Figure 1.

The SF of Na_vRh contains two Na⁺ binding sites. (A) The structure of the Na_vRh SF (PDB code: 4DXW). The SF vestibule is constituted by the side groups of Ser180/Ser181/Glu183, and carbonyl oxygen atoms of Thr178 and Leu179. A Ca²⁺ ion is bound at an inner site of SF. For visual clarity, only two diagonal protomers are shown. (B) The SF sequence of Na_vRh is similar to those of the eukaryotic Na_v channels. Sequences of Na_vRh, Na_vAb, NaChBac and human Na_v1.4 and Ca_v1.3 were aligned using ClustalW. The residues in the negatively charged outer and inner rings are shaded red and blue, respectively. Ser181 in Na_vRh and its corresponding residues in other homologs including Tyr407 in hNa_v1.4I are shaded yellow. (C) The simulated structures of SF when Na⁺ binds to site 1 (upper panel), site 2 (middle panel) or both (lower panel). The snapshots are taken from simulation 1 in Supplementary information, Table S1. Na⁺ ions are shown as brown spheres. Arrows indicate the corresponding positions of SF structures in free energy profile. (D) Left panel, the free energy estimated from the negative logarithm of Na⁺ density in the MD simulation of Na_vRh pore domain as a function of the axial position of SF, derived from simulation 1. Right panel, the single ion PMF of Na⁺ as a function of the SF axis. All structure figures were prepared with PyMol⁶⁴.