Local anesthetic and antiepileptic drug access and binding to a bacterial voltage-gated sodium channel

Abstract

Voltage-gated sodium (Nav) channels are important targets in the treatment of a range of pathologies. Bacterial channels, for which crystal structures have been solved, exhibit modulation by local anesthetic and anti-epileptic agents, allowing molecular-level investigations into sodium channel-drug interactions. These structures reveal no basis for the "hinged lid"-based fast inactivation, seen in eukaryotic Nav channels. Thus, they enable examination of potential mechanisms of use- or state-dependent drug action based on activation gating, or slower pore-based inactivation processes. Multimicrosecond simulations of NavAb reveal high-affinity binding of benzocaine to F203 that is a surrogate for FS6, conserved in helix S6 of Domain IV of mammalian sodium channels, as well as low-affinity sites suggested to stabilize different states of the channel. Phenytoin exhibits a different binding distribution owing to preferential interactions at the membrane and water-protein interfaces. Two drug-access pathways into the pore are observed: via lateral fenestrations connecting to the membrane lipid phase, as well as via an aqueous pathway through the intracellular activation gate, despite being closed. These observations provide insight into drug modulation that will guide further developments of Nav inhibitors.

Keywords: bacterial sodium channel; drug binding.

Fig. 1.

(A) Simulation system showing Na_vAb (two of four subunits as ribbons, with voltage sensor domain (VSD) S1 and S2 in blue and S3 and S4 in green, pore domain (PD) S5 in yellow and S6 in red, P-loop P1, P2, and selectivity filter (SF; orange) in a hydrated lipid bilayer (chains as gray lines; water as red/white sticks; NaCl as yellow/cyan balls) and PHT (dark gray sticks). (B and C) Stick representations of BZC (B) and PHT (C).

Fig. 2.

(A) Sequence alignment of segment S6 of Na_vAb to other Na_v channels (showing only FS6-containing domain IV; see SI Appendix, Fig. S1, for full comparison). F201, F203, F207, and N211 in Na_vAb and F1764 in Na_v1.2 are indicated. Amino acids were colored with Jalview (46, 47) using the Zappo scheme. (B) Aligned structures (showing three subunits) of Na_vAb and model Na_v1.2 (based on Na_vAb) comparing Na_vAb F201, F203, and F207 to Na_v1.2 F1764. Conserved N211 (Na_vAb numbering) is also indicated.

Fig. 3.

Free energy surfaces (Left) and binding sites (Right) for BZC (A–D) and PHT (E–H), with color scale as inset. For clarity, B for BZC includes only −10 < y < 10 Å (full data in SI Appendix, Fig. S5). Binding sites for BZC (C and D) and PHT (G and H), identified from free energy minima, are shown as spheres (colored for clarity, not by free energy).

Fig. 4.

Interactions involved in binding sites A_b to G_b of BZC, with segments colored as in Fig. 1. (Insets) Arrows point directly at the BZC molecule.

Fig. 5.

Interactions involved in the binding sites A_p to F_p of PHT. (Insets) Arrows point directly at the PHT molecule.

Fig. 6.

Drug-binding pathways. (A) Side view of the channel showing movement of BZC from water to the central cavity along lipophilic and aqueous pathways (dotted curves). The full PD and only one VSD subunit are shown. (B and C) The free energy profile for BZC movement through a fenestration (B) and those for BZC (red) and PHT (blue) movements through the activation gate (C). The free energy is set to zero in water, using SI Appendix, Fig. S2A to set the membrane reference for B.