Ion Channel Characterization using Current Voltage Resonance Spectroscopy

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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.