Pore opening of mutants in VSD _I and VSD _II during MD simulations. (A and B) Each panel depicts the time series of the minimum radius of the aqueous pathway of gating pore through VSDs during simulations. A 1.5-Å–dashed line represents the potential pore opening threshold for water and ion permeation through VSDs. WT traces are presented in black, R1 mutants (R219Q and R808Q) in blue, R2 mutants (R222Q and R811Q) in green, and R3 mutants (R225Q and R814Q) in red. (C and D) Each panel shows molecular images of VSD_I (C) and VSD_II (D) WT and mutants (S3 removed for clarity). In each snapshot, gating charges (blue), countercharges (red), HCS (green) residues are illustrated in licorice representation. Sodium (yellow) and chloride (gray) ions are depicted in spheres. Snapshots are selected at the time points indicated by black arrows in A and B. The water molecules are illustrated as a transparent red surface. The black-dashed circles indicate the non-accessible solvent region, separating the extracellular and intracellular water milieu.