Fractional availability of current in the presence of bbTBA exhibits voltage independence at negative potentials. (A) Slo1 currents from excised inside-out patches were activated by the indicated voltage protocol (top; prepulse voltages from −300 to +100 mV with 40-mV increments) with 4 µM of internal Ca²⁺ and 20 µM bbTBA. The peak amplitude of the rapidly decaying current exhibits little change until prepulse voltages of 0 mV or more positive. Red trace corresponds to a −300-mV prepulse and blue to a +60-mV prepulse. (B) In the same patch, currents were activated with 4 µM Ca²⁺ along with 75 µM bbTBA. Red trace, −300-mV prepulse; blue trace, −20-mV prepulse. (C) The peak current amplitudes are plotted as a function of prepulse potential for both 20 µM (open circles) and 75 µM (filled circles) bbTBA. Red lines correspond to fits of a Boltzmann function with V_h = 64.9 ± 54.4 mV (z = 0.62 ± 0.33 e) and 22.9 ± 34.9 mV (z = 0.46 ± 0.13 e) for 20 and 75 µM bbTBA, respectively. (D) Normalized fractional availability was determined based on the fit of the Boltzmann function in C, better illustrating the leftward shift in availability with the increase in bbTBA. (E) The same protocol was used to activate currents in 100 µM Ca²⁺ (a different patch) with 20 µM bbTBA. The peak current at −100 mV (blue trace) shows some reduction compared with −300 mV (red trace). (F) The same patch was exposed to 75 µM bbTBA, producing more rapid and complete block. (G) The peak current is plotted as a function of prepulse potential either with 20 µM bbTBA (V_h = −39.4 ± 3.3 mV and z = 0.73 ± 0.06 e) or 75 µM bbTBA (V_h = −72.7 ± 4.5 mV and z = 0.72 ± 0.09 e). (H) The normalized fractional availability curves for the data in G are replotted.