Concentration dependence of paxilline inhibition at four different conditions of channel Po. (A) Traces show currents activated by brief steps to 160 mV under equilibration conditions of −70 mV and 300 µM Ca²⁺ with 5, 20, 50, and 200 nM paxilline. Red line shows the identical level of peak current for each paxilline test. (B) Traces show currents activated by steps to 160 mV under equilibration conditions of 40 mV and 300 µM Ca²⁺ for 200 nM, 500 nM, and 5 µM paxilline, all from the same patch. (C) The onset of inhibition of current activated at 160 mV with 300 µM Ca²⁺ at −70-mV equilibration condition from traces as in A is shown for five paxilline concentrations along with single-exponential fits (red). (D) Onset of inhibition with single-exponential fits (red) is shown for the higher Po equilibrium condition (B). (E) The fraction of unblocked BK current is plotted as a function of paxilline for four different equilibration conditions (−70, 0, 40, and 70 mV, all with 300 µM Ca²⁺) from experiments as in A and B. Dotted red lines correspond to fits of a Hill function, with IC₅₀ values of 11.7 ± 1.9 nM, 58.4 ± 2.9 nM, 469.8 ± 94.9 nM, and 5.37 ± 1.0 µM, from left to right. Solid lines correspond to the fit of Scheme 1, with K_p = 11.85 nM and E_p = 0.002, with n = 1.09. (F) The inhibition of NPo at −80 mV and either 10 or 300 µM Ca²⁺ is displayed from Fig. 5 B, with the dotted red line corresponding to the best fit from Scheme 1 (E) (K_p = 11.85 nM). (G) IC₅₀s from E are plotted as a function of O/C and fit with log(IC₅₀) = m(O/C) + log(K_cp), where m (0.33 ± 0.02) is the slope of the relationship and K_p (12.1 ± 1.1 nM) is the extrapolated IC₅₀ with all channels closed. This provides a model-independent estimate of paxilline inhibition of closed channels. (H) IC₅₀ estimates are replotted as a function of Po along with the fit in F. The extrapolation of the line to Po of ∼1 provides a model-independent estimate of paxilline inhibition of open channels.