Mutant bacterial sodium channels as models for local anesthetic block of eukaryotic proteins

Abstract

Voltage gated sodium channels are the target of a range of local anesthetic, anti-epileptic and anti-arrhythmic compounds. But, gaining a molecular level understanding of their mode of action is difficult as we only have atomic resolution structures of bacterial sodium channels not their eukaryotic counterparts. In this study we used molecular dynamics simulations to demonstrate that the binding sites of both the local anesthetic benzocaine and the anti-epileptic phenytoin to the bacterial sodium channel NavAb can be altered significantly by the introduction of point mutations. Free energy techniques were applied to show that increased aromaticity in the pore of the channel, used to emulate the aromatic residues observed in eukaryotic Nav1.2, led to changes in the location of binding and dissociation constants of each drug relative to wild type NavAb. Further, binding locations and dissociation constants obtained for both benzocaine (660 μM) and phenytoin (1 μM) in the mutant channels were within the range expected from experimental values obtained from drug binding to eukaryotic sodium channels, indicating that these mutant NavAb may be a better model for drug binding to eukaryotic channels than the wild type.

Keywords: NavAb; benzocaine; drug binding; local anesthetic; phenytoin; sodium channel.

Figure 1.

Sequence alignments of the S6 domains of NavAb (Arcobacter butzleri), NavAe1 (Alkalilimnicola ehrlichei), NavMs (Magnetococcus marinus), NavRh (Rickettsiales sp HIMB114) and NaChBac (Bacillus haldurans), as well as from domains I to IV of human Nav1.2, are shown as determined using ClustalW2. F1764 and Y1771 in Nav1.2 DIV (bright blue) are implicated in the binding of local anesthetic drugs. The corresponding aromatic residues in each sequence alignment are indicated with pale blue boxes and the NavAb residues, T206 and V213, are indicated in the black outlined box. It should be noted that 3 of the 4 domains in Nav1.2 have an aromatic residue at the position equivalent to 1771 in DIV. All other aromatic residues are indicated by a lilac box.

Figure 2.

Most populated clusters of the drugs benzocaine and phenytoin obtained from 300 ns of molecular dynamics simulation where blue, red, yellow and cyan represent progressively less populated clusters. While the full pore forming domain of NavAb (residues 115–221) was used in these simulations, for clarity, only selected residues (200–221) from 3 of the 4 S6 helices are shown in this figure. A: In the 1S system benzocaine remained in the activation gate. B: In the 4S system benzocaine was not able to pass the bulky aromatic residues and remained in the vicinity of the fenestrations. The blue cluster is pi-stacking with F206. C: In the 1S system phenytoin was able to sit in the center of the cavity (red) and move and rotate in the vicinity of the fenestrations (blue, yellow and cyan clusters). D: In the 4S system phenytoin is more restricted in its movement and remains in a pocket formed by the aromatic residues and M209 (blue) or straddles F206 in the fenestration (red).

Figure 3.

Free energy surfaces obtained from metadynamics simulation for the 1S (800 ns - A and B) and 4S systems (400 ns - C and D) in the presence of benzocaine (A and C) and phenytoin (B and D). The surfaces are integrated onto a 2D surface and viewed looking down the axis of the pore from the extracellular space. Two quadrants are shown for the 1S systems where the upper quadrant was obtained opposite to the site of the mutation and the lower quadrant was obtained at the site of the mutation. A single quadrant is shown for the 4S systems as the mutations introduced into each subunit maintain the 4 fold symmetry of the protein. Contours are shown every 1 kcal/mol. The full pore forming domain of NavAb (residues 115–221) was used in these simulations.

Figure 4.

Free energy surfaces obtained from metadynamics simulation for the 1S (800 ns - A and B) and 4S systems (400 ns - C and D) in the presence of benzocaine (A and C) and phenytoin (B and D) viewed from in the plane of the membrane. Contours are shown every 1 kcal/mol.

Figure 5.

Snapshots from the equilibrium simulations representing the 2 most favored positions of benzocaine and phenytoin in each system. While the full pore forming domain of NavAb (residues 115–221) was used in these simulations, for clarity, only selected residues from the S6 helices (residues 200–221) and the region between the S5 and S6 helices (residues 170–178) are shown in this figure. A and B: Benzocaine in the 1S system in the region of the activation gate. C and D: benzocaine in the 4S system with the amine oriented toward Y213 (C) and pi-stacking with F206 (D). E and F: phenytoin in the 1S system occupying the central cavity above Y213 (E) and in the fenestration (F). G and H: phenytoin in the 4S system with (G) the amine oriented toward Y213 while the benzyl rings are surrounded by the bulky residues M209, F206 and Y213; or (H) straddling F206 and with one benzyl ring protuding into the fenestration.

Figure 6.

Interaction energies between each drug and the residues lining the channel lumen for each of the most populated clusters. (A) Benzocaine in the 1S system where cluster 1 and 2 are located above and in the activation gate respectively. (B) Benzocaine in the 4S system in which Clusters 1 to 4 are all located in the fenestrations but the drug is oriented in different directions. (C) Phenytoin in the 1S system in which clusters 1, 3 and 4 are all located in the fenestrations while cluster 2 is located in the activation gate. (D) Phenytoin in the 4S system. Only regions of the sequence with significant interactions are shown.

Figure 7.

A schematic representation of the preferred drug binding positions (red) in the pore forming domain of NavAb (represented in gray) with the residues mutated in this study (206 and 213) shown explicitly for 2 of the 4 domains in each case. The fenestrations are indicated with black arrows in panel (A). Panels (A), (B) and (C) show the wild type, 1S, 4S channels in the presence of benzocaine while panels (D), (E) and (F) show the wild type, 1S, and 4S channels in the presence of phenytoin. Including increased aromaticity moves both drugs away from the activation gate site, and toward the fenestrations.