MEASURING KINETICS OF COMPLEX SINGLE-ION CHANNEL DATA USING MEAN-VARIANCE HISTOGRAMS

The measurement of single ion channel kinetics is difficult when those channels exhibit subconductance events. When the kinetics are fast, a nd when the current magnitudes are small, as is the case for Na+, Ca2, and some K+ channels, these difficulties can lead to serious errors in the estimation of channel kinetics. I present here a method, based on the construction and analysis of mean-variance histograms, that can overcome these problems. A mean-variance histogram is constructed by calculating the mean current and the current variance within a brief ' 'window'' (a set of N consecutive data samples) superimposed on the di gitized raw channel data. Systematic movement of this window over the data produces large numbers of mean-variance pairs which can be assemb led into a two-dimensional histogram. Defined current levels (open, cl osed, or sublevel) appear in such plots as low variance regions. The t otal number of events in such low variance regions is estimated by cur ve fitting and plotted as a function of window width. This function de creases with the same time constants as the original dwell time probab ility distribution for each of the regions. The method can therefore b e used: 1) to present a qualitative summary of the single channel data from which the signal-to-noise ratio, open channel noise, steadiness of the baseline, and number of conductance levels can be quickly deter mined; 2) to quantify the dwell time distribution in each of the level s exhibited.In this paper I present the analysis of a Na+ channel reco rding that had a number of complexities. The signal-to-noise rato was only about 8 for the main open state, open channel noise, and fast fli ckers to other states were present, as were a substantial number of su bconductance states. ''Standard'' half-amplitude threshold analysis of these data produce open and closed time histograms that were well fit ted by the sum of two exponentials, but with apparently erroneous time constants, whereas the mean-variance histogram technique provided a m ore credible analysis of the open, closed, and subconductance times fo r the patch. I also show that the method produces accurate results on simulated data in a wide variety of conditions, whereas the half-ampli tude method, when applied to complex simulated data shows the same err ors as were apparent in the real data. The utility and the limitations of this new method are discussed.