Steady-state block of BK currents by bbTBA. (A) BK currents were activated by steps to +180 mV with 0 Ca²⁺ (1), 4 µM Ca²⁺ (2), or 100 µM Ca²⁺ (3), along with 0, 1, 5, or 20 µM bbTBA. (B) GV curves generated from steady-state current levels were determined for each Ca²⁺ concentration (1, 0 µM; 2, 4 µM; 3, 100 µM) in the presence of various concentrations of bbTBA, and all curves were fit simultaneously with a strict open-channel blocking model (Eq. 12). Details of fitting are given in Materials and methods and Results. The optimal fit (red lines) for the open-channel block (OB) assumption yielded K_bo = 4.0 ± 0.32 µM with z_b = 0.06 ± 0.01 e. (C) The same GV curves displayed in B were simultaneously fit to Eq. 13 with the constraint that K_bo = K_bc and z_o = z_c. In this case, K_bo = K_bc = 4.7 ± 0.3 µM with z_b = 0.07 ± 0.1 e. Blue ovals highlight regions of the curves in which the fitted curves deviate markedly from the observed results, with the OB model doing poorly at low Ca²⁺, and the CB–OB model being inadequate at higher Ca²⁺.