Characterizing fenestration size in sodium channel subtypes and their accessibility to inhibitors

Abstract

Voltage-gated sodium channels (Nav) underlie the electrical activity of nerve and muscle cells. Humans have nine different subtypes of these channels, which are the target of small-molecule inhibitors commonly used to treat a range of conditions. Structural studies have identified four lateral fenestrations within the Nav pore module that have been shown to influence Nav pore blocker access during resting-state inhibition. However, the structural differences among the nine subtypes are still unclear. In particular, the dimensions of the four individual fenestrations across the Nav subtypes and their differential accessibility to pore blockers is yet to be characterized. To address this, we applied classical molecular dynamics simulations to study the recently published structures of Nav1.1, Nav1.2, Nav1.4, Nav1.5, and Nav1.7. Although there is significant variability in the bottleneck sizes of the Nav fenestrations, the subtypes follow a common pattern, with wider DI-II and DIII-IV fenestrations, a more restricted DII-III fenestration, and the most restricted DI-IV fenestration. We further identify the key bottleneck residues in each fenestration and show that the motions of aromatic residue sidechains govern the bottleneck radii. Well-tempered metadynamics simulations of Nav1.4 and Nav1.5 in the presence of the pore blocker lidocaine also support the DI-II fenestration being the most likely access route for drugs. Our computational results provide a foundation for future in vitro experiments examining the route of drug access to sodium channels. Understanding the fenestrations and their accessibility to drugs is critical for future analyses of diseases mutations across different sodium channel subtypes, with the potential to inform pharmacological development of resting-state inhibitors and subtype-selective drug design.

Figure 1

Overview of voltage-gated sodium channel structure and function. (A) Cut-surface view of a eukaryotic sodium channel pore module (voltage sensors not shown here) in different states during an action potential: resting (red), activated (green), and fast-inactivated (purple); also, highlighting the location of pore cavity (yellow box) and fenestrations. (B) Topology of the pseudotetrameric eukaryotic sodium channel alpha-subunit showing each of the domains (DI-DIV) consisting of six transmembrane helices each (S1-S6). (C) Top-down view of the entire sodium channel depicting the voltage sensors (VS) wrapping around the pore module (PM) in a domain-swapped manner; fenestrations labeled by arrows.

Figure 2

Simulations capture dynamics of fenestration shape and dimensions. Comparison of a representative fenestration, DI-II in Nav1.4, as seen in the cryo-EM structure (A) and two snapshots sampled from the MD simulation (B and C); a transverse view of the fenestration in sphere representation (top) and an axial view from outside the pore module (bottom) depict the bottleneck positions and key fenestration-lining residues. Overall tunnel profile time series (D) shows the changes to fenestration shape over the 500 ns simulation. The range of bottleneck radius sampled across the three replicate simulations is shown as a combined distribution (E); radius of cryo-EM structure indicated by gray dotted line.

Figure 3

Distributions of fenestration bottleneck radius for each fenestration and each human Nav subtype. Data from all three replicates of equilibrium MD simulations were combined to produce the overall bottleneck distributions for each fenestration (DI-II, DII-III, DIII-IV, and DI-IV) of Nav1.1 (blue), Nav1.2 (orange), Nav1.4 (green), Nav1.5 (red), and Nav1.7 (purple). Colored horizontal dashed lines indicate the bottleneck radius of each fenestration in the starting structures of each Nav subtype.

Figure 4

Fenestration bottleneck residues on S6. Sequence alignment of S6 helices by domain and subtype (aligned to NavAb), highlighting the conserved position of hydrophobic bottleneck residues in the four fenestrations. Bottleneck forming residues are contained in a box whose color represents the fenestration which they line (blue, DI-II; red, DII-III; cyan, DIII-IV; magenta, DI-IV). The 29 residues of S6 numbered sequentially in the top row to aid in identifying common positions between subtypes. Bottleneck residues on the upper and lower portion of S6DIII line different fenestrations.

Figure 5

F1459 (in the 15th position on S6) is responsible for gating the DII-III fenestration of hNav1.5. Chi1(χ₁) dihedral angle of F1459 (purple) plotted alongside bottleneck radius (red) over time with a moving average window of 20 ns (A); bottleneck radius distributions resultant from individual sidechain deletions of bottleneck residues (B) showed that deletion of the F₁₅ aromatic sidechain had a profound effect on creating a consistently wide bottleneck radius; representative snapshots for F1459 being in the “down” configuration resulting in a wide fenestration bottleneck radius (C); compared with F1459 being in the “up” configuration, resulting in a narrowed fenestration bottleneck radius (D).

Figure 6

Accessibility of Nav1.4 fenestrations. The two-dimensional (2D) FES of lidocaine’s occupancy within the pore cavity and fenestrations of Nav1.4, reconstructed from the hills potentials deposited during metadynamics simulations, where warmer/darker red colors indicate energetically favorable regions and cooler/darker blue regions represent significant barriers to lidocaine. (A) Top-down polar view of the FES (obtained by integrating out z, keeping r and θ) with the MFEP through fenestrations DI-II, DII-III, DIII-IV, and DI-IV, colored blue, red, cyan, and magenta, respectively, ending at the minimum free energy binding site at the mouth of DII-III. (B) Four transverse views of each individual fenestration extending radially from the central pore axis at r = 0 (obtained by integrating out θ, keeping z and r, using 50° segments of the 3D FES for each corresponding fenestration, as indicated in (A) with DI-II spanning the blue segment, DII-III spanning the red, DIII-IV spanning the cyan, and DI-IV spanning the magenta); MFEPs also mapped along relevant sections of the transverse fenestration axes. (C) Free energy value along the MFEPs along each fenestration, colored accordingly, showing the most favorable pathway along fenestration DI-II.

Figure 7

Accessibility of Nav1.5 fenestrations. The 2D FES of lidocaine’s occupancy within the pore cavity and fenestrations of Nav1.5, reconstructed from the hills potentials deposited during metadynamics simulations. (A) The top-down polar view (obtained by integrating out z, keeping r and θ) with the MFEP through each fenestration, colored blue, red, cyan, and magenta, ending at the minimum free energy binding site at the mouth of DI-II (note that the last section of the cyan and red pathways overlap with the magenta line). (B) Transverse views of each fenestration extending radially from the central pore axis (obtained by integrating out θ, keeping z and r, for each corresponding fenestration segment, spanning 50°, as depicted in A); MFEPs also mapped along relevant sections of the transverse fenestration axes. (C) Free energy value along the MFEPs, colored according to each fenestration, also showing the most favorable pathway along fenestration DI-II.