Figure 3.

Na⁺ and Ca²⁺ bind to site 2 differently. (A) Ca²⁺ coming from intracellular side stays at site 2 but cannot penetrate into the extracellular solution. Free energy estimated from the negative logarithm of ion density at site 2 as a function of the axial position of SF is indicated for Na⁺ (orange) and Ca²⁺ (green). The approximate positions of carbonyl oxygen atoms (O) of Thr178 and Leu179 are labeled. (B) Numbers of coordinating atoms are shown for Na⁺ (orange) and Ca²⁺ (green). The scheme of presentation is identical to that in Figure 2B. (C) The simulated structures of Na_vRh SF when Na⁺ (brown) or Ca²⁺ (green) binds at site 2. Two perpendicular views are shown. The distances between the ions and the surrounding carbonyl oxygens are indicated in dashed line and labeled in the unit of Å. (D) The 2D free energies estimated from the negative logarithm of ion density in the SF are plotted for Na⁺ (upper panel) and Ca²⁺ (lower panel) in unit of kcal/mol. Thr178 and Leu179 are labeled along the SF axis. (E) Coordination numbers contributed by Thr178 (O) or Leu179 (O) are indicated in dashed and solid lines, respectively, in the same color scheme as B. The data of B and E are derived from ABF simulation (left panel) and equilibrium simulations 1 and 8 (right panel), respectively. (F) Cytosolic Ca²⁺ ions block Na⁺ ion conductance through the channel pore. The relationship between average normalized current density from cells expressing the NaChBac (black traces), the NaChBac/Na_vRh-filter chimera (green traces) and human Na_v1.1 (purple traces) channels are plotted as a function of intracellular free calcium concentration (± SEM; n = 5 to 6 cells each) and are fit to the Hill equation. The estimated IC₅₀ values are 74 μM, 61 μM and 98 μM for NaChBac, the NaChBac/Na_vRh-filter chimera and hNa_v1.1, respectively. Inset: representative inward Na⁺ current density records from whole cell patches of HEK-293T cells expressing sodium channels in the presence of the indicated intracellular free calcium concentrations. Channels were activated by 0.5 s or 0.05 s (hNa_v1.1) depolarizations from −140 mV (holding potential) to 0 mV with 150 mM NaCl in the bath.