Structure of a bacterial voltage-gated sodium channel pore reveals mechanisms of opening and closing

Abstract

Voltage-gated sodium channels are vital membrane proteins essential for electrical signalling; in humans, they are key targets for the development of pharmaceutical drugs. Here we report the crystal structure of an open-channel conformation of NavMs, the bacterial channel pore from the marine bacterium Magnetococcus sp. (strain MC-1). It differs from the recently published crystal structure of a closed form of a related bacterial sodium channel (NavAb) by having its internal cavity accessible to the cytoplasmic surface as a result of a bend/rotation about a central residue in the carboxy-terminal transmembrane segment. This produces an open activation gate of sufficient diameter to allow hydrated sodium ions to pass through. Comparison of the open and closed structures provides new insight into the features of the functional states present in the activation cycles of sodium channels and the mechanism of channel opening and closing.

Figure 1. Sequence alignment of the NavMs pore construct and the equivalent region of NavAb.

Alignments are based on their respective crystal structures, with coloured boxes indicating the locations of the S5 and S6 transmembrane segments (magenta and blue, respectively), the turret loop (cyan), the P1- and P2-helix regions (green and yellow, respectively), the SF (red) and the beginning of the C-terminal domain (the latter, with residue names indicated in grey, is not visible in either of the crystal structures). The black underlined region indicates where the deviation between the S6 helices in the open and closed forms occurs.

Figure 2. Crystal structure of the NavMs pore.

(a) The crystal structure of the NavMs pore depicted in ribbon representation. The regions in one of the four monomers are coloured according to the scheme in Fig. 1 (blue for the S6 helix, magenta for the S5 helix, green for P1, yellow for P2 and red for the SF), with the other three monomers depicted in grey for clarity. (b) Electrostatic representation of the NavMs pore (from the membrane normal direction), showing that the entire surface is very hydrophobic. (c) Overlaid structures of the NavMs (red) and NavAb (blue) pores depicted in ribbon representations, showing that they are very similar in all regions except the C-terminal end of S6 (closest to the intracellular surface), where they deviate significantly. The directions of the motions are indicated by the yellow arrows. A video morphing between the open and closed structures is included as Supplementary Movie 1. (d) Comparison of the electrostatic surfaces of the NavMs symmetric pore model (based on a tetramer constructed from the most bent monomer) and the NavAb pore, viewed along the membrane normal and sliced through the middle of the tetramer (top) and from the cytoplasmic surface (bottom). (e) Plot of residue number versus RMSD (root mean square deviation) differences between NavMs (chain A) and NavAb (chain A) C_α positions in the S6 helix, showing that the major differences occur starting at residue Thr209 (indicated by the yellow box). The colours of the names of the residues identified on the model are reflected in the colour of the points on the plot. (f) Stereo electron density map (2Fo–Fc, 1 σ-contour) of the open-channel conformation (red) backbone atoms overlaid on the closed-channel conformation backbone atoms (blue), which clearly shows the deviation of the structures at the bottom of the S6 helix.

Figure 3. Features of the NavMs pore open-channel structure.

(a) Left: the accessible inner surface (in grey) of the NavMs symmetric pore model, showing that there is a continuous pathway from the extracellular to the cytoplasmic surface of the membrane, which is of sufficient size to enable the passage of a fully hydrated sodium ion (except around the SF, where only a hemi-hydrated ion will fit). In this figure the NavMs (red) and NavAb (blue) structures are overlaid as ribbon diagrams. Right: plot of channel radius versus distance along the channel direction for the open NavMs (red) and closed NavAb (blue) structures, showing that in the extracellular half (including the SF and the central cavity), the diameters of the open and closed forms are nearly identical, but that in the region of the activation gate there is a substantial difference in the diameter of the cavity of the open versus closed forms. (b) Schematic model for the opening and closing of bacterial sodium channels, showing changes in the orientations of the bottom half of the S6 helices and the compensating changes required in the disordered regions of the C-terminal domain. Note only two monomers are shown for clarity.

Figure 4. Linker movement required to open the pore.

Overlay showing the residues (in space-filling representation) involved in potential clashes at the end of the open NavMs (red) with those in the S4–S5 linker hinge region (yellow) of the closed NavAb form (blue). This suggests that in the open form of the full-length NavMs channel, the N-terminal end of S5 and the linker must be in a slightly different position from that present in the NavAb closed structure.