Voltage-gated ion channels serve a critical function in the nervous system. These transmembrane proteins allow neurons to propagate action potentials via an influx of positively charged ions, which is the basis of neural signal transmission. The structure and function of voltage-gated ion channels is highly conserved across the animal kingdom (1). The voltage-gated sodium channel NaVPaS (PDB ID: 6A90), isolated from Periplaneta americana (American cockroach) is an important protein in nervous system function and a target for multiple neurotoxins.  This type of protein is particularly significant because it is critical for transmitting signals in the nervous system, and inhibition of proper function can result in death (2).

            Na_VPaS is a single-subunit, transmembrane ion channel of molecular weight 183700.94 Da and isoelectric point 5.98 (3). Shen and colleagues recently elucidated the NaVPaS structure using cryo-EM. Much of the protein has an α-helical secondary structure, with some random coils and 3/10 helices connecting the transmembrane domains.  The protein also contains two pairs of antiparallel β-strands in domain I on opposite sides of the membrane (2). It consists of four domains (I-IV) with six transmembrane segments each (S1-S6). In each domain, segments S1-S4 comprise a voltage-sensitive region, and segments S5-S6 form part of the pore area. The domains together create a pore to allow ions to diffuse into the cell. The S3b-S4 region of each domain is a helix-turn-helix structure that drives the voltage sensitivity of the channel, called a voltage-sensor paddle. Its importance in the voltage-gating action of the protein makes the voltage-sensor paddle a prime target for toxins that disrupt ion channel function (4).

            Several molecules interfere with voltage-gated ion channel functions. These neurotoxins can be useful because they help researchers to learn about the structural and functional aspects of ion channels (5). Tetrodotoxin(TTX) was originally characterized in poisonous pufferfish, and has since been found in several deadly venoms, including those of the blue-ringed octopus and the predatory moon snail. TTX is not produced by these animals themselves; instead, endosymbiotic bacteria synthesize the toxin (2). TTX is an aminoperhydroquinazoline, with molecular formula C₁₁H₁₇N₃O₈, which binds to the pore domain of voltage-gated sodium channels (6). The molecule contains one guanidium, two ethers, several hydroxyl groups, and an oxygen anion (2). TTX functions as a pore blocker, which blocks the pore of the ion channel and physically prevent ions from moving across the membrane. The association of TTX with NaVPaS (PDB ID: 6A95) is stabilized by electrostatic interactions between TTX and the outer part of the pore domain of Na_VPaS, and by hydrogen bonding between Asp-375 and Glu-701in this region of Na_VPaS and the polar groups of TTX (2).

Saxitoxin (STX) is a neurotoxin that causes shellfish poisoning in Homo sapiens when ingested. Like TTX, STX is produced by endosymbiotic bacteria, and it is a pore blocker that associates with the outer region of the pore domain of NaVPaS (2). It contains a reduced purine ring system and has molecular formula C₁₀H₁₇N₇O₄ (7). The compound contains two guanidium groups (1,2,3- and 7,8,9-), two C12 hydroxyl groups, and a C13 carbamoyl. The STX 1,2,3-guanidium binds toTyr-376 in the first loop of the pore domains in Na_VPaS domain I. The STX C12 hydroxyls interact with Na_VPaS domain IV, and the N7 of STX forms a hydrogen bond with Glu-701 of Na_VPaS (2).

            The small peptide Dc1a (PDB ID: 2MI5) is another neurotoxin that interferes with voltage-gated ion channel function. Shen and colleagues recently described the structure of Dc1a, isolated from Diguetia canities (Desert bush spider) venom, complexed with Na_VPaS (PDB ID: 6A90). Rather than blocking the pore, like TTX and STX, Dc1a associates with the voltage-sensor paddle of domain II and pore domains of Na_VPaS and forces the channel to remain active. Dc1a consists of 57 residues arranged in five N-terminal beta sheets and a C-terminal cysteine knot (2). 

Dc1a associates with the third extracellular helix of NaVPaS domain II by Dc1a-Tyr-33 interaction with Na_VPaS-His-1032 and Dc1a-Asp-56 interaction with Na_VPaS-Arg-1027. Dc1a also associates with Na_VPaS domain II extracellular S1-S2 loop by Dc1a-Arg-41 interaction with Na_VPaS-Asp-542. The Dc1a β3-β4 hairpin inserts into the NaVPaS voltage-sensing domain II (VSDII) cavity, where Dc1a-Phe-47 and Dc1a-Phe-48 interact with the nonpolar residues in the region. Dc1a-Phe-48 also interacts with Na_VPaS -Arg-613, which is a critical Na_VPaS gating charge residue, and Na_VPaS -Gln-1002. Interactions between Dc1a-Lys-44 and Na_VPaS -Gln-1002 further stabilizes the Dc1a β3-β4 hairpin and VSDII cavity binding. Together, these interactions maintain the voltage-sensing region in a stable active state (2).

            The Na_VPaS protein was isolated from the American cockroach, but it shares a great degree of homology with voltage-gated sodium channels in other species (1). The Dali server is a website that compares protein tertiary structures by calculating similarity of interatomic distance in a protein’s folded conformation. The server rated several proteins as similar to Na_VPaS, including the brain isoform of the human voltage-gated sodium channel NaV1.2 (PDB ID: 2KAV), which was given a Z-score of 11.9 in comparison with Na_VPaS (8), where a high score of 52 denotes an identical structure, and a low score of 0 denotes a total lack of common structures. In terms of sequence homology, NCBI PSI-BLAST, which compares primary structures of proteins for homology, yielded an E-value of 2e-22, where a low score of 0 denotes a 100% homologous protein (9). Both voltage-gated sodium channels perform the same function of allowing neurons to propagate action potentials in different species (2,10).

            The critical regions for TTX, STX, and Dc1a binding are conserved in NaVPaS and NaV1.2, showing conserved binding patterns.  There are several variants of the C-terminal cytoplasmic region of Na_V1.2 which are not directly involved in neurotoxin binding but do have significant implications in human health and disease. This region, which spans Na_V1.2 residues 1777-1882, is made up of three parts: residues 1777-1789, which are part of a disordered region, Leu-1790 to Glu-1868, which is comprised of four α-helices and two short antiparallel β-strands, and Ser-1869 to Arg-1882, which constitute a less-ordered helical region.  Mutations in Phe-1795, Phe-1798, or Tyr-1799 in helix I and Leu-1855, Ile-1857, or Leu-1858 in helix IV alter the kinetics of inactivating the channel, which can cause cardiac arrhythmias and neurological problems like epilepsy (10).

Other voltage-gated ion channels share a similar structure with Na_VPaS, like the mammalian voltage-gated calcium channel CaV1.1 (PDB ID: 5GJV) isolated from Oryctolagus cuniculus (European rabbit). Ca_V1.1, at 1873 residues, is a larger protein than Na_VPaS, at 1596 residues (2,11). The Dali server rated Ca_V1.1 with a Z-score of 22.1, meaning its tertiary structure is more similar to that of Na_VPaS than Na_V1.2 (8). The psi-blast software also rated Ca_V1.1 and Na_VPaS as highly homologous, with an E-value of 6e-145 (9).

There are structural differences in the extracellular domain of the proteins that make them specific to either sodium ions (Na_VPaS) or calcium ions (Ca_V1.1). The long, extracellular loops of the pore domain of Ca_V1.1 are stabilized by disulfide bonds and form a selectivity filter, which ensures that only molecules of the correct size (i.e. Ca²⁺) can pass through the pore when the channel is activated (11). This selectivity filter would likely interfere with binding of neurotoxins to the pore domain of the protein.

            The structure of the voltage-gated sodium channel NaVPaS gives great insight into how it functions under normal conditions. The structure of complexes formed by Na_VPaS and neurotoxins like TTX, STX, and Dc1a show how interactions with a few critical residues can completely inhibit normal function. Comparing the structures of Na_VPaS and Na_V1.2 shows how similar proteins in different species can perform the same function and be susceptible to various mutations. Even proteins that carry out slightly different functions, like Na_VPaS and Ca_V1.1, which allow transmembrane diffusion of sodium ions and calcium ions, respectively, can have similar structures with modified features like the selectivity filter of Ca_V1.1. Voltage dependent ion channels are critical proteins for nervous system function. Small perturbations in the function of these proteins can have wide-ranging and often deadly effects, as in the case of animal neurotoxins like TTX, STX, and Dc1a. It is crucial to know how the structure of these proteins facilitate their function under normal conditions to understand what happens when toxins disrupt the process and how disaster can be avoided when they do.