Figure 5. Analysis of the effect of ruthenium red on the amplitude of the single-channel current. (A) Single channel currents recorded in the presence of either 10 µM (left) or 40 µM (right) ruthenium red. The graph below each single-channel current shows the amplitude distribution of open channel current. Histograms were constructed by including data points from 5–20 channel openings during an experiment, excluding periods when the channel closed to the zero current level. The amplitude distributions were fit as the sum of three Gaussian components by a maximum likelihood fitting routine. (B) Plot of the occupancy probability of the fully open state, O, and subconductance states S1 and S2. Occupancy probabilities were measured as the integral of each Gaussian component divided by the total area under all three curves. Data were fit straight line by linear regression. (Inset) Plot of the amplitude of the fully open state, O, as a function of ruthenium red concentration in the patch electrode. The data were fit with a simple expression for binding to a single site, with K1/2 = 49 µM.