Rigby John R, Poelzing Steven
Bioengineering, University of Utah, USA.
J Vis Exp. 2011 Feb 8(48):2361. doi: 10.3791/2361.
Presently, there are no established methods to measure multiple ion channel types simultaneously and decompose the measured current into portions attributable to each channel type. This study demonstrates how impedance spectroscopy may be used to identify specific frequencies that highly correlate with the steady state current amplitude measured during voltage clamp experiments. The method involves inserting a noise function containing specific frequencies into the voltage step protocol. In the work presented, a model cell is used to demonstrate that no high correlations are introduced by the voltage clamp circuitry, and also that the noise function itself does not introduce any high correlations when no ion channels are present. This validation is necessary before the technique can be applied to preparations containing ion channels. The purpose of the protocol presented is to demonstrate how to characterize the frequency response of a single ion channel type to a noise function. Once specific frequencies have been identified in an individual channel type, they can be used to reproduce the steady state current voltage (IV) curve. Frequencies that highly correlate with one channel type and minimally correlate with other channel types may then be used to estimate the current contribution of multiple channel types measured simultaneously.
Voltage clamp measurements were performed on a model cell using a standard voltage step protocol (-150 to +50 mV, 5mV steps). Noise functions containing equal magnitudes of 1-15 kHz frequencies (zero to peak amplitudes: 50 or 100mV) were inserted into each voltage step. The real component of the Fast Fourier transform (FFT) of the output signal was calculated with and without noise for each step potential. The magnitude of each frequency as a function of voltage step was correlated with the current amplitude at the corresponding voltages.
In the absence of noise (control), magnitudes of all frequencies except the DC component correlated poorly (|R|<0.5) with the IV curve, whereas the DC component had a correlation coefficient greater than 0.999 in all measurements. The quality of correlation between individual frequencies and the IV curve did not change when a noise function was added to the voltage step protocol. Likewise, increasing the amplitude of the noise function also did not increase the correlation. Control measurements demonstrate that the voltage clamp circuitry by itself does not cause any frequencies above 0 Hz to highly correlate with the steady-state IV curve. Likewise, measurements in the presence of the noise function demonstrate that the noise function does not cause any frequencies above 0 Hz to correlate with the steady-state IV curve when no ion channels are present. Based on this verification, the method can now be applied to preparations containing a single ion channel type with the intent of identifying frequencies whose amplitudes correlate specifically with that channel type.
目前，尚无既定方法可同时测量多种离子通道类型，并将测得的电流分解为各通道类型的贡献部分。本研究展示了如何利用阻抗谱来识别与电压钳实验中测得的稳态电流幅度高度相关的特定频率。该方法包括将包含特定频率的噪声函数插入电压阶跃协议中。在本文所呈现的工作中，使用模型细胞来证明电压钳电路不会引入高相关性，并且当不存在离子通道时，噪声函数本身也不会引入任何高相关性。在将该技术应用于含有离子通道的标本之前，这种验证是必要的。所呈现协议的目的是展示如何表征单一离子通道类型对噪声函数的频率响应。一旦在单个通道类型中识别出特定频率，就可以用它们来重现稳态电流 - 电压（IV）曲线。与一种通道类型高度相关且与其他通道类型相关性最小的频率，随后可用于估计同时测量的多种通道类型的电流贡献。
使用标准电压阶跃协议（-150至 +50 mV，步长5 mV）对模型细胞进行电压钳测量。将包含1 - 15 kHz频率等幅度（零至峰值幅度：50或100 mV）的噪声函数插入每个电压阶跃中。对于每个阶跃电位，分别计算有无噪声时输出信号的快速傅里叶变换（FFT）的实部。将每个频率的幅度作为电压阶跃的函数与相应电压下的电流幅度进行相关性分析。
在无噪声（对照）情况下，除直流分量外，所有频率的幅度与IV曲线的相关性都很差（|R| < 0.5），而在所有测量中直流分量的相关系数均大于0.999。当在电压阶跃协议中添加噪声函数时，各个频率与IV曲线之间的相关质量并未改变。同样，增加噪声函数的幅度也不会增加相关性。对照测量表明，电压钳电路本身不会使任何高于0 Hz的频率与稳态IV曲线高度相关。同样，在存在噪声函数的情况下进行的测量表明，当不存在离子通道时，噪声函数不会使任何高于0 Hz的频率与稳态IV曲线相关。基于此验证，该方法现在可应用于含有单一离子通道类型的标本，旨在识别其幅度与该通道类型特异性相关的频率。
