Voltage dependency of conductance and VSD activation in specific split channels. (A) Representative currents of the wild-type, L341 split, Y347 split, and Δ342–348 split recorded with the indicated voltage protocol, with the exception of L341 split where the holding potential was −20 mV. (B) Normalized conductance plotted against voltage (mean ± SEM; n = 7–10). The curves are Boltzmann fits with the following V_1/2 values (mean ± SD): −28.0 ± 1.8 mV (L341 split), 21.4 ± 1.7 mV (WT), and 21.7 ± 2.1 mV (Y347 split). Fitting was not done for the Δ342–348 split because the conductance did not saturate in the voltage range tested. (C) Representative voltage-dependent fluorescence changes reported by I319C-TMRM. Only traces elicited by the voltage steps of −160 (blue), −60 (green), and 60 (red) mV are shown. (D) Normalized fluorescence changes plotted against voltage (mean ± SEM; n = 5–10); asterisk indicates statistical significance (P < 0.05 in t test with Bonferroni correction) with respect to the continuous channel. The curves are Boltzmann fits. Inset contains V_1/2 values of the Boltzmann fits (mean ± SD): −54.3 ± 2.8 mV (L341 split), −57.7 ± 3.0 mV (WT), −49.0 ± 3.6 mV (Y347 split), and 35.6 ± 3.4 mV (Δ342–348 split).