Differences in fractional unblock of BK channels by bbTBA between 4 and 300 µM Ca²⁺ are not supportive of fully state-independent block. (A) From measurements of macroscopic conductance (Fig. 1), the fractional unblock (=G(bbTBA/G(control)) with 5 µM bbTBA was determined over the full range of voltages for both 300 µM Ca²⁺ (red circles) or 4 µM Ca²⁺ (blue circles) and compared with GVs for current activation. Over the range of +20 to +60 mV, the fractional unblock at 4 µM Ca²⁺ is larger than that for 300 µM Ca²⁺, whereas fractional unblock with 300 µM Ca²⁺ approaches 1.0 at the most negative activation voltages. (B) Predictions for simple open-channel block (Scheme 1a) are illustrated. GV curves at 4 (open circle) and 300 (closed circle) µM Ca²⁺ are plotted along with GV curves in the presence of bbTBA (blue with 4 µM Ca²⁺ and red with 300 µM Ca²⁺). Fractional unblock is also plotted for 4 µM (blue circle) and 300 µM (red circle) at each voltage. (C) Similar plots for currents generated with Scheme 2a show that fractional unblock is identical for both 4 and 300 µM Ca²⁺ at each voltage. (D) The relationships for Scheme 2b, in which closed-channel block is voltage independent, are shown. The limiting fractional unblock at negative voltages reflects the voltage independence of closed-channel block. (E) Block and fractional unblock were determined from currents simulated using blocking constants derived from simultaneous fitting of GV curves to Scheme 2b (Fig. 4 G, with K_bc of ∼3 K_bo). (F) Predictions derived from currents simulated with Scheme 3a are plotted. (G) Predictions based on Scheme 3b (z_c = 0.0 e) are plotted. (H) Predicted block and fractional unblock are shown based on currents simulated with blocking constants from simultaneous fitting of GV curves to Scheme 3b (Fig. 4 H, with K_bo = 3 K_bc).