Voltage‐gated sodium channels (Na V channels) are transmembrane protein complexes that are constituted of an α‐subunit of ~260 kDa and up to 4 auxiliary β‐subunits (β1–4) of 30–40 kDa. The pore‐forming α‐subunit alone is sufficient to obtain sodium current; however, coexpression of β‐subunits modifies expression level, kinetics, and voltage dependence of channel gating (1). The α‐subunit is organized in 4 homologous domains (DI–IV). Each domain consists of 6 putative transmembrane segments (S1–S6) connected by extracellular or intracellular loops. The S4 segments are the most conserved segments, and they contain a basic residue, either Lys or Arg, in every third position. These positively charged S4 segments are believed to function as voltage sensors. They transport gating charges by moving outward on membrane depolarization, thus initiating the voltage‐dependent activation, which results in the opening of the channel. The selectivity filter and pore are formed by the transmembrane segments S5 and S6 along with the reentrant segments that are part of the loop that connects the S5 and S6 of each domain. The short intracellular linker that connects the DIII and DIV contains a highly conserved sequence of 3 hydrophobic residues (Ile, Phe, and Met) or IFM motif. Sodium channel inactivation is mediated by this hydrophobic motif, since it serves as an inactivation gate crucial for causing fast inactivation by binding to a receptor. This inactivation gate receptor is located near or within the intracellular mouth of the sodium channel pore.

Sea anemones have become “medicinal chemists” of unprecedented skills by evolving a unique peptide biochemistry and neuropharmacology to develop components that ensure complete shutdown of the nervous system of their prey. Although a number of sea anemone toxins have been isolated and characterized, these animals remain understudied in comparison with other venomous animals, such as scorpions, spiders, cone snails, and snakes. Sea anemones are a known pharmacological treasure of biological active compounds acting on a diverse panel of ion channels, such as Na V and voltage‐gated potassium (K V ) channels, TRPV1 channels, or acid‐sensing ion channels (ASICs) (2–4). Out of this group, toxins that target sodium channels are the best studied so far, with >100 known toxins (5). Sea anemone sodium channel toxins can be divided into 3 structural classes, based on their structural differences and activity profile (4, 6–8). All toxins belonging to these three classes exert the same pharmacological activity. They are believed to bind to the channel at receptor site 3. On binding at site 3, these toxins trap the voltage‐sensing segment S4 of DIV in its inward position: this prevents the normal outward movement of the voltage sensors and herewith the conformational changes necessary for fast inactivation (9).

To date, 7 toxins from Anthopleura elegantissima have been isolated and characterized: APE1‐1, APE1‐2, APE2‐2, and ApC, which are type 1 sodium channel toxins; and APETx1, a modifier of the human ether a go‐go related gene (hERG) K+ channel; APETx2, which specifically inhibits ASIC3; and APEKTx1, a potent and selective K V 1.1 inhibitor (8, 10–13). APETx1 has been well characterized, and it was first reported to be a selective modulator of hERG channels with an EC 50 value of 34 nM, although it also exerts some activity over other K V channels at higher concentrations (Supplemental Fig. S1 and ref. 10). It is thought that APETx1 binds to the outer vestibule of the hERG channel. Toxin binding causes a shift in the voltage dependence of both activation and inactivation curves, resulting in a blockage of the potassium current. Note that full inhibition is not observed, even at high concentrations. Its interaction with hERG channels is voltage dependent, and it suggests that APETx1 exerts a preferential affinity for the closed state of the channel (10, 14). Further structure‐function studies have highlighted amino acids on the hERG channel, which are key residues for APETx1 interaction (15).

APETx2 was found to be a potent and selective inhibitor of ASIC3 channels. The current through these ASIC3 channels is reversibly inhibited by APETx2 with an IC 50 value of 63 nM (8). ASIC3 channels are mainly expressed in sensory neurons and have been identified to play an important role in acid‐induced hyperalgesia and in high‐intensity pain stimuli (8, 16–18). As such, APETx2 has been a valuable tool to study these channels. In vivo studies have shown that administration of APETx2 prevents postoperative acid‐induced inflammatory pain (19), (20). Recently, it was found that APETx2 could substantially inhibit tetrodotoxin (TTX)‐resistant currents and to a lesser extend TTX‐sensitive currents in rat DRG neurons. It was shown that the observed inhibition of Na V 1.8 channels resulted from a rightward shift in the voltage dependence of activation and a reduction in the maximal macroscopic conductance (21). Similar to ASIC3 channels, Na V 1.8 channels are also expressed in sensory neurons, and they are involved in inflammatory pain. Na V 1.8 inhibitors can produce analgesic effects in vivo (22–24). The knowledge that APETx2 can exhibit target promiscuity indicates that part of the observed analgesic effect of APETx2 in vivo may result from an inhibition of the Na V 1.8 conductance (21).

Since these experiments were done in DRG neurons, little information on the Na V subtype‐ or phyla‐selectivity is available for APETx2.

Here we present the biochemical analysis and electrophysiological characterization of APETx3, a natural occurring mutant of APETx1 differing by a Thr to Pro substitution at position 3. APETx3 is a novel sodium channel toxin, structurally unrelated to the existing classes of sea anemone toxins targeting sodium channels. Furthermore, we re‐evaluated the selectivity of APETx1 and APETx2 to show that they are not as selective as previously thought.