。然而，应用阻抗频谱校正主导信号是一种同时定量跨肌膜离子通道的新方法。这是一个重要的工具，因为它将允许研究人员在通道受到并发活动通道产生的电压影响时，最终量化其自然环境中的电流。这一提议的成功完成将允许同时量化任何可兴奋细胞的肌膜电流，而不仅仅是心肌细胞。与公共健康相关：离子通道描述了可兴奋的心肌细胞的电压分布。当离子通道功能在心脏的不同区域之间发生变化或变得不同时，个体对心脏性猝死的易感性显著增加，而心脏性猝死是美国的主要死亡原因。这项建议试图开发一种方法，在生理动作电位期间同时量化心肌细胞中的多个功能离子通道，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.