PMC

Ion permeation profiles for sodium and potassium. (A) Free-energy profile (Left) constructed from the total Na⁺ positions (Right) sampled during a 1 μs simulation (4,000 frames spaced 0.25 ns apart), showing the free-energy barriers and minima (corresponding to the sites S0–S4). (B) Free-energy profile (Left) constructed from the K⁺ positions (Right) sampled during a 1 μs simulation The high free-energy barrier for potassium is clearly visible as a population gap in the SF between S1 and S3. Because many conductance events require more than one ion, these are multi-ion free-energy profiles.

Occupancy of the fenestrations. (A) The fenestrations that were initially empty rapidly filled with the lipid fatty acid chains. The conformational space sampled by lipids (tails in gray, head groups in red) viewed from the cytoplasmic surface shows that they entered the cavity over the course of the simulation. Each fenestration is occupied by at least one lipid tail for 90% of the time. Nevertheless, there is frequent exchange of lipids in the fenestrations with the bulk bilayer. (B) Image in A rotated 90° showing that only lipids from the cytoplasmic bilayer leaflet enter the fenestrations.

MD Simulations of NavMs. (A) System used for simulation: NavMs (in green ribbons) embedded in a POPC lipid bilayer (in gray). Sodium ions are red, chloride ions green, water molecules blue, and lipid head groups orange. (B) Effects of simulations on the protein structure: Overlay of the conformational space sampled by NavMs during 250 ns of a 1-μs simulation. The backbone C_α atoms of the transmembrane helices were weakly restrained to retain the open conformation of the channel, whereas the SF was not restrained. (C) (Left) The accessibility of the internal cavity of the initial (crystal) and final (simulation) structures, Upper and Lower, respectively. (Right) Plots showing the internal dimensions of the crystal and final simulation structures.

Sodium and potassium ion permeation parameters. (A) Cumulative ion translocation over 1 μs at selected voltages. The calculations used 0.5 M concentrations of NaCl and KCl. All curves show linear behavior for the ion permeation events. (B) Calculated I-V curves for Na⁺ (red) and K⁺ (blue) used for calculation of single-channel conductances. There is no voltage lag for Na⁺, allowing for a simple linear fit, but K⁺ has a 70-mV voltage lag and is fitted to the expression I = aΔV/[1 − exp(−bΔV)]; ΔV = V − V₀ (). Dashed lines indicate the 60% confidence interval, and error bars are from block averaging over three blocks. (C) Sample whole-cell current traces from NavMs-transfected HEK 293T cells elicited by depolarizing pulses from a −180-mV holding potential. Currents were recorded from the same cell in the presence of either 150 mM NaCl (black) or KCl (blue). The resulting current–voltage relationship (Right) was normalized to the peak of Na⁺ current for each cell (n = 9). The Nernst equation reversal potential for Na⁺ in these conditions is 41 mV, which is close to the averaged measured values of 43 ± 1 in the extracellular sodium condition. The expected reversal potential for K⁺ under these conditions was 128 mV. (D, left) Sample single-channel events recorded from inside-out patches where sodium is the charge carrier. Channel-opening events were triggered by variable depolarizations from −180 mV. The resulting event amplitudes are plotted (right) as a function of voltage, and the conductance was estimated by fitting the data to a linear relationship (n = 4).

Ion-binding sites in the selectivity filter. (A) Five Na⁺-binding sites, labeled S0–S4, were identified in the equilibrium simulations. These sites were identical for all simulations, irrespective of applied voltage. S0 is at the vestibule of the SF, contacting Ser54 and Met57 side chains; S1 (corresponding to the site designated “HFS” in ref. ) is the only site within the SF that is off the symmetry axis of the channel due to strong contact of the ion with one Glu53; S2 is a minor site just beyond the contact distance of the Glu side chains; S3 (corresponding to site “CEN” in ref. ) and S4 (corresponding to site “IN” in ref. ) are at the height of Leu52 and Thr51 backbone carbonyls, respectively. S0, S2, S3, and S4 are stabilized by full water-hydration shells. S1 has a partial water hydration shell. (B) Representative positions of individual permeating Na⁺ (varying colors) along the membrane normal (x axis) versus simulation time (first 300 ns, 332 mV). (C) Multi-ion number density histogram over the entire simulation showing the five ion-binding sites. Ions enter the SF either directly or loiter at the external site S0 before being stopped at site S1. The ions then move to the lower end of the SF at S3 and S4. Some ions also call transiently at the intermediate site S2, but S1 and S2 are never populated at the same time due to their proximity. Once leaving S3/S4, the ions extensively explore the cavity and can rebind to S3/S4 before ultimately leaving through the open gate. (D) Total number of ions in the SF versus time. There are typically one to two ions in the SF at any given time. (E) Average occupancy of the SF throughout the simulations. Most commonly (∼70%), there are two ions, one at S0–S1 and another at S2–S4. At 0.5 M NaCl, the channel is never observed to be empty. (F) Overlay of the calculated ion positions and the experimental (crystallographic) electron density map. The calculated atom positions (S0–S4) are indicated as red Xs. The 2Fo-Fc electron density map (no ions were included in the map calculation, contoured at 1.5σ) is colored in blue, and the Fo-Fc omit map (contoured at 3σ) is colored in green. The electron density matches the S2 and S3/S4 sites seen in the simulations.