Characterization of VSD motions of Ci-VSP K364 mutants by VCF of TMRM labeled on S3-S4 linker. (A) Superimposed representative normalized VCF data of the WT and six K364 Ci-VSP mutants (shown in different colors) upon a step to 0, 50, and 100 mV. Traces are normalized by the fluorescence level at the baseline and the size of maximum change of fluorescence across mutants and the WT. (B) F-V curves for WT and K364 mutants. Data are presented as mean ± SE. Curves are Boltzmann fits. (C) Voltage that gives half of the maximum fluorescence change (VHalf), acquired by fitting the F-V curve with the equation (FL(V)/FLMAX = 1/[1 + exp{-ZF(V-VHalf)/RT}], where F is Faraday constant, R is the gas constant, T is the absolute temperature, Z is the effective valency, V is voltage, and FLMAX is the maximum fluorescence change. The number of recordings is shown at the bottom of the graph. Data are presented as mean ± SE. ∗p < 0.05; ∗∗p < 0.01; n.s., statistically not significant, one-way ANOVA followed by Dunnett’s test. (D) Left: the time constant by fitting the activation phase of TMRM fluorescence with a single exponential function from 0 to 40 mV, and with a double exponential function more than 50 mV of K364 mutants and the WT (top). The proportion of the slow component is shown at the bottom. Right: the time constant of the deactivation phase fitted with a single exponential function. Data are presented as mean ± SE.