A systematic method of noise analysis for channels with single-channel conductance of fA level

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Single channel conductance of the voltage-gated proton channel was expected to be below pS. The noise analysis method for evaluating such small conductance channels was examined in this study. Whole-cell recordings of the voltage-gated proton channel from microglial cells were performed. The time course of activation at depolarization potential was very slow for the proton channel and long recording time was necessary to collect enough numbers of current traces for depolarizing pulses. For the analysis we found that the most critical step was detrend upon the selection of current traces. In the long time of recordings current traces change their shape. Current traces showing these systematic changes, rather than stochastic changes, must be eliminated from the analysis. Several criteria for selecting current traces were imposed. Ensemble average was performed from those selected traces. Fluctuating components were extracted and power-spectrum and non-stationary noise analysis were performed at various experimental conditions. Double-Lorentzian fit was done for the power spectral density. The cut-off frequency of 5 kHz was set for the non-stationary noise analysis. The variance-mean plot was drawn and we found that this plot gave valuable criteria for the selection of traces. The process from selecting traces with different criteria to the variance-mean plot was repeated until the fitted parameters were converged. From variance-mean plots amplitudes of single channel current, numbers of active channels and maximal open probability were estimated. The estimated single channel current for the voltage-gated proton channel was 12 fA. Underlying mechanisms for improvements of the data analysis using selected traces were discussed.