Systematic Study of Binding of μ-Conotoxins to the Sodium Channel NaV1.4

Voltage-gated sodium channels (Na_V) are fundamental components of the nervous system. Their dysfunction is implicated in a number of neurological disorders, such as chronic pain, making them potential targets for the treatment of such disorders. The prominence of the Na_V channels in the nervous system has been exploited by venomous animals for preying purposes, which have developed toxins that can block the Na_V channels, thereby disabling their function. Because of their potency, such toxins could provide drug leads for the treatment of neurological disorders associated with Na_V channels. However, most toxins lack selectivity for a given target Na_V channel, and improving their selectivity profile among the Na_V1 isoforms is essential for their development as drug leads. Computational methods will be very useful in the solution of such design problems, provided accurate models of the protein-ligand complex can be constructed. Using docking and molecular dynamics simulations, we have recently constructed a model for the Na_V1.4-µ-conotoxin-GIIIA complex and validated it with the ample mutational data available for this complex. Here, we use the validated Na_V1.4 model in a systematic study of binding other µ-conotoxins (PIIIA, KIIIA and BuIIIB) to Na_V1.4. The binding mode obtained for each complex is shown to be consistent with the available mutation data and binding constants. We compare the binding modes of PIIIA, KIIIA and BuIIIB to that of GIIIA and point out the similarities and differences among them. The detailed information about Na_V1.4-µ-conotoxin interactions provided here will be useful in the design of new Na_V channel blocking peptides.     

Toxins Targeting the KV1.3 Channel: Potential Immunomodulators for Autoimmune Diseases

Autoimmune diseases are usually accompanied by tissue injury caused by autoantigen-specific T-cells. K_V1.3 channels participate in modulating calcium signaling to induce T-cell proliferation, immune activation and cytokine production. Effector memory T (T_EM)-cells, which play major roles in many autoimmune diseases, are controlled by blocking K_V1.3 channels on the membrane. Toxins derived from animal venoms have been found to selectively target a variety of ion channels, including K_V1.3. By blocking the K_V1.3 channel, these toxins are able to suppress the activation and proliferation of T_EM cells and may improve T_EM cell-mediated autoimmune diseases, such as multiple sclerosis and type I diabetes mellitus.     

Spider-venom peptides as therapeutics

Spiders are the most successful venomous animals and the most abundant terrestrial predators. Their remarkable success is due in large part to their ingenious exploitation of silk and the evolution of pharmacologically complex venoms that ensure rapid subjugation of prey. Most spider venoms are dominated by disulfide-rich peptides that typically have high affinity and specificity for particular subtypes of ion channels and receptors. Spider venoms are conservatively predicted to contain more than 10 million bioactive peptides, making them a valuable resource for drug discovery. Here we review the structure and pharmacology of spider-venom peptides that are being used as leads for the development of therapeutics against a wide range of pathophysiological conditions including cardiovascular disorders, chronic pain, inflammation, and erectile dysfunction.     

Spider Neurotoxins Targeting Voltage-Gated Sodium Channels

The voltage-gated sodium (Na_v) channel is a target for a number of drugs, insecticides, and neurotoxins. These bind to at least seven identified neurotoxin binding sites and either block conductance or modulate sodium channel gating and/or kinetics. A number of polypeptide toxins from the venoms of araneomorph and mygalomorph spiders have been isolated and characterized that interact with several of these sites. Certain huwentoxins and hainantoxins appear to target site 1 to block Na_v channel conductance. The δ -atracotoxins and Magi 4 slow Na_v-channel inactivation via an interaction with neurotoxin site 3. The δ -palutoxins, and most likely μ -agatoxins and curtatoxins, target site 4. However, their action is complex with the μ -agatoxins causing a hyperpolarizing shift in the voltage-dependence of activation, an action analogous to scorpion β -toxins, but with both δ -palutoxins and μ -agatoxins slowing Na_v channel inactivation, a site 3-like action. Many spider toxins target undefined sites, while others are likely to cross-react with other ion channels due to conserved structures within domains of voltage-gated ion channels. It is already clear, however, that many spider toxins represent highly potent and specific molecular tools to define novel links between sites modulating channel activation and inactivation. Other spider toxins show phyla specificity and are being considered as lead compounds for the development of biopesticides. Others display tissue specificity via interactions with specific Na_v channel subtypes and should provide useful tools to delineate the molecular determinants to target ligands to these channel subtypes. These studies are being greatly assisted by the determination of the pharmacophore of these toxins, but without precise identification of their binding site and mode of action their potential in the mentioned areas remains underdeveloped.     

Seven novel modulators of the analgesic target NaV 1.7 uncovered using a high-throughput venom-based discovery approach

Background and Purpose

Chronic pain is a serious worldwide health issue, with current analgesics having limited efficacy and dose-limiting side effects. Humans with loss-of-function mutations in the voltage-gated sodium channel Na_V1.7 (hNa_V1.7) are indifferent to pain, making hNa_V1.7 a promising target for analgesic development. Since spider venoms are replete with Na_V channel modulators, we examined their potential as a source of hNa_V1.7 inhibitors.

Experimental Approach

We developed a high-throughput fluorescent-based assay to screen spider venoms against hNa_V1.7 and isolate ‘hit’ peptides. To examine the binding site of these peptides, we constructed a panel of chimeric channels in which the S3b-S4 paddle motif from each voltage sensor domain of hNa_V1.7 was transplanted into the homotetrameric K_V2.1 channel.

Key Results

We screened 205 spider venoms and found that 40% contain at least one inhibitor of hNa_V1.7. By deconvoluting ‘hit’ venoms, we discovered seven novel members of the NaSpTx family 1. One of these peptides, Hd1a (peptide μ-TRTX-Hd1a from venom of the spider Haplopelma doriae), inhibited hNa_V1.7 with a high level of selectivity over all other subtypes, except hNa_V1.1. We showed that Hd1a is a gating modifier that inhibits hNa_V1.7 by interacting with the S3b-S4 paddle motif in channel domain II. The structure of Hd1a, determined using heteronuclear NMR, contains an inhibitor cystine knot motif that is likely to confer high levels of chemical, thermal and biological stability.

Conclusion and Implications

Our data indicate that spider venoms are a rich natural source of hNa_V1.7 inhibitors that might be useful leads for the development of novel analgesics.     

Isolation,synthesis and characterization of ω-TRTX-Cc1a,a novel tarantula venom peptide that selectively targets L-type CaV channels

Spider venoms are replete with peptidic ion channel modulators, often with novel subtype selectivity, making them a rich source of pharmacological tools and drug leads. In a search for subtype-selective blockers of voltage-gated calcium (Ca_V) channels, we isolated and characterized a novel 39-residue peptide, ω-TRTX-Cc1a (Cc1a), from the venom of the tarantula Citharischius crawshayi (now Pelinobius muticus). Cc1a is 67% identical to the spider toxin ω-TRTX-Hg1a, an inhibitor of Ca_V2.3 channels. We assembled Cc1a using a combination of Boc solid-phase peptide synthesis and native chemical ligation. Oxidative folding yielded two stable, slowly interconverting isomers. Cc1a preferentially inhibited Ba²⁺ currents (I_Ba) mediated by L-type (Ca_V1.2 and Ca_V1.3) Ca_V channels heterologously expressed in Xenopus oocytes, with half-maximal inhibitory concentration (IC₅₀) values of 825 nM and 2.24 μM, respectively. In rat dorsal root ganglion neurons, Cc1a inhibited I_Ba mediated by high voltage-activated Ca_V channels but did not affect low voltage-activated T-type Ca_V channels. Cc1a exhibited weak activity at Na_V1.5 and Na_V1.7 voltage-gated sodium (Na_V) channels stably expressed in mammalian HEK or CHO cells, respectively. Experiments with modified Cc1a peptides, truncated at the N-terminus (ΔG1–E5) or C-terminus (ΔW35–V39), demonstrated that the N- and C-termini are important for voltage-gated ion channel modulation. We conclude that Cc1a represents a novel pharmacological tool for probing the structure and function of L-type Ca_V channels.     

Three Peptide Modulators of the Human Voltage-Gated Sodium Channel 1.7, an Important Analgesic Target,from the Venom of an Australian Tarantula

Voltage-gated sodium (Na_V) channels are responsible for propagating action potentials in excitable cells. Na_V1.7 plays a crucial role in the human pain signalling pathway and it is an important therapeutic target for treatment of chronic pain. Numerous spider venom peptides have been shown to modulate the activity of Na_V channels and these peptides represent a rich source of research tools and therapeutic lead molecules. The aim of this study was to determine the diversity of Na_V1.7-active peptides in the venom of an Australian Phlogius sp. tarantula and to characterise their potency and subtype selectivity. We isolated three novel peptides, μ-TRTX-Phlo1a, -Phlo1b and -Phlo2a, that inhibit human Na_V1.7 (hNa_V1.7). Phlo1a and Phlo1b are 35-residue peptides that differ by one amino acid and belong in NaSpTx family 2. The partial sequence of Phlo2a revealed extensive similarity with ProTx-II from NaSpTx family 3. Phlo1a and Phlo1b inhibit hNa_V1.7 with IC₅₀ values of 459 and 360 nM, respectively, with only minor inhibitory activity on rat Na_V1.2 and hNa_V1.5. Although similarly potent at hNa_V1.7 (IC₅₀ 333 nM), Phlo2a was less selective, as it also potently inhibited rNa_V1.2 and hNa_V1.5. All three peptides cause a depolarising shift in the voltage-dependence of hNa_V1.7 activation.     

Expression,Purification and Refolding of a Human NaV1.7 Voltage Sensing Domain with Native-like Toxin Binding Properties

The voltage-gated sodium channel Na_V1.7 is an important target for drug development due to its role in pain perception. Recombinant expression of full-length channels and their use for biophysical characterization of interactions with potential drug candidates is challenging due to the protein size and complexity. To overcome this issue, we developed a protocol for the recombinant expression in E. coli and refolding into lipids of the isolated voltage sensing domain (VSD) of repeat II of Na_V1.7, obtaining yields of about 2 mg of refolded VSD from 1 L bacterial cell culture. This VSD is known to be involved in the binding of a number of gating-modifier toxins, including the tarantula toxins ProTx-II and GpTx-I. Binding studies using microscale thermophoresis showed that recombinant refolded VSD binds both of these toxins with dissociation constants in the high nM range, and their relative binding affinities reflect the relative IC₅₀ values of these toxins for full-channel inhibition. Additionally, we expressed mutant VSDs incorporating single amino acid substitutions that had previously been shown to affect the activity of ProTx-II on full channel. We found decreases in GpTx-I binding affinity for these mutants, consistent with a similar binding mechanism for GpTx-I as compared to that of ProTx-II. Therefore, this recombinant VSD captures many of the native interactions between Na_V1.7 and tarantula gating-modifier toxins and represents a valuable tool for elucidating details of toxin binding and specificity that could help in the design of non-addictive pain medication acting through Na_V1.7 inhibition.     

Pharmacology and biochemistry of spider venoms.

Spider venoms represent an incredible source of biologically active substances which selectively target a variety of vital physiological functions in both insects and mammals. Many toxins isolated from spider venoms have been invaluable in helping to determine the role and diversity of neuronal ion channels and the process of exocytosis. In addition, there is enormous potential for the use of insect specific toxins from animal sources in agriculture. For these reasons, the past 15-20 years has seen a dramatic increase in studies on the venoms of many animals, particularly scorpions and spiders. This review covers the pharmacological and biochemical activities of spider venoms and the nature of the active components. In particular, it focuses on the wide variety of ion channel toxins, novel non-neurotoxic peptide toxins, enzymes and low molecular weight compounds that have been isolated. It also discusses the intraspecific sex differences in given species of spiders.     

Actions of sea anemone type 1 neurotoxins on voltage-gated sodium channel isoforms

As voltage-gated Na⁺ channels are responsible for the conduction of electrical impulses in most excitable tissues in the majority of animals (except nematodes), they have become important targets for the toxins of venomous animals, from sea anemones to molluscs, scorpions, spiders and even fishes. During their evolution, different animals have developed a set of cysteine-rich peptides capable of binding different extracellular sites of this channel protein. A fundamental question concerning the mechanism of action of these toxins is whether they act at a common receptor site in Na⁺ channels when exerting their different pharmacological effects, or at distinct receptor sites in different Na_v channels subtypes whose particular properties lead to these pharmacological differences. The α-subunits of voltage-gated Na⁺ channels (Na_v1.x) have been divided into at least nine subtypes on the basis of amino acid sequences. Sea anemones have been extensively studied from the toxinological point of view for more than 40 years. There are about 40 sea anemone type 1 peptides known to be active on Na_v1.x channels and all are 46-49 amino acid residues long, with three disulfide bonds and their molecular weights range between 3000 and 5000 Da. About 12 years ago a general model of Na_v1.2-toxin interaction, developed for the α-scorpion toxins, was shown to fit also to action of sea anemone toxin such as ATX-II. According to this model these peptides are specifically acting on the type 3 site known to be between segments 3 and 4 in domain IV of the Na⁺ channel protein. This region is indeed responsible for the normal Na⁺ currents fast inactivation that is potently slowed by these toxins. This fundamental “gain-of-function” mechanism is responsible for the strong increase in the action potential duration. They constitute a class of tools by means of which physiologists and pharmacologists can study the structure/function relationships of channel proteins. As most of the structural and electrophysiological studies were performed on type 1 sea anemone sodium channel toxins, we will present a comprehensive and updated review on the current understanding of the physiological actions of these Na channel modifiers.     

Spider Venoms and Spider Toxins

Abstract

Spider venoms and toxins are useful tools for the study of ion channels and synaptic functions of neurons in vertebrates and invertebrates. The components of spider venom, such as proteins, peptides, polyamines and bioamines, are species-specific. The various functions of these toxins are reviewed in this paper.     

Conotoxins That Could Provide Analgesia through Voltage Gated Sodium Channel Inhibition

Chronic pain creates a large socio-economic burden around the world. It is physically and mentally debilitating, and many sufferers are unresponsive to current therapeutics. Many drugs that provide pain relief have adverse side effects and addiction liabilities. Therefore, a great need has risen for alternative treatment strategies. One rich source of potential analgesic compounds that has emerged over the past few decades are conotoxins. These toxins are extremely diverse and display selective activity at ion channels. Voltage gated sodium (Na_V) channels are one such group of ion channels that play a significant role in multiple pain pathways. This review will explore the literature around conotoxins that bind Na_V channels and determine their analgesic potential.     

Molecular Surface of JZTX-V (β-Theraphotoxin-Cj2a) Interacting with Voltage-Gated Sodium Channel Subtype NaV1.4

Voltage-gated sodium channels (VGSCs; Na_V1.1–Na_V1.9) have been proven to be critical in controlling the function of excitable cells, and human genetic evidence shows that aberrant function of these channels causes channelopathies, including epilepsy, arrhythmia, paralytic myotonia, and pain. The effects of peptide toxins, especially those isolated from spider venom, have shed light on the structure–function relationship of these channels. However, most of these toxins have not been analyzed in detail. In particular, the bioactive faces of these toxins have not been determined. Jingzhaotoxin (JZTX)-V (also known as β-theraphotoxin-Cj2a) is a 29-amino acid peptide toxin isolated from the venom of the spider Chilobrachys jingzhao. JZTX-V adopts an inhibitory cysteine knot (ICK) motif and has an inhibitory effect on voltage-gated sodium and potassium channels. Previous experiments have shown that JZTX-V has an inhibitory effect on TTX-S and TTX-R sodium currents on rat DRG cells with IC₅₀ values of 27.6 and 30.2 nM, respectively, and is able to shift the activation and inactivation curves to the depolarizing and the hyperpolarizing direction, respectively. Here, we show that JZTX-V has a much stronger inhibitory effect on Na_V1.4, the isoform of voltage-gated sodium channels predominantly expressed in skeletal muscle cells, with an IC₅₀ value of 5.12 nM, compared with IC₅₀ values of 61.7–2700 nM for other heterologously expressed Na_V1 subtypes. Furthermore, we investigated the bioactive surface of JZTX-V by alanine-scanning the effect of toxin on Na_V1.4 and demonstrate that the bioactive face of JZTX-V is composed of three hydrophobic (W5, M6, and W7) and two cationic (R20 and K22) residues. Our results establish that, consistent with previous assumptions, JZTX-V is a Janus-faced toxin which may be a useful tool for the further investigation of the structure and function of sodium channels.     

Precious Natural Peptides from Spider Venoms: New Tools for Studying Potassium Channels

Among the large variety of animal toxins that target potassium channels, spider peptides constitute an unique class of voltage-dependent K⁺ (Kv) current inhibitors according to their structure and pharmacological properties. Spider toxins that block Kv currents are small basic peptides the include three disuflide bridges and belong to the family of inhibitor cystine knot (ICK) molecules. Unlike snake, bee, scorpion, or sea anemone toxins that block Kv1 or Kv3 channels, ICK spider toxins target Kv2 and Kv4 channels, which are expressed in the central nervous system (CNS) and in the cardiovascular system. Their selective affinities for Kv2 and/or Kv4 subfamilies are very useful for dissecting these currents in neuronal and cardiac cells and for the determination of their contribution in physiological processes. Their mode of action is also original, since they induce a shift of channel opening to more depolarized potentials that alter the voltage-dependent properties of K currents. Then they are called gating modifiers. Structure-function studies of these gating modifiers were recently facilitated by solving their tridimensional structure together with the crystallization of prokaryotic K⁺ channels. Spider toxins present an active molecular surface, including a hydrophobic patch surrounded by charged residues, which are important for their binding on Kv channels. Gating modifiers interact with important residues in the S3C-S4 external loop via both hydrophobic and electrostatic interactions. Several dynamic interaction models were proposed, but all of them remain putative.     

A bifunctional sea anemone peptide with Kunitz type protease and potassium channel inhibiting properties

Sea anemone venom is a known source of interesting bioactive compounds, including peptide toxins which are invaluable tools for studying structure and function of voltage-gated potassium channels. APEKTx1 is a novel peptide isolated from the sea anemone Anthopleura elegantissima, containing 63 amino acids cross-linked by 3 disulfide bridges. Sequence alignment reveals that APEKTx1 is a new member of the type 2 sea anemone peptides targeting voltage-gated potassium channels (K_Vs), which also include the kalicludines from Anemonia sulcata. Similar to the kalicludines, APEKTx1 shares structural homology with both the basic pancreatic trypsin inhibitor (BPTI), a very potent Kunitz-type protease inhibitor, and dendrotoxins which are powerful blockers of voltage-gated potassium channels. In this study, APEKTx1 has been subjected to a screening on a wide range of 23 ion channels expressed in Xenopus laevis oocytes: 13 cloned voltage-gated potassium channels (K_V1.1–K_V1.6, K_V1.1 triple mutant, K_V2.1, K_V3.1, K_V4.2, K_V4.3, hERG, the insect channel Shaker IR), 2 cloned hyperpolarization-activated cyclic nucleotide-sensitive cation non-selective channels (HCN1 and HCN2) and 8 cloned voltage-gated sodium channels (Na_V1.2–Na_V1.8 and the insect channel DmNa_V1). Our data show that APEKTx1 selectively blocks K_V1.1 channels in a very potent manner with an IC₅₀ value of 0.9 nM. Furthermore, we compared the trypsin inhibitory activity of this toxin with BPTI. APEKTx1 inhibits trypsin with a dissociation constant of 124 nM. In conclusion, this study demonstrates that APEKTx1 has the unique feature to combine the dual functionality of a potent and selective blocker of K_V1.1 channels with that of a competitive inhibitor of trypsin.     

Spider peptide toxins as leads for drug development

Venomous animals use a highly complex cocktails of proteins, peptides and small molecules to subdue and kill their prey. As such, venoms represent highly valuable combinatorial peptide libraries, displaying an extensive range of pharmacological activities, honed by natural selection. Modern analytical technologies enable us to take full advantage of this vast pharmacological cornucopia in the hunt for novel drug leads. Spider venoms represent a resource of several million peptides, which selectively target specific subtypes of ion channels. Structure–function studies of spider toxins are leading not only to the discovery of novel molecules, but also to novel therapeutic routes for cardiovascular diseases, cancer, neuromuscular diseases, pain and to a variety of other pathological conditions. This review presents an overview of spider peptide toxins as candidates for therapeutics and focuses on their applications in the discovery of novel mechanisms of analgesia.     

Purification and characterization of Ts15, the first member of a new α-KTX subfamily from the venom of the Brazilian scorpion Tityus serrulatus

Voltage-gated potassium channel toxins (KTxs) are basic short chain peptides comprising 23-43 amino acid residues that can be cross-linked by 3 or 4 disulfide bridges. KTxs are classified into four large families: α-, β-, γ- and κ-KTx. These peptides display varying selectivity and affinity for K_v channel subtypes. In this work, a novel toxin from the Tityus serrulatus venom was isolated, characterized and submitted to a wide electrophysiological screening on 5 different subtypes of Na_V channels (Na_V1.4; Na_V1.5; Na_V1.6; Na_V1.8 and DmNa_V1) and 12 different subtypes of K_V channels (K_V1.1 - K_V1.6; K_V2.1; K_V3.1; K_V4.2; K_V4.3; Shaker IR and ERG). This novel peptide, named Ts15, has 36 amino acids, is cross-linked by 3 disulfide bridges, has a molecular mass of 3956 Da and pI around 9. Electrophysiological experiments using patch clamp and the two-electrode voltage clamp techniques show that Ts15 preferentially blocks K_V1.2 and K_V1.3 channels with an IC₅₀ value of 196 ± 25 and 508 ± 67 nM, respectively. No effect on Na_V channels was observed, at all tested concentrations. Since Ts15 shows low amino acid identity with other known KTxs, it was considered a bona fide novel type of scorpion toxin. Ts15 is the unique member of the new α-Ktx21 subfamily and therefore was classified as α-Ktx21.1.     

The Dragon’s Paralysing Spell: Evidence of Sodium and Calcium Ion Channel Binding Neurotoxins in Helodermatid and Varanid Lizard Venoms

Bites from helodermatid lizards can cause pain, paresthesia, paralysis, and tachycardia, as well as other symptoms consistent with neurotoxicity. Furthermore, in vitro studies have shown that Heloderma horridum venom inhibits ion flux and blocks the electrical stimulation of skeletal muscles. Helodermatids have long been considered the only venomous lizards, but a large body of robust evidence has demonstrated venom to be a basal trait of Anguimorpha. This clade includes varanid lizards, whose bites have been reported to cause anticoagulation, pain, and occasionally paralysis and tachycardia. Despite the evolutionary novelty of these lizard venoms, their neuromuscular targets have yet to be identified, even for the iconic helodermatid lizards. Therefore, to fill this knowledge gap, the venoms of three Heloderma species (H. exasperatum, H. horridum and H. suspectum) and two Varanus species (V. salvadorii and V. varius) were investigated using Gallus gallus chick biventer cervicis nerve–muscle preparations and biolayer interferometry assays for binding to mammalian ion channels. Incubation with Heloderma venoms caused the reduction in nerve-mediated muscle twitches post initial response of avian skeletal muscle tissue preparation assays suggesting voltage-gated sodium (Na_V) channel binding. Congruent with the flaccid paralysis inducing blockage of electrical stimulation in the skeletal muscle preparations, the biolayer interferometry tests with Heloderma suspectum venom revealed binding to the S3–S4 loop within voltage-sensing domain IV of the skeletal muscle channel subtype, Na_V1.4. Consistent with tachycardia reported in clinical cases, the venom also bound to voltage-sensing domain IV of the cardiac smooth muscle calcium channel, Ca_V1.2. While Varanus varius venom did not have discernable effects in the avian tissue preparation assay at the concentration tested, in the biointerferometry assay both V. varius and V. salvadorii bound to voltage-sensing domain IV of both Na_V1.4 and Ca_V1.2, similar to H. suspectum venom. The ability of varanid venoms to bind to mammalian ion channels but not to the avian tissue preparation suggests prey-selective actions, as did the differential potency within the Heloderma venoms for avian versus mammalian pathophysiological targets. This study thus presents the detailed characterization of Heloderma venom ion channel neurotoxicity and offers the first evidence of varanid lizard venom neurotoxicity. In addition, the data not only provide information useful to understanding the clinical effects produced by envenomations, but also reveal their utility as physiological probes, and underscore the potential utility of neglected venomous lineages in the drug design and development pipeline.     

Sea anemone venom as a source of insecticidal peptides acting on voltage-gated Na+ channels.

Sea anemones produce a myriad of toxic peptides and proteins of which a large group acts on voltage-gated Na+ channels. However, in comparison to other organisms, their venoms and toxins are poorly studied. Most of the known voltage-gated Na+ channel toxins isolated from sea anemone venoms act on neurotoxin receptor site 3 and inhibit the inactivation of these channels. Furthermore, it seems that most of these toxins have a distinct preference for crustaceans. Given the close evolutionary relationship between crustaceans and insects, it is not surprising that sea anemone toxins also profoundly affect insect voltage-gated Na+ channels, which constitutes the scope of this review. For this reason, these peptides can be considered as insecticidal lead compounds in the development of insecticides.