Figure 2.

Ca²⁺ binds to site 1 and blocks Na⁺ entry from the extracellular side. (A) Free energy estimated from the negative logarithm of ion density at site 1 is shown for Na⁺ (orange) and Ca²⁺ (green), respectively, as a function of the axial position of SF. The approximate positions of Ser181 and Glu183 are labeled. (B) Numbers of coordinating atoms are indicated for Na⁺ (orange)/Ca²⁺ (green) in left panel, or for Na⁺ in the presence of Ca²⁺ bound at site 1 (blue) in right panel. The total coordination numbers and the contribution from water molecules are shown as dashed and solid lines, respectively. (C) Coordination numbers contributed by Ser181 (solid line) or Glu183 (dashed line) are indicated in the same scheme as in B. The data shown in A-C are derived from simulations 1, 3 and 10, respectively. (D) The simulated structures of SF when Na⁺ or Ca²⁺ binds to site 1. Na⁺ (brown sphere) bound to Glu183 and Ser181 (upper left panel), Ser181 (upper right panel), and Ca²⁺ (green) bound to Glu183 (lower left panel) are shown. Na⁺ is stably coordinated by Glu183 and Ser181 when Ca²⁺ is bound to Glu183 in the non-adjacent protomer (lower right panel). The subunits of Na_vRh are shown in four colors to highlight the intrinsic asymmetry. The same color scheme is applied to all the figures if not otherwise indicated. (E) The 2D free energy estimated from radial-axial density in SF is plotted for Na⁺ (upper panel) and Ca²⁺ (lower panel) in unit of kcal/mol. Ser181 and Glu183 are labeled along the SF axis. (F) PMF values of Na⁺ (red) and Ca²⁺ (green) as a function of the SF axis illustrate that Ca²⁺ has to overcome a huge energy barrier to reach site 2.