VSD relaxation in the isolated VSD of Ci-VSP. (A, left) Cartoon representation of the full-length WT Ci-VSP and the truncated mutant Δ239–576 that lacks the phosphatase domain (red) attached to the VSD (blue). (right) Sensing current traces (I_s) of the truncation mutant Δ239–576 recorded using the indicated activation protocol. (B) QV curves for the mutant Δ239–576 (red) and WT Ci-VSP (black) obtained by integrating the activating I_s from pulse protocols shown in A and Fig. 7 A, respectively. (C) A superposition of I_sOFF current traces at −20 mV after 10-ms (black) and 3-s (red) depolarizations to 80 mV is displayed. Note the slowing in I_sOFF decay and the reduction in the fast component amplitude of the red trace. The inset shows the normalized tracings to better highlight the slowing in I_sOFF decay. (D) Kinetics of activating I_sON (open triangles) of the mutant Δ239–576 obtained from different activation voltage pulses (similar to the protocol shown in A) and of deactivating I_sOFF of the same mutant obtained using 80-mV prepulses of the indicated durations from 3 ms to 3 s (colored symbols) and pulsing to different deactivation voltages. (E) Weighted time constant of I_sOFF decay measured at −20 mV as a function of the duration of the conditioning prepulse at 80 mV. The obtained curve was fitted to a single exponential function and yielded a time constant of τ = 76 ± 11 ms (n = 5). (F) For both the full-length Ci-VSP (left) and the truncation mutant Δ239–576 (right), the normalized amount of sensing charges moved during the conditioning prepulse to 80 mV (Q_ON, left axis) was superimposed on the depolarization-induced slowing in the kinetics of I_sOFF decay (τ_w I_sOFF, right axis). Fitting the normalized charge displacement as a function of depolarization duration with a single exponential function yielded τQ_ON^WT = 61.7 ± 0.1 ms (n = 4) for full-length Ci-VSP and τQ_ON^Δ239–576 = 18.8 ± 0.1 ms (n = 5) for the truncation mutant. Error bars represent SEM.