Paxilline reduces NPo at conditions where voltage sensors are largely inactive, and does not influence the voltage-sensor equilibrium. (A; top) The trace shows example channel activity at −80 mV and 300 µM Ca²⁺ for a macropatch with many channels. 4-ms steps to 100 mV (not depicted) were also used to monitor paxilline inhibition. (Middle and bottom) These traces show activity under the same conditions but with 5 and 100 nM paxilline, respectively. NPo was determined from activity at −80 mV. (B) The NPo measured in paxilline for 3 s of activity normalized by the NPo measured in the absence of paxilline is plotted as a function of paxilline. The solid line corresponds to a fit of $NPo(pax)/NPo=11+([PAX]IC50)n,$ with IC₅₀ = 5.5 ± 1.1 nM with n = 1. Red dotted line corresponds to a fit of the same function with n = 2. (C) Gating currents measured with an activation step to 160 mV from −80 mV with 0 µM Ca²⁺ are shown in the absence and presence of 500 nM paxilline. (D) The Q_on integrated over 1 ms for steps to the indicated potentials are plotted for control and 500-nM paxilline solutions. Fitted lines correspond to: without paxilline, z = 0.56 ± 0.050 e with V_h = 137.6 ± 14.3 mV; with paxilline, z = 0.54 ± 0.025 e with V_h = 131.4 ± 2.1 mV. (E) Q_off is plotted as a function of previous command potential for 0 and 500 nM paxilline. Without paxilline, z = 0.66 ± 0.07 e with V_h = 142.7 ± 14.3 mV; with paxilline, z = 0.56 ± 0.035 e with V_h = 130.2 ± 9.1 mV.