Fig. 2. Dependence of PF-05089771 inhibition on prepulse voltage. (A) Voltage dependence of occupancy of fast and slow inactivated states in absence of drug. Occupancy of fast inactivated states (open circles) was assayed by sodium current (INa) evoked by a 20-millisecond test step to −20 mV after 100-millisecond prepulses to varying voltages normalized to the current evoked from −140 mV. Solid curve: best fit by the Boltzmann Eq. 1/(1 + exp ((Vm − Vh)/k)), wherein Vm is conditioning potential, Vh is voltage of half-maximal inactivation, and k is the slope factor, with Vh = −66.5 mV and k = 8.2; collected values Vh = −65 ± 8 mV, and k = 8 ± 2.4 mV (mean ± S.D., n = 23). Slow inactivation (filled circles) was measured with a 5-second depolarization to varying voltages, returning to −120 mV for 100 milliseconds to allow recovery from fast inactivation and then assaying channel availability with a test pulse to −20 mV. Relative sodium current was normalized to that with a conditioning pulse to −140 mV. Data are fit by a modified Boltzmann equation (Carr et al., 2003): I/Imax = (1 − Iresid)/((1 + exp ((Vm − Vh)/k)) + Iresid, with Vh = −31 ± 9 mV, k = 16 ± 6 mV, and Iresid = 0.7 ± 0.2 (mean ± S.D., n = 8). (B) Time course of inhibition by 200 nM PF-05089771 using stimulation prototcols as in Fig. 1 with 4-second prepulses to −80, −40, or 0 mV. (C) Collected results for inhibition produced by 10 minutes of drug exposure with protocols using prepulses to each voltage. Open circles show results from individual cells, and closed symbols show mean ± S.D., n = 6 for each prepulse voltage. (D) Collected results for time course of inhibition with prepulses to each voltage. Open circles show results from individual cells, and closed symbols show mean ± S.D., n = 6 for each prepulse voltage. The time constant for best fit of a single exponential decay to steady state, with prepulses to −80 mV (284 ± 52 seconds, n = 5), −40 mV (167 ± 31 seconds, n = 6), or 0 mV (151 ± 12 seconds, n = 6) (0 mV vs. −40 mV, P = 0.68; 0 mV vs. −80 mV, P = 0.008, two-tailed Mann-Whitney Test). Open circles indicate individual experiments, and filled circles show mean ± S.D. from collected results.