Figure 3.

Single-channel kinetics corresponds with macroscopic measurements. (A) With the method shown in Fig. 2, on (filled circles) and off (open circles) rate constants for C₁₀ block were determined at various membrane potentials from six inside-out patches, each containing a single BK channel. Each measurement was made from 60 to 100 s of single-channel recording, and each dwell time histogram included 120,000 to 250,000 events. Each point represents the mean ± SEM. Voltage dependence of the rate constants was fitted (solid lines) with Eq. 1 (see text). Values for the fitting in this figure are: k(0) = 5.18 ms⁻¹, δ = 0.06 (block rate) and k(0) = 10.00 ms⁻¹, δ = 0.10 (unblock rate). Rate constants measured at more negative potentials than 20 mV clearly deviated from the shown voltage dependence. For reasons specified in the text, those values were not shown or included in the fitting. (B) Remaining fractions of steady-state macroscopic BK currents in the presence of 10 (open black squares, n = 15) or 100 μM C₁₀ (filled black squares, n = 6) are plotted against membrane potential. Error bars representing SEM are plotted with the mean values, but are often smaller than the symbols. Voltage dependence in the remaining fraction of macroscopic currents was fitted (solid lines) with Eq. 2 (see text). Values at 20 mV and below could not be measured reliably due to very small currents at these potentials, therefore they were not included in the fitting. Indeed the measurements at these potentials apparently deviate from the fitting of voltage dependence. Values used to fit the data are as following: K(0) = 22.6 μM, δ = 0.14 (10 μM C₁₀) and K(0) = 29.9 μM and δ = 0.19 (100 μM C₁₀). Gray symbols in B represent the predicted fraction of remaining steady-state currents with a two-state: O↔B blocking reaction using average on and off rates in A. Note in this prediction no dependence of block on open probability was taken into consideration. In the case of 100 μM C₁₀, 10 times the on rate in 10 μM C₁₀ was used.