Structurally homologous and evolutionarily related families of voltage-gated sodium and calcium channels show a conserved asparagine in S6 and the presence of four hydrated cavities between S6 and the S4–S5 linker. (A) Sequence logos and per-position hydrophobicity plots of the S6 helix for voltage-gated sodium and calcium channels (Nav/Cav), bacterial voltage-gated sodium channels (NavBac), and TRP channels (TRP; Palovcak et al., 2015; Kasimova et al., 2016). In the sequence logos, the height of each residue at a given position is proportional to its frequency at this position, and the height of the overall stack of residues decreases linearly with Shannon entropy (Crooks et al., 2004). Aromatic residues (F, W, Y) are shown in orange; hydrophobic not aromatic (A, C, I, L, M, V) in black; hydrophilic (N, Q, S, T) in green; positively (H, K, R) and negatively (D, E) charged in blue and red, respectively; and G and P in purple. In the hydrophobicity plots, positions with positive $ΔG$ are hydrophilic, and those with negative $ΔG$ are hydrophobic. The error bars represent SD. The red shaded area highlights the residues lining the central cavity (between Y671 and I679). (B) Orientation of the conserved asparagine with respect to the pore in different Nav and Cav structures (Payandeh et al., 2011, 2012; Zhang et al., 2012; Tsai et al., 2013; Tang et al., 2014, 2016; Ahuja et al., 2015; Wu et al., 2015, 2016; Lenaeus et al., 2017; Shen et al., 2017; Sula et al., 2017; Yan et al., 2017; Irie et al., 2018). The side chain position in the plane perpendicular to the pore axis is shown: the Cα-atom is represented as a black dot and is located at (0,0), and the terminal Cγ-atom (Nζ in one of the NavPas subunits where the conserved asparagine is substituted for a lysine) is shown as colored symbols (different for each Nav/Cav channel). The x-axis is aligned with the vector connecting the Cα-atom and the center of the pore. The red shaded area highlights the pore region. The two insets show the pore domains of the NavMs structure (1, top and side views) and of the NavPas structure (2, top view). The conserved asparagine points toward the S4–S5 linker in NavMs and toward the pore in NavPas. In NavMs, the water molecules inside the cavity located between the S6 helix and the S4–S5 linker are shown in blue.