3.1. Structural Types of NaTxsToday, it is quite clear that the wide range of modulating action of NaTxs and the effect on the action potential duration of NaVs are due to their structural characteristics, surface electrostatic potential, and dipole moment [81,87]. Sodium channel toxins produced by sea anemones, neurotoxins, are currently the best characterized [1,2,4,7,13,14,22,35,37,39,58,80,88,89]. Traditionally all NaTxs studied to date have been classified into four structural types, according to amino acid sequences and a different Cys residues distribution pattern (Figure 2, Figure 3, Figure 4 and Figure 5) [2,7,80,81,90]. The experimental data have demonstrated that the toxic and paralyzing action of sea anemone venoms is associated with the profound effects of neurotoxins type 1 and 2 (though with different toxic activity in the concentration range from a few ng to hundreds of μg) on prey, with which animals communicate in natural conditions [2,3,21,22,23,24,25,26,27,28,29,30,31,32,33,34,80,81,82,91,92,93,94].Almost all type 1 and 2 natural NaTxs produced by sea anemones possess from 46 to 49 amino acid residues linked by three disulphide bridges, following the similar connectivity pattern (C1-C5, C2-C4, C3-C6) not yet found in any other toxin (Figure 2 and Figure 3). The only exception is type 1 NaTxs isolated from the sea anemone Actinia equina, Ae1, which has 54 amino acid residues [33]. In addition to Ae1, a number of its highly identical amino acid sequences (Ae2-1, Ae3-1, Ae4-1, Ae2-2, Ae2-3) have been derived from A. equina mRNA sequences [95].Today, more than 40 type 1 NaTxs are known (Figure 2); most of them have been isolated from the species belonging to the Actiniidae family (Anthopleura, Anemonia, Condylactis, Bunodosoma, Actinia, Antheopsis genera) [2,81]. Besides several A. equina neurotoxins, some amino acid sequences of putative homologs of the native neurotoxin Av2 have been derived from the sea anemone A. viridis gDNA sequence [88,95]. Gene cloning, which became a traditional method of studying sea anemone neurotoxins by the end of the last century, made it possible to detect up to a dozen or even more highly homologous amino acid sequences in each of the studied species, which significantly expanded the arsenal of neurotoxins for further structural and functional studies [5,14,16,39]. The majority of the studied sea anemone neurotoxins to date belong to the most common structural type 1: ATX-Ia, -II, -V from Anemonia sulcata [21,22,23,24,26,82,93,94,95,96,97,98], ApA and ApB from Anthopleura xanthogrammica [25,30,99], Am-III from Antheopsis maculata [100], Rc-1 from Radianthus crispus [101], CgNa from Condylactis gigantea [102], Gigantoxin-2 from Stichodactyla gigantea [103], BcIII from Bunodosoma caissarum [104], BgII, BgIII from Bunodosoma granulifera [105], and cangitoxins from Bunodosoma cangicum [106,107,108]. Figure 2. Multiple alignment of the amino acid sequences of the type 1 sea anemone NaTxs: ApA (UniProt ID: P01530) [25], ApB (P01531) [30], PCR1-2 (P0C5F8), PCR2-1 (P0C5G0), PCR2-5 (P0C5F9), PCR2-10 (P0C5G1), PCR3-6 (P0C5G2), PCR3-7 (P0C5G3) [99] from A. xanthogrammica; Am-III (P69928) [100] from A. maculata; Rc-1 (P0C5G5) [101] from Heteractis crispa (=R. crispus); CgNa (P0C280) [85,102] from C. gigantea; Cp1 (P0CH42) and Cp2 (P0C280) [32] from Condylactis passiflora; Gigantoxin-2 (Q76CA3) [103] from S. gigantea; AETX-1 (P69943) [34] from Anemonia erythraea; ATX-Ia (=ATX-I) (P01533) [96], ATX-Ib (A0A0S1M165), ATX-II (P01528) [23], ATX-V (P01529) [97] from A. sulcata; Av2 (P0DL52) [95,98] from Anemonia viridis (previously known as A. sulcata) and Av6, Av9 (sequences, deduced from A. viridis genomic DNA [95]; BcIII (Q7M425) [104] from B. caissarum; BgII (P0C1F4), BgIII (P0C1F5) [105] from B. granulifera; CGTX (P82803), CGTX-II (P0C7P9), CGTX-III (P0C7Q0) [106], Bcg1a (P86459), Bcg1b (P86460) [107,108] from B. cangicum; APE1-1 (P0C1F0), APE1-2 (P0C1F1) [109], APE2-1 (=ApC) (P01532) [110], APE2-2 (P0C1F3) [109] from Anthopleura elegantissima; Hk2a (P0C5F4), Hk7a (P0C5F5), Hk8a (P0C5F6), Hk16a (P0C5F7) [111] from Anthopleura sp.; AFT-I (P10453), AFT-II (P10454) from Anthopleura fuscoviridis [112]; Bca1a (GenBank accession number: KY789430) [113] from Bunodosoma capense; AdE-1 (E3P6S4) [114] from Aiptasia diaphana; Ae1 (=Ae1-1) (Q9NJQ2) [33] from A. equina, Ae2-1 (B1NWU2), Ae2-2 (B1NWU3), Ae2-3 (B1NWU4), Ae3-1 (B1NWU5), and Ae4-1 (B1NWU6) derived from A. equina genomic DNA [95]. The disulfide bridges are shown above the alignment. The sequence similarity is shown as a dark (high) and light (low) gray background, the multiple sequence alignment was performed using the Vector NTI Advance 11.0 software. Figure 2. Multiple alignment of the amino acid sequences of the type 1 sea anemone NaTxs: ApA (UniProt ID: P01530) [25], ApB (P01531) [30], PCR1-2 (P0C5F8), PCR2-1 (P0C5G0), PCR2-5 (P0C5F9), PCR2-10 (P0C5G1), PCR3-6 (P0C5G2), PCR3-7 (P0C5G3) [99] from A. xanthogrammica; Am-III (P69928) [100] from A. maculata; Rc-1 (P0C5G5) [101] from Heteractis crispa (=R. crispus); CgNa (P0C280) [85,102] from C. gigantea; Cp1 (P0CH42) and Cp2 (P0C280) [32] from Condylactis passiflora; Gigantoxin-2 (Q76CA3) [103] from S. gigantea; AETX-1 (P69943) [34] from Anemonia erythraea; ATX-Ia (=ATX-I) (P01533) [96], ATX-Ib (A0A0S1M165), ATX-II (P01528) [23], ATX-V (P01529) [97] from A. sulcata; Av2 (P0DL52) [95,98] from Anemonia viridis (previously known as A. sulcata) and Av6, Av9 (sequences, deduced from A. viridis genomic DNA [95]; BcIII (Q7M425) [104] from B. caissarum; BgII (P0C1F4), BgIII (P0C1F5) [105] from B. granulifera; CGTX (P82803), CGTX-II (P0C7P9), CGTX-III (P0C7Q0) [106], Bcg1a (P86459), Bcg1b (P86460) [107,108] from B. cangicum; APE1-1 (P0C1F0), APE1-2 (P0C1F1) [109], APE2-1 (=ApC) (P01532) [110], APE2-2 (P0C1F3) [109] from Anthopleura elegantissima; Hk2a (P0C5F4), Hk7a (P0C5F5), Hk8a (P0C5F6), Hk16a (P0C5F7) [111] from Anthopleura sp.; AFT-I (P10453), AFT-II (P10454) from Anthopleura fuscoviridis [112]; Bca1a (GenBank accession number: KY789430) [113] from Bunodosoma capense; AdE-1 (E3P6S4) [114] from Aiptasia diaphana; Ae1 (=Ae1-1) (Q9NJQ2) [33] from A. equina, Ae2-1 (B1NWU2), Ae2-2 (B1NWU3), Ae2-3 (B1NWU4), Ae3-1 (B1NWU5), and Ae4-1 (B1NWU6) derived from A. equina genomic DNA [95]. The disulfide bridges are shown above the alignment. The sequence similarity is shown as a dark (high) and light (low) gray background, the multiple sequence alignment was performed using the Vector NTI Advance 11.0 software. In addition to these neurotoxins, 14 NaTxs type 2 representatives from several species of the family Stichodactylidae (mainly Stichodactyla and Radianthus (=Heteractis) genera) have also been isolated to date [27,28,29,115,116,117,118,119,120]. Recently, three new representatives of type 2 neurotoxins, δ-TLTX-Hh1x (Hh X), δ-TLTX-Ca1a (Ca I), and δ-TLTX-Ta1a (Ta I), toxic to crabs, have been discovered in sea anemones belonging to the Thalassianthidae family species (Heterodactyla hemprichii, Cryptodendrum adhaesivum, and Thalassianthus aster) (Figure 3) [119]. The complete amino acid sequences of type 2 neurotoxins have been established using protein structural chemistry (neurotoxin sequencing by Edman degradation) and molecular biology (cloning and sequencing of genes coding some neurotoxins). Figure 3. Multiple alignment of the amino acid sequences of type 2 sea anemone NaTxs: RpII (UniProt ID: (P01534) [27], RpIII (P08380) [91] from Heteractis magnifica (Radianthus paumotensis); RTX-I (P30831) [28], RTX-II (P30783) [115], RTX-III (P30832) [29], RTX-IV (P30784), and RTX-V (P30785) [116] from Heteractis crispa (earlier Radianthus macrodactylus); SHTX-IV (B1B5I9) [117] from Stichodactyla haddoni; ShI (P19651) [31,118] from Stichodactyla helianthus; Gigantoxin-3 (Q76CA0) [103] from S. gigantea; Ca I (D2KX90) from C. adhaesivum, Hh X (D2KX91) from H. hemprichii, Ta I (D2KX92) [119] from T. aster. The disulfide bridges are shown above the alignment. The sequence similarity is shown as a dark (high) and light (low) gray background, the multiple sequence alignment was performed using the Vector NTI software. Figure 3. Multiple alignment of the amino acid sequences of type 2 sea anemone NaTxs: RpII (UniProt ID: (P01534) [27], RpIII (P08380) [91] from Heteractis magnifica (Radianthus paumotensis); RTX-I (P30831) [28], RTX-II (P30783) [115], RTX-III (P30832) [29], RTX-IV (P30784), and RTX-V (P30785) [116] from Heteractis crispa (earlier Radianthus macrodactylus); SHTX-IV (B1B5I9) [117] from Stichodactyla haddoni; ShI (P19651) [31,118] from Stichodactyla helianthus; Gigantoxin-3 (Q76CA0) [103] from S. gigantea; Ca I (D2KX90) from C. adhaesivum, Hh X (D2KX91) from H. hemprichii, Ta I (D2KX92) [119] from T. aster. The disulfide bridges are shown above the alignment. The sequence similarity is shown as a dark (high) and light (low) gray background, the multiple sequence alignment was performed using the Vector NTI software. The NaTxs belonging to types 1 and 2 are believed to have the same ancestral gene [2]. The representatives of these types have proved to differ immunologically as antigenic cross-reactivity has not been revealed for them [27,31]. It has been found that both NaTx types, 1 and 2, have the same binding site (so-called ’receptor site 3‘) [120], the extracellular region of NaV voltage-sensor domain IV (VSD-IV) [77,121]. It is one from the eight known NaV binding sites. It is also the binding site of scorpion α-toxins [24]. However, the interaction of type 2 NaTxs with the site 3 (Figure 1), in contrast to that of type 1 NaTxs, does not block the binding of α-toxins to this site [110]. Moran et al. (2007) believe that this site is a heterogeneous receptor for many so-called site-3 toxins, including sea anemone neurotoxins [122,123].Unlike types 1 and 2 neurotoxins, five shorter NaTxs (with 27–32 amino acid residues and toxicity for crabs) were mainly isolated from the sea anemones of the Actiniidae family (Figure 4a) and assigned to type 3 [110]. Thus, the shortest neurotoxin ATX-III (=Av3, 27 aa) isolated earlier from A. sulcata (=A. viridis) [70,124,125], is stabilized by three disulfide bonds (C1-C5, C2-C4, C3-C6). While four others longer neurotoxins of this type, PaTX (31 aa) from Entacmaea quadricolor (=Parasicyonis actinostoloides) [126], Da-I and Da-II (30 aa) from Dofleinia armata, and Er-I (32 aa) from Entacmaea ramsayi [127], were observed to contain four disulfide bonds. These five toxic peptides are unrelated to each other and evidently have different structural folds and properties [57,122]. According to 1H-NMR data and the secondary structure elements analysis, ATX-III forms an unusual compact structural motif with the complete absence of regular α-helices and β-strands, but only with β- and γ-turns [128]. The residues localized on the molecular surface form a hydrophobic region and may represent a NaV binding surface [129]. Like NaTx types 1 and 2, short type 3 neurotoxins also slow down the kinetics of NaV channel inactivation [82,110]. They were shown to be inactive [21,26] on mammalian cells and very active on that of crustaceans and insects [65,70,122,130,131]. The recombinant form of neurotoxin Av3 (=ATX-III) was also inactive on mammalian rNaV1.2a, rNaV1.4, and hNaV1.5 channels [122]. It is still unknown how other type 3 neurotoxins interact with insect and mammalian Navs.The small sea anemone Calliactis parasitica (Hormathiidae family), inhabiting marine biocenosis in symbiosis with hermit crabs, produces two highly identical toxins, calitoxins I and II (CLX-1 and CLX-2) (46 aa, 95% identity), classified as type 4 NaTxs (Figure 4b) [132,133]. Their residues are cross-linked by three disulphide bridges, the topology of which is similar to that of types 1 and 2 NaTxs but, despite this, they are attributed to the separate type 4 due to differences in amino acid sequences with those of types 1 and 2 representatives. Both of the peptides differ in the substitution of only one amino acid residue at the position 6 (Glu/Lys). Neurotoxins of types 3 and 4, whose amino acid sequences differ significantly from those of types 1 and 2, appear to be less abundant in sea anemone venoms [31,57,134].In addition to these four types, two native neurotoxins are described: Halcurin isolated from the sea anemone Halcurias carlgreni (a primitive specie belonging to the Halicuridae family [135]) and Nv1 from Nematostella vectensis (from the small starlet sea anemone of the Edwardsiidae family) showing higher specificity for insect channels [136]. The amino acid sequences of both neurotoxins (47 aa) are highly homologous to those of NaTxs types 1 and 2 (Figure 4c). Figure 4. Multiple alignment of the amino acid sequences of: (a) type 3 sea anemone NaTxs: ATX-III (=Av3) (UniProt ID: P01535) from A. sulcata [124], PaTX (P09949) from E. quadricolor (=P. actinostoloides) [126], Er-I (P09949/P09949-1) from E. ramsayi, Da-I (a major form) and Da-II (P0DMZ2/P0DMZ2-1) from D. armata [127]; (b) type 4 NaTxs CLX-1 (UniProt ID: P14531) and CLX-2 (P49127) from C. parasitica [132]; and (c) Halcurin (UniProtKB: P0C5G6) from H. carlgreni [135] and Nv1 (B1NWS1) from N. vectensis [137]. The sequences of two Nv1 isoforms with C-terminal Q (B1NWS1-1) and K (B1NWS1-2) are shown [https://www.uniprot.org/uniprot/B1NWS1] [138]. C-terminal residues of both isoforms are underlined. The disulfide bridges are shown above the alignment. The sequence similarity is shown as a dark (high) and light (low) gray background, the multiple sequence alignment was performed using the Vector NTI software. Figure 4. Multiple alignment of the amino acid sequences of: (a) type 3 sea anemone NaTxs: ATX-III (=Av3) (UniProt ID: P01535) from A. sulcata [124], PaTX (P09949) from E. quadricolor (=P. actinostoloides) [126], Er-I (P09949/P09949-1) from E. ramsayi, Da-I (a major form) and Da-II (P0DMZ2/P0DMZ2-1) from D. armata [127]; (b) type 4 NaTxs CLX-1 (UniProt ID: P14531) and CLX-2 (P49127) from C. parasitica [132]; and (c) Halcurin (UniProtKB: P0C5G6) from H. carlgreni [135] and Nv1 (B1NWS1) from N. vectensis [137]. The sequences of two Nv1 isoforms with C-terminal Q (B1NWS1-1) and K (B1NWS1-2) are shown [https://www.uniprot.org/uniprot/B1NWS1] [138]. C-terminal residues of both isoforms are underlined. The disulfide bridges are shown above the alignment. The sequence similarity is shown as a dark (high) and light (low) gray background, the multiple sequence alignment was performed using the Vector NTI software. The high activity of Nv1 has been established on arthropods NaVs in a way that this neurotoxin inhibits the inactivation of DmNaV1/TipE channel of drosophila by binding to its site 3. At the same time, Nv1 is almost inactive on mammalian channels (rNaV1.4-β-1, hNaV1.5-β-1). It is completely inactive on the rNav1.2a-β-1 channel [137]. The dozen of putative Nv1 neurotoxins derived from the cluster of genomic DNA was presented by Moran et al. [95]. Furthermore, some genes encoded identical precursors of this neurotoxin. Moreover, some more peptides belonging to the Nv1 family (Nv4–Nv8) encoded by highly homologous genes at different N. vectensis developmental stages were obtained later [17]. The toxicity of different representatives of this family varied for different organisms, zebrafish larvae, and cherry shrimps [17]. The residues of these neurotoxins are cross-linked by three disulphide bridges similarly to the bridges of the type 1 and 2 representatives (C1-C5, C2-C4, C3-C6). A high degree of Halcurin sequence homology with the type 2 neurotoxins (49–74%) is revealed. This neurotoxin has several residues conserved only for type 1 NaTxs, and also has 47% sequence identity with Nv1. Halcurin has been observed to be very toxic to crabs (with LD50 of 5.8 μg/kg), but it has not been lethal to mice.Besides these sea anemone representatives, there are neurotoxins with lethality to crabs, AETX-II and AETX-III (Figure 5), isolated from the small sea anemone Anemonia erythraea (Actiniidae family) (an organism with a body wall diameter of 2–5 cm) inhabiting tropical ocean regions [34]. Their amino acid sequences include 59 residues (with more than 90% identity) and a distinct fold is stabilized by five disulfide bonds located at the positions 5, 11, 12, 19, 27, 28, 36, 44, 46, 60, a disulfide pattern of which is not determined yet. Although the mechanism of modulating action of these neurotoxins has not yet been studied, the sequence homology (40%) with the toxin Tx1 from the Phoneutria nigriventer spider indicates the likelihood of the similar modulating effect of AETX-II and AETX-III towards voltage-gating NaVs. It has been established that neurotoxins are inactive on mammalian channels. Moreover, the LD50 values of AETX-II and AETX-III against crabs have been estimated to be 0.53 and 0.28 µg/kg, respectively [34]. Figure 5. Amino acid sequences of AETX-II, AETX-III from A. erythraea [34]. The sequence similarity is shown as a dark (high) and light (low) gray background. Figure 5. Amino acid sequences of AETX-II, AETX-III from A. erythraea [34]. The sequence similarity is shown as a dark (high) and light (low) gray background. Three new toxins, AnmTX Cj 1a-1 (48 aa), AnmTX Cj 1b-1 (46 aa), and AnmTX Cj 1c-1 (49 aa), have recently been identified from the cold-water sea anemone Cnidopus japonicus by high-throughput proteomics and bioinformatics methods [139]. Both toxins, AnmTX Cj 1c-1 and AnmTX Cj 1a-1, are homologous to NaTxs types 2 and 1, gigantoxin-2 (44 aa), and gigantoxin-3 (49 aa) from S. gigantea with 52 and 50% homology, respectively (Figure 6a). Apparently, the pattern of their disulfide bonds is similar to that of type 1 and 2 neurotoxins. Moreover, the AnmTX Cj 1b-1 toxin is homologous to BDS-I (43 aa) from the sea anemone A. sulcata [140] (Figure 6a) and their sequences homology with each other is 48%.To date, the specific targets of gigantoxin-3 and gigantoxin-2, as well as AnmTX Cj 1a-1 and AnmTX Cj 1c-1, have not yet been identified. However, AnmTX Cj 1a-1 and AnmTX Cj 1c-1 homology to some types 1 and 2 neurotoxins supports their functional activity as modulators of sodium channels inactivation. It has recently been found that BDS-I, known as a specific inhibitor of the KV3 family of voltage-gated potassium channels [48] (Figure 6), is also able to bind to human NaV1.7 channels [140] with very high binding efficiency (similar to that of the neurotoxin 1 type CGTX-II from B. cangicum (Figure 6b) [106]).In addition to the above sea anemone neurotoxins, two new toxic peptides, BcIV (41 aa) [141] and Am-II (46 aa) [100], were found in the venoms of the sea anemones B. caissarum and A. maculata, respectively. They turned out to be homologous to the peptide toxins APETx1 (42 aa) [41] and APETx2 (42 aa) [42] from A. elegantissima, as well as BDS-I (43 aa) [140], and BDS-II (43 aa) [142] toxins from A. sulcata (Figure 6c).Both peptides, BcIV and Am-II, are characterized by a β-defensin-like fold similar to that of NaTx neurotoxins and APETx-like peptides, which have the same pattern of disulfide bonds. However, the identity of the amino acid sequences of BcIV and Am-II with the sequences of known NaTxs (Figure 2) and APETx-like (Figure 6c) representatives is somewhat lower (BcIV has 45 and 48% identity with APETx1 and APETx2 as well as 42% with Am-II and BDS-I, BDS-II (Figure 6a–c)).It has been shown that BcIV is able to bind NaV channels because, like the type 1 neurotoxin BcIII [104], it has a paralyzing effect in vivo [141], albeit in a much lower minimal lethal dose comparing to BcIII (2000 μg/kg vs 219 µg/kg, respectively); while the main targets of APETx1 and APETx2 peptides, as known, are the human acid-sensing ion ASIC3 channel and human cardiac potassium channel, hERG [41,42]. It is known that APETx2 effectively inhibits, besides ASIC3, NaV1.2 (IC50 of 114 ± 25 nM) and NaV1.8 channels (IC50 of 55 ± 10 nM).The new Am-II peptide also has a paralytic effect (LD50 420 µg/kg) six times lower than its homolog from A. maculata, Am-III (LD50 of 70 µg/kg), the representative of NaTxs type 1, and twice higher than Am-I (LD50 of 830 µg/kg) structurally similar to PaTX-like NaTxs type 3 (Figure 4a) [100]. However, unlike BDS-I and BDS-II, which highly specifically blocks the activity of the potassium channel KV3.4 and has a weak effect on TTX-sensitive NaV of neuroblastoma and cardiac and skeletal muscle cells, Am-II, very likely, acts on NaV channels as it is lethal to crabs [100]. Therefore, BcIV and Am-II are structurally new peptide toxins with a putative species-specific action. 3.3. Some Key Functionally Significant Amino Acid Residues of NaTxsIn the 1960–1980s, it was found that sea anemone neurotoxins (such as ApA, ApB, ATX-II, ATX-III) were toxic to arthropods/insects and could also have cardiostimulatory and neuromuscular effects on mammals [21,22,23,24,25,26,30], whereas their specific effect on NaV channels was later observed to manifest itself in a wide range of concentrations [34,65,66,82,83,85,93,94,103,123,150,151].Although a large number of sea anemone neurotoxins are now known, only a few of them have been studied in sufficient detail. High sequence identity and the presence of conservative, functionally important amino acid residues, indicate the preservation of the high stability of a NaTxs structure and function [152]. The analysis of the structure-functional relationships of some NaTxs type 1 representatives, and their functionally significant and conserved amino acid residues identified by site-directed mutagenesis, has been demonstrated in a number of experimental papers and reviews [2,37,80,81].While most type 1 neurotoxins contain 47 amino acid residues (except ATX-I and CgNa with 46 aa ApA and ApB with 49 aa, AFT-I, AFT-II and Gigantoxin with 48 aa) all type 2 neurotoxins described to date have 48 amino acid residues (Figure 2 and Figure 3). Within each of the four types of neurotoxins (1–4), there is a high (up to 98%) degree of structural homology.A characteristic of the type 2 neurotoxins is the presence of only two mainly hydrophobic residues at the N-terminus of a sequence and of three-four basic residues at the C-terminus, which distinguishes them from type 1 neurotoxins with three N-terminal and two C-terminal residues (with the exception of ATX-V with one C-terminal Lys (Figure 2)). In addition, the amino acid sequences of both types contain different amounts of charged and aromatic residues. Therefore, the type 2 neurotoxins have seven positively and negatively charged residues (excluding RTX-I and RpII with six of them) in a sequence, and their N-terminal fragments contain much more negatively charged residues (including the conservative tripeptide 6DDD/E8), and C-terminal fragments include more positively charged ones. At the same time, almost all representatives of these NaTxs are characterized by the presence of a unique C-terminal 4-membered fragment, 45RKKK48. Therefore, the positive charge of type 2 neurotoxins is significantly higher than that of type 1.

It should also be noted that there are significant differences (playing, as known, an important role in protein-protein interactions) in the content of NaTx aromatic residues. Both neurotoxin types have one highly conserved Trp at position 28; it is unique for type 2 neurotoxins (an exception is RpII with one more Trp at position 24). While type 1 neurotoxins either have only one Phe and/or Tyr residue and two or three Trp residues, type 2 neurotoxins have two Phe and from one to five Tyr residues. This, together with a large number of charged residues at N-terminus (4, 6–8 positions) and C-terminus (45–48 positions), may cause their greater affinity to their targets, NaV channels of various subtypes (due to the emergence of several intermolecular π-π interactions between them).

Currently, many type 1 neurotoxins (for example, ATX-II, ATF-II, Bc-III, Cp1, CgNa, CGTX-II, Bcg1a, and Bcg1b) have electrophysiologically been characterized in terms of their modulating activity and selectivity towards the mammals and insects sodium channels of various subtypes, NaV1.1–NaV1.9 [59,62,77,81,82,83,84,90,92,94,102,105,110,120,123,150,153,154,155,156]. So far, not more than two or three NaTxs of structure type 2 have been investigated, using this approach. It is quite obvious that point substitutions in types 1 and 2 neurotoxin sequences and, in particular, the different distribution of positively and negatively charged residues can significantly change the value of the peptides dipole moment, which results in their specific interaction with certain targets, various NaV subtypes [81,87].The first isolated neurotoxins (ApA, ApB, ATX-II, ATX-III, RTX-III, RpII [21,22,23,24,25,26,27,28,29,30,31,32,33,34]) showed high toxicity to mammals and/or arthropods (Table 1). Their further electrophysiological studies indicated a high activity in relation to various NaV subtypes when these neurotoxins slowed down process of the channel inactivation. ApA was selective for the mammalian NaV1.5 channel, and ApB had high affinity for the variety of NaV channel subtypes [147,157]. Those neurotoxins differed in seven amino acid residues and showed a 20–50-fold preference for cardiac channels over neuronal ones [158,159]. Several other neurotoxins were powerful insectotoxins (type 1 ATX-I [21,22] and type 3 ATX-III (=Av3) from A. sulcata (=A. viridis) [122]) while some NaTxs acted on the channels of both mammals and arthropods (for example, ATX-II from A. sulcata [110], RTX-I–RTX-V from R. macrodactylus [28,29,115,116]). Moreover, while ATX-II was characterized by a high insecticidal activity, the toxicity of highly homologous RTX-I–RTX-V (Figure 3) was higher for mammals, but it differed by 2–3 orders of magnitude [28,29,115,116,160,161]. A comparative study of the toxicity of types 1 and 2 NaTxs, CgNa (CgII) (C. gigantea), Cp1 (C. parasitica), and ShI (S. helianthus), respectively, showed that they were lethal to crustaceans, moderately toxic to insects, and non-toxic to mammals [85]. Since lots of sea anemones neurotoxins are highly toxic to insect channels, they can be considered a promising source of new insecticides, and the leading position in this direction belongs to the recombinant neurotoxins Av1 (A. viridis) and Nv1 (N. vectensis) [37,95]. A distinctive feature of the modulating effect of sea anemone neurotoxins on insect NaV channels is the almost complete absence of channel inactivation after the toxin application, which is not typical for mammalian channels [37,80,150].It is interesting that the results of mutagenesis and electrophysiological testing of a number of NaTxs show that the amino acid residues that are functionally important for the binding of neurotoxins to various mammalian and arthropod NaVs sometimes differ. For different representatives they often contradict each other. Thus, while the positively charged residues of ATX-II, Lys36, Arg14, and the negatively charged ones, Asp7, Asp9, and C-terminal Gln47 (Figure 8a), have been observed to play an important role for the functional activity as their substitutions, resulting in the abolishing of the neurotoxins effect [162]; while according to Moran et co-workers (2006), the replacement of Asp7Ala is not critical for the Av2 (=ATX-II) activity [163]. However, while Lys14, a conservative residue almost in all type 1 neurotoxins, was not essential for the ApA activity, it and positively charged Lys37 and Lys49 (Figure 8b) were observed to be functionally important for the activity or the affinity of ApB [164,165,166,167] with some subtypes of NaV heart and neurons.

Table 1. Toxicity of NaTxs and binding to synaptosomes.

Table 1. Toxicity of NaTxs and binding to synaptosomes.

See Anemone SpeciesNeurotoxinLethal Dose,
(μg/kg) aBinding to Synaptosomes of Rat Brain,
KD (μM) bRef.LD50 (Mice)LD100 (Crabs)A. sulcataATX-I
ATX-II
ATX-III
ATX-V4000
100
18,000
19.04.4
3.7
6.7
10.47.0
0.15
10.0
0.05[21,23,26,97,153]A. xanthogrammicaApA
ApB66.0174
8.022.0
78.00.12
0.035[30]
[25,26]S. giganteaGigantoxin II200014.010.0[26,103]R. paumotensis
(=H. magnífica)RpI
RpII
RpIII
RpIV145
4200
53.0
40.036.0
15.0
10.0
90.00.9
10.0
0.3
10.0[27]
[27]
[91]
[27]S. helianthusShI20,0000.6 [31,118]P. actinostoloidesPaTX20,00010.0 [126,130,134]H. crispaRTX-I
RTX-II
RTX-III
RTX-IV
RTX-V
δ-SHTX-Hcr1f3000
1650
25.0
40.0
350
42003.5
4.0
82.0
4.4
12.0
15.0 [28]
[115]
[29]
[116]
[116]
[168]

Figure 8. Functionally significant amino acid residues of NaTxs (a–c,e) and ATX-II putative binding site (d). The homology models of ATX-II (based on ApA, PDB ID: 1Ahl) (a), and PD-I with VSD-IV of rNaV1.2 channel (based on hNaV1.2, PDB ID: 6J8E) (d), and 3D structure of ApB (1Apf) (b), and ATX-III (1ANS) (e). The ribbon diagram of the neurotoxins is shown, key residues presented as sticks and colored blue (basic), red (acidic), green (aliphatic), orange (polar), yellow (proline) and pink (aromatic). The ribbon diagram of rNaV1.2 fragment is colored from N- (blue) to C-termini (red), key ATX-II interacting residues are colored as described above. (c) Multiple alignment of the amino acid sequences of the type 1 NaTxs; key residues are colored as described above, and positively (ATX-III, ApA, ApB) or negatively (CgNa) charged motif is underlined.

Figure 8. Functionally significant amino acid residues of NaTxs (a–c,e) and ATX-II putative binding site (d). The homology models of ATX-II (based on ApA, PDB ID: 1Ahl) (a), and PD-I with VSD-IV of rNaV1.2 channel (based on hNaV1.2, PDB ID: 6J8E) (d), and 3D structure of ApB (1Apf) (b), and ATX-III (1ANS) (e). The ribbon diagram of the neurotoxins is shown, key residues presented as sticks and colored blue (basic), red (acidic), green (aliphatic), orange (polar), yellow (proline) and pink (aromatic). The ribbon diagram of rNaV1.2 fragment is colored from N- (blue) to C-termini (red), key ATX-II interacting residues are colored as described above. (c) Multiple alignment of the amino acid sequences of the type 1 NaTxs; key residues are colored as described above, and positively (ATX-III, ApA, ApB) or negatively (CgNa) charged motif is underlined.