Research Article

Structural basis for the modulation of voltage-gated sodium channels by animal toxins

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Science  19 Oct 2018:
Vol. 362, Issue 6412, eaau2596
DOI: 10.1126/science.aau2596

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Structures of voltage-gated sodium channels

In “excitable” cells, like neurons and muscle cells, a difference in electrical potential is used to transmit signals across the cell membrane. This difference is regulated by opening or closing ion channels in the cell membrane. For example, mutations in human voltage-gated sodium (Nav) channels are associated with disorders such as chronic pain, epilepsy, and cardiac arrhythmia. Pan et al. report the high-resolution structure of a human Nav channel, and Shen et al. report the structures of an insect Nav channel bound to the toxins that cause pufferfish and shellfish poisoning in humans. Together, the structures give insight into the molecular basis of sodium ion permeation and provide a path toward structure-based drug discovery.

Science, this issue p. eaau2486, p. eaau2596

Structured Abstract


Almost all venoms contain toxins that modulate the activity of voltage-gated sodium (Nav) channels in order to incapacitate prey or predators. The single-chain eukaryotic Nav channels comprise four homologous repeats. The central pore domain is constituted by the carboxyl-terminal segments from all four repeats, and each repeat also has a voltage-sensing domain (VSD). Toxins are broadly divided into two categories—pore blockers that physically occlude the channel pore and gating modifiers that alter channel gating by interfering with the VSDs. Whereas small-molecule neurotoxins such as tetrodotoxin (TTX) and saxitoxin (STX) function as pore blockers, most peptidic Nav channel toxins are gating modifiers that trap the channel in a particular stage of the gating cycle through interactions with one or more VSDs. In neither case is the structural basis of channel modulation fully understood.


Dc1a is a peptidic gating modifier toxin (GMT) from venom of the desert bush spider Diguetia canities that specifically binds to VSDII of insect Nav channels to promote channel opening. We showed through biochemical analysis that Dc1a interacts with NavPaS, a Nav channel from the American cockroach Periplaneta americana, for which a cryo–electron microscopy (cryo-EM) structure was recently determined at 3.8-Å resolution. We therefore sought to solve the structure of the complex between NavPaS and Dc1a. As Dc1a occupies a distinctly different channel binding site to pore blockers, we also attempted to supplement the complex with TTX or STX to obtain structures of the ternary complexes.


The cryo-EM structure of NavPaS-Dc1a was determined to an overall resolution of 2.8 Å in the presence of 300 mM NaCl, whereas those of NavPaS-Dc1a-TTX and NavPaS-Dc1a-STX were resolved at 2.6 Å and 3.2 Å, respectively, in the presence of 150 mM NaCl.

VSDII constitutes the primary docking site for Dc1a, which undergoes considerable structural rearrangement upon binding to the channel. The toxin inserts into the cleft between VSDII and the pore region, making intimate contacts with both domains. The network of intermolecular interactions seen in the cryo-EM structure was validated through examination of the effect of toxin and channel mutations using the orthologous NavBg channel from the German cockroach Blattella germanica.

Four residues, Asp/Glu/Lys/Ala (DEKA), at a corresponding locus in the selectivity filter (SF) of each repeat confer Na+ selectivity. A Na+ ion was observed in the same position in the structures of NavPaS-Dc1a and NavPaS-Dc1a-TTX, coordinated by the Asp and Glu residues in the DEKA motif of the SF, and an invariant Glu on the P2 helix in repeat II, a helix in the entryway to the SF on the extracellular side. Both TTX and STX form extensive electrostatic interactions with residues in the outer electronegative ring that attracts cations into the SF and Asp and Glu in the DEKA motif, completely blocking access of Na+ ions to the SF.


The structure of the NavPaS-Dc1a complex suggests that the network of interactions between Nav channels and GMTs is more complex than previously anticipated. Therefore, caution has to be applied when using isolated Nav channel VSDs for drug discovery or for understanding the molecular basis of GMT action. The current structures elucidate the molecular basis for the insect selectivity of Dc1a and the subtype-specific binding of TTX or STX to Nav channels. Unambiguous structural elucidation of the bound TTX and STX, whose molecular weights are both around 300 Da, showcases the power of cryo-EM and its potential for structure-aided drug discovery.

Structural basis for specific binding of GMT Dc1a and guanidinium pore blockers TTX and STX by NavPaS.

(A) Dc1a inserts into the extracellular cavity between VSDII and the pore elements of repeat III. (B) Molecular mechanism for pore blockade by TTX and STX. Top: The carboxylate groups of Asp (D) and Glu (E) residues in the DEKA motif and an invariant Glu on P2II together constitute a potential Na+ binding site (designated the DEE site). Bottom: TTX and STX block access of Na+ to the DEE site from the extracellular side. A semitransparent presentation of the electrostatic surface potential of the entrance to the SF viewed from the extracellular side is shown. CTD, C-terminal domain; R, Arg; L, Leu; Y, Tyr; K, Lys.


Animal toxins that modulate the activity of voltage-gated sodium (Nav) channels are broadly divided into two categories—pore blockers and gating modifiers. The pore blockers tetrodotoxin (TTX) and saxitoxin (STX) are responsible for puffer fish and shellfish poisoning in humans, respectively. Here, we present structures of the insect Nav channel NavPaS bound to a gating modifier toxin Dc1a at 2.8 angstrom-resolution and in the presence of TTX or STX at 2.6-Å and 3.2-Å resolution, respectively. Dc1a inserts into the cleft between VSDII and the pore of NavPaS, making key contacts with both domains. The structures with bound TTX or STX reveal the molecular details for the specific blockade of Na+ access to the selectivity filter from the extracellular side by these guanidinium toxins. The structures shed light on structure-based development of Nav channel drugs.

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