Crystal structure of an orthologue of the NaChBac voltage-gated sodium channel

Abstract

Voltage-gated sodium (Na(v)) channels are essential for the rapid depolarization of nerve and muscle, and are important drug targets. Determination of the structures of Na(v) channels will shed light on ion channel mechanisms and facilitate potential clinical applications. A family of bacterial Na(v) channels, exemplified by the Na(+)-selective channel of bacteria (NaChBac), provides a useful model system for structure-function analysis. Here we report the crystal structure of Na(v)Rh, a NaChBac orthologue from the marine alphaproteobacterium HIMB114 (Rickettsiales sp. HIMB114; denoted Rh), at 3.05 Å resolution. The channel comprises an asymmetric tetramer. The carbonyl oxygen atoms of Thr 178 and Leu 179 constitute an inner site within the selectivity filter where a hydrated Ca(2+) resides in the crystal structure. The outer mouth of the Na(+) selectivity filter, defined by Ser 181 and Glu 183, is closed, as is the activation gate at the intracellular side of the pore. The voltage sensors adopt a depolarized conformation in which all the gating charges are exposed to the extracellular environment. We propose that Na(v)Rh is in an 'inactivated' conformation. Comparison of Na(v)Rh with Na(v)Ab reveals considerable conformational rearrangements that may underlie the electromechanical coupling mechanism of voltage-gated channels.

Fig. 1. The structure of Na_vAP exhibits a closed conformation

(a) Na_vAP exhibits an asymmetric tetramer in the structure. The green sphere indicates the bound ion within the selectivity filter. Leu219, which occludes the activation gate, is shown in light purple sticks in the cytoplasmic view. (b) Na_vAP is closed at both the activation gate and the entrance to the selectivity filter. The channel passage (left panel) is indicated by cyan dots. The pore radii (right panel) of Na_vAP (green) are compared with those of Na_vAb (grey). The residues that constitute the constriction sites, Ser181 at the entrance to the selectivity filter and Leu219 at the activation gate, are shown in sticks in periplasmic and cytoplasmic views, respectively. (c) A semi-transparent surface illustration of the periplasmic entrance to the selectivity filter in Na_vAP and Na_vAb.

Fig. 2. A Ca²⁺ ion is bound in the asymmetric selectivity filter of Na_vAP

(a) A side view of the selectivity filter vestibule of Na_VAP. (b) The carboxylate groups of Na_vAP-E183 and Na_vAb-E178 are positioned similarly in spite of their distinct Cα locations within the selectivity filter. The residues that are important for slow inactivation of Na_v1.4 are colored red in the sequence alignment. (c) The Na_vAP selectivity filter is sodium-selective. (d) Current-voltage relationships for NaChBac and the NaChBac/Na_vAP-filter chimera, and rates of inactivation (tau, τ) of I_Na. (e) A Ca²⁺ ion is bound at an inner site within the selectivity filter. The distances between the ion and the surrounding groups are indicated in angstroms (Å). (f) Progressive reduction in I_Na by the addition of Ca²⁺ or Cd²⁺. The potency of I_Na block was estimated (right; see Methods).

Fig. 3. The VSDs of Na_vAP exhibit a depolarized conformation

(a) Superimposition of the four protomers in Na_vAP structure. An enlarged view of S3–S4 linkers is shown on the right to highlight their conformational distinctions. (b) The S1 to S4 segments of the four VSDs exhibit similar conformations with all the gating charges pointing to the extracellular surface. The gating charges (R1–R4) on the S4 segment, as well as Phe55 and Glu58 on the S2 segment, are shown as sticks. (c) The coordination of the gating charges in the four VSDs of Na_vAP. Hydrogen bonds are represented by red (Mol A–C) or green (Mol D) dashed lines. The residues that mediate invariant interactions between gating charges and the external-negative cluster are labeled in red.

Fig. 4. Molecular basis of charge transfer of VSDs

(a) Superimposition of the structures of Na_vAP (green), Na_vAb (grey), and Kv1.2 (brown), relative to the pore domains. Cytoplasmic views are shown. Ca²⁺ and K⁺ are shown in green and purple spheres. (b) There is a one helical turn shift toward the extracellular side of Na_vAP-S4 compared to Na_vAb-S4 when the charge transfer centers (CTC) are superimposed. Note that the segment of Na_vAP-S4 containing R1 and R2 is an α-helix, whereas the corresponding segment of Na_vAb-S4 is still a 3₁₀-helix. (c) Structural comparison of the S4 segments from Na_vAP, Kv1.2, and the paddle chimera. These structures are superimposed against the CTC. (d) A schematic illustration of the process of one charge (R4) transfer across the occluding residue, Phe, within the CTC. A structure-based animation is shown in Supplementary movie S1.