Hoťka Matej, Zahradník Ivan
Department of Muscle Cell Research, Institute of Molecular Physiology and Genetics, Centre of Biosciences, Slovak Academy of Sciences, Bratislava, Slovak Republic.
Department of Biophysics, Faculty of Science, Pavol Jozef Šafárik University, Košice, Slovak Republic.
PLoS One. 2017 Nov 22;12(11):e0188452. doi: 10.1371/journal.pone.0188452. eCollection 2017.
Correct detection of membrane currents under whole-cell patch-clamp conditions is limited by the transfer function of a recording system. The low-pass output filter of a recording amplifier alters the time course of membrane current and causes errors in relevant descriptors. To solve these problems, we developed a practical procedure for reconstruction of the time course of membrane currents based on deconvolution of recorded currents in frequency domain. The procedure was tested on membrane capacitance estimates from current responses to step voltage pulses. The reconstructed current responses, in contrast to original current records, could be described exactly by an adequate impedance model of a recorded cell. The reconstruction allowed to increase the accuracy and reliability of membrane capacitance measurements in wide range of cell sizes and to suppress the cross-talk errors well below the noise. Moreover, it allowed resolving the instabilities in recording conditions arising from parasitic capacitance and seal resistance variation. Complex tests on hardware models, on simulated data sets, and on living cells confirmed robustness and reliability of the deconvolution procedure. The aptitude of the method was demonstrated in isolated rat cardiac myocytes by recording of spontaneous vesicular events, by discerning the formation of a fusion pore, and by revealing artefacts due to unstable seal resistance.
在全细胞膜片钳条件下，膜电流的准确检测受到记录系统传递函数的限制。记录放大器的低通输出滤波器会改变膜电流的时间进程，并在相关描述符中产生误差。为了解决这些问题，我们开发了一种基于频域中记录电流的去卷积来重建膜电流时间进程的实用方法。该方法在根据阶跃电压脉冲的电流响应进行膜电容估计时进行了测试。与原始电流记录相比，重建后的电流响应可以通过记录细胞的适当阻抗模型精确描述。这种重建提高了在广泛细胞大小范围内膜电容测量的准确性和可靠性，并将串扰误差抑制到远低于噪声水平。此外，它还能解决由寄生电容和封接电阻变化引起的记录条件不稳定性问题。在硬件模型、模拟数据集和活细胞上进行的复杂测试证实了去卷积方法的稳健性和可靠性。通过记录自发性囊泡事件、辨别融合孔的形成以及揭示由于不稳定封接电阻引起的伪迹，该方法在分离的大鼠心肌细胞中得到了验证。
