Paxilline inhibits channels at conditions of low Po, but not under conditions of high Po. (A) An inside-out patch bathed with 300 µM Ca²⁺ was initially held at −80 mV. The patch potential was then stepped to holding potentials of −40, 0, 40, and 80 mV (from left to right), and 1.6-ms steps to 160 mV were used to monitor available BK current, while 100 nM paxilline was applied after the 10th step at a given holding potential. Paxilline was washed out and full recovery from block was obtained between tests at different holding potentials. At the most negative potentials, block is substantial, whereas at 40 and 0 mV, there is little development of block. (B) The peak outward current from the traces in A is plotted as a function of elapsed experimental time. (C) The time course of reduction of current during sustained depolarization to 80 mV is shown, establishing that the reduction of current at the positive holding potentials (B) does not reflect paxilline block, but an intrinsic slow reduction in BK conductance with prolonged depolarization. (D) All traces are from the same single-channel patch, stimulated with the protocol shown on the top. The patch was constantly exposed to 300 µM cytosolic Ca²⁺. (1) Control activity in the absence of paxilline. (2) The holding potential was changed to −70 mV, and 100 nM paxilline was applied. The trace was taken after 30 s in paxilline. (3) The trace was taken ∼30 s after the patch had been returned to 70 mV while in the constant presence of 100 nM paxilline. (4) The trace was taken ∼30 s after the patch was again returned to −70 mV while in paxilline. (5) The trace was obtained after washout of paxilline while holding the patch at 70 mV. (6) The trace was taken ∼30 s after 100 nM paxilline was applied while holding at 70 mV.