The mutants G214C-C363S and G214C-R253A-R254A-C363S were expressed in oocytes and labeled with TMRM. Changes in the fluorescence emission of TMRM observed from the mutant G214C-R253A-R254A-C363S (B) were slower than those observed from the mutant G214C-C363S (A) when a conditioning pulse to +80 mV was applied for 5 s to produce the relaxation of the VSD. The differences in the time constant of the fluorescence were significant for all the potentials tested below −10 mV (C), reaching up to fourfold below −100 mV. The voltage dependence of the normalized amplitude of the fluorescence change showed no difference between G214C-C363S (D; filled squares) and G214C-R253A-R254A-C363S (D; open squares) when the HP = −60 mV. However, the amplitude of the fluorescence change from the G214C-R253A-R254A-C363S mutant showed a clear shift toward negative potentials (D; open circles) with respect to the G214C-C363S mutant (D; filled circles) when the membrane was conditioned to +80 mV for 5 s. Likewise, the voltage dependence of the charge movement displays a small shift toward positive potentials for the mutant G214C-R253A-R254A-C363S (E; open squares) with respect to the mutant G214C-C363S (E; filled squares). Prepulsing the membrane to +80 mV for 5 s produces a deeper relaxation of the VSD of the mutant G214C-R253A-R254A-C363S, as reflected by a larger shift toward negative potentials (E; open circles) with respect to the shift observed in the mutant G214C-C363S (E; filled circles).