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Background and Purpose Voltage-gated sodium (Na-V) channels are expressed de novo in carcinomas where their activity promotes invasiveness. Breast and colon cancer cells express the neonatal splice variant of Na(V)1.5 (nNa(V)1.5), which has several amino acid substitutions in the domain I voltage-sensor compared with its adult counterpart (aNa(V)1.5). This study aimed to determine whether nNa(V)1.5 channels could be distinguished pharmacologically from aNa(V)1.5 channels. Experimental Approach Cells expressing either nNa(V)1.5 or aNa(V)1.5 channels were exposed to low MW inhibitors, an antibody or natural toxins, and changes in electrophysiological parameters were measured. Stable expression in EBNA cells and transient expression in Xenopus laevis oocytes were used. Currents were recorded by whole-cell patch clamp and two-electrode voltage-clamp, respectively. Key Results Several clinically used blockers of Na-V channels (lidocaine, procaine, phenytoin, mexiletine, ranolazine, and riluzole) could not distinguish between nNa(V)1.5 or aNa(V)1.5 channels. However, two tarantula toxins (HaTx and ProTx-II) and a polyclonal antibody (NESOpAb) preferentially inhibited currents elicited by either nNa(V)1.5 or aNa(V)1.5 channels by binding to the spliced region of the channel. Furthermore, the amino acid residue at position 211 (aspartate in aNa(V)1.5/lysine in nNa(V)1.5), that is, the charge reversal in the spliced region of the channel, played a key role in the selectivity, especially in antibody binding. Conclusion and Implications We conclude that the cancer-related nNa(V)1.5 channel can be distinguished pharmacologically from its nearest neighbour, aNa(V)1.5 channels. Thus, it may be possible to design low MW compounds as antimetastatic drugs for non-toxic therapy of nNa(V)1.5-expressing carcinomas.

Arthropod peptide toxins rich in disulfide bonds are one of the potential sources of bioactive substances. Due to their structure, toxins have increased stability and are able to bind to ion channels, blocking them or changing the gating mechanism. Some spider toxins bind to different types of calcium channels. Calcium ions, in turn, play an important role in many cellular processes, namely, apoptosis. The aim of this paper is to investigate the effect of a number of toxins – arachnid ion-channel blockers in – on intracellular processes associated with the induction of apoptosis in mammalian cells. Materials and Methods. Toxins ω-hexatoxin-Hv1a, ω-theraphotoxin-Hhn2a were used in the study, as they are inhibitors of L- and P/Q-type calcium channels, respectively. Apoptosis was induced using the AC-1001H3 peptide. The authors used fluorescence microscopy to study the effect of toxins on the apoptosis level, oxidative stress, and mitochondrial potential in CHO-K1 cells. Results. The authors observed that incubation of cells with toxins (10 nM) and AC-1001H3 peptide led to increased ROI intracellular concentration, which should have induced apoptotic mechanisms. However, the effect was the opposite. In addition, there was an increase in the mitochondrial potential level. Despite this, the used toxins blocked apoptosis caused by AC-1001H3 and reduced the natural apoptosis level in the CHO-K1 cells. Conclusion. The study demonstrated the antiapoptotic effect of some arthropod peptide toxins. The studied toxins can be used in the treatment of pathologies associated with the activation of apoptotic mechanisms. Keywords: apoptosis, spider toxin, peptide. Пептидные токсины членистоногих, богатые дисульфидными связями, являются одним из потенциальных источников биоактивных веществ. За счет своей структуры токсины обладают повышенной стабильностью и способны связываться с ионными каналами, блокируя их или изменяя механизм стробирования. Ряд токсинов пауков способен связываться с кальциевыми каналами разных типов. Ионы кальция в свою очередь играют важную роль во многих процессах в клетке, одним из которых является апоптоз. Цель работы – исследовать влияние ряда токсинов – блокаторов ионных каналов паукообразных – на внутриклеточные процессы, связанные с индукцией апоптоза в клетках млекопитающих. Материалы и методы. В исследовании использовались токсины ω-hexatoxin-Hv1a, ω-theraphotoxin-Hhn2a, которые являются ингибиторами кальциевых каналов L- и P/Q-типов соответственно. Индукция апоптоза проводилась с использованием пептида AC-1001H3. Изучалось влияние токсинов на уровень апоптоза, оксидативного стресса и митохондриального потенциала в клетках линии CHO-K1 с использованием методов флуоресцентной микроскопии. Результаты. Было установлено, что инкубация клеток с токсинами в концентрации 10 нМ и индуктором апоптоза AC-1001H3 приводила к росту внутриклеточной концентрации активных форм кислорода, что должно индуцировать апоптотические механизмы, однако эффект был противоположным. Кроме того, происходило повышение уровня митохондриального потенциала. Несмотря на это использованные токсины блокировали апоптоз, вызванный AC-1001Н3, и снижали уровень естественного апоптоза в культуре клеток CHO-K1. Выводы. Проведенное исследование продемонстрировало антиапоптотический эффект ряда пептидных токсинов членистоногих. Изученные токсины могут найти применение при лечении патологии, связанной с активацией апоптотических механизмов. Ключевые слова: апоптоз, токсин паука, пептид.

Jingzhaotoxin-I, -III, -IV, -XIII, and −35 (JZTX-I, -III, -IV, -XIII, and −35), gating modifier toxins isolated from the venom of the Chinese tarantula Chilobrachys Jingzhao, were reported to act on cardiac sodium channels and Kv channels. JZTX-I and JZTX-XIII inhibited the hERG channel with the IC50 value of 626.9 nM and 612.6 nM, respectively. JZTX-III, -IV, and −35 share high sequence similarity with JZTX-I and JZTX–XIII, but they showed much lower affinity on the hERG channel compared with JZTX-I and JZTX-XIII. The inhibitory potency of the above five toxins on the hERG channel was not in accordance with their affinity on the Nav1.5 and Kv2.1 channels, indicating that the bioactive surfaces of the five toxins interacting with hERG, Nav1.5 and Kv2.1 are at least in part different. Structure-function analysis of the gating modifier toxins suggested that the functional bioactive surface binding to the hERG channel consists of a conserved hydrophobic patch, surrounding acidic residues (Glu10 in JZTX-XIII, Glu11 in JZTX-I), and basic residues which may be different from residues binding to the Kv2.1 channel.

Nicotinic acetylcholine receptors (nAChRs) are members of the “cys-loop” ligand-gated ion channel superfamily that play important roles in both the peripheral and central system. At the neuromuscular junction, the endplate current is induced by ACh binding and nAChR activation, and then, the current declines to a small steady state, even though ACh is still bound to the receptors. The kinetics of nAChRs with high affinity for ACh but no measurable ion conductance is called desensitization. This adopted desensitization of nAChR channel currents might be an important mechanism for protecting cells against uncontrolled excitation. This study aimed to show that Grammostola spatulata toxin (GsMTx4), which was first purified and characterized from the venom of the tarantula Grammostola spatulata (now genus Phixotricus), can facilitate the desensitization of nAChRs in murine C2C12 myotubes. To examine the details, muscle-type nAChRs, which are expressed heterologously in HEK293T cells, were studied. A single channel current was recorded under the cell-attached configuration, and the channel activity (NPo) decayed much faster after the addition of GsMTx-4 to the pipette solution. The channel kinetics were further analyzed, and GsMTx-4 affected the channel activity of nAChRs by prolonging the closing time without affecting channel conductance or opening activity. The interaction between nAChRs embedded in the lipid membrane and toxin inserted into the membrane may contribute to the conformational change in the receptor and thus change the channel activity. This new property of GsMTx-4 may lead to a better understanding of the desensitization of ligand-gated channels and disease therapy.

Voltage-gated sodium channels are critical for the generation and propagation of action potentials. Gating modifier toxins from spider venom can modulate the gating mechanism of sodium channels and thus have potential as drug leads. Here, we established expression of the gating modifier toxin PaurTx-3, a sodium channel inhibitor found in the venom of the spider Phrixotrichus auratus. Whole-cell voltage-clamp recordings indicated that recombinant PaurTx-3 (rPaurTx-3) inhibited Nav1.4, Nav1.5, and Nav1.7 currents with IC50 values of 61 nM, 72 nM, and 25 nM, respectively. Furthermore, rPaurTx-3 irreversibly inhibited Nav1.7 currents, but had 60–70% recovery in Nav1.4 and Nav1.5 after washing with a bath solution. rPaurTx-3 also hyperpolarized the voltage-dependent steady-state inactivation curve and significantly slowed recovery from fast inactivation of Nav1.7. Current-clamp recordings showed that rPaurTx-3 suppressed small DRG neuron activity. The biological activity assay findings for rPaurTx-3 support its potent pharmacological effect in Nav1.7 and small DRG neurons.

Pain is a significant public health burden in the United States, and current treatment approaches rely heavily on opioids, which often have limited efficacy and can lead to addiction. In humans, functional loss of the voltage-gated sodium channel Nav1.7 leads to pain insensitivity without deficits in the central nervous system. Accordingly, discovery of a selective Nav1.7 antagonist should provide an analgesic without abuse liability and an improved side-effect profile. Huwentoxin-IV, a component of tarantula venom, potently blocks sodium channels and is an attractive scaffold for engineering a Nav1.7-selective molecule. To define the functional impact of alterations in huwentoxin-IV sequence, we produced a library of 373 point mutants and tested them for Nav1.7 and Nav1.2 activity. We then combined favorable individual changes to produce combinatorial mutants that showed further improvements in Nav1.7 potency (E1N, E4D, Y33W, Q34S–Nav1.7 pIC50 = 8.1 ± 0.08) and increased selectivity over other Nav isoforms (E1N, R26K, Q34S, G36I, Nav1.7 pIC50 = 7.2 ± 0.1, Nav1.2 pIC50 = 6.1 ± 0.18, Nav1.3 pIC50 = 6.4 ± 1.0), Nav1.4 is inactive at 3 μm, and Nav1.5 is inactive at 10 μm. We also substituted noncoded amino acids at select positions in huwentoxin-IV. Based on these results, we identify key determinants of huwentoxin's Nav1.7 inhibition and propose a model for huwentoxin-IV's interaction with Nav1.7. These findings uncover fundamental features of huwentoxin involved in Nav1.7 blockade, provide a foundation for additional optimization of this molecule, and offer a basis for the development of a safe and effective analgesic.

Peptides isolated from spider venoms are of pharmacological interest due to their neurotoxic activity, acting on voltage-dependent ion channels present in different types of human body tissues. Three peptide toxins titled as Ap2, Ap3 and Ap5 were purified by RP-HPLC from Acanthoscurria paulensis venom. They were partially sequenced by MALDI In-source Decay method and their sequences were completed and confirmed by transcriptome analysis of the venom gland. The Ap2, Ap3 and Ap5 peptides have, respectively, 42, 41 and 46 amino acid residues, and experimental molecular masses of 4886.3, 4883.7 and 5454.7 Da, with the Ap2 peptide presenting an amidated C-terminus. Amongst the assayed channels – NaV1.1, NaV1.5, NaV1.7, CaV1.2, CaV2.1 and CaV2.2 – Ap2, Ap3 and Ap5 inhibited 20–30 % of CaV2.1 current at 1 μM concentration. Ap3 also inhibited sodium current in NaV1.1, Nav1.5 and Nav1.7 channels by 6.6 ± 1.91 % (p = 0.0276), 4.2 ± 1.09 % (p = 0.0185) and 16.05 ± 2.75 % (p = 0.0282), respectively. Considering that Ap2, Ap3 and Ap5 belong to the 'U'-unknown family of spider toxins, which has few descriptions of biological activity, the present work contributes to the knowledge of these peptides and demonstrates this potential as channel modulators.

The naturally occurred peptide toxins from animal venoms are valuable pharmacological tools in exploring the structure-function relationships of ion channels. Herein we have identified the peptide toxin κ-LhTx-1 from the venom of spider Pandercetes sp (the Lichen huntsman spider) as a novel selective antagonist of the KV4 family potassium channels. κ-LhTx-1 is a gating-modifier toxin impeded KV4 channels’ voltage sensor activation, and mutation analysis has confirmed its binding site on channels’ S3b region. Interestingly, κ-LhTx-1 differently modulated the gating of KV4 channels, as revealed by toxin inhibiting KV4.2/4.3 with much more stronger voltage-dependence than that for KV4.1. We proposed that κ-LhTx-1 trapped the voltage sensor of KV4.1 in a much more stable resting state than that for KV4.2/4.3 and further explored the underlying mechanism. Swapping the non-conserved S3b segments between KV4.1(280FVPK283) and KV4.3(275VMTN278) fully reversed their voltage-dependence phenotypes in inhibition by κ-LhTx-1, and intensive mutation analysis has identified P282 in KV4.1, D281 in KV4.2 and N278 in KV4.3 being the key residues. Furthermore, the last two residues in this segment of each KV4 channel (P282/K283 in KV4.1, T280/D281 in KV4.2 and T277/N278 in KV4.3) likely worked synergistically as revealed by our combinatorial mutations analysis. The present study has clarified the molecular basis in KV4 channels for their different modulations by κ-LhTx-1, which have advanced our understanding on KV4 channels’ structure features. Moreover, κ-LhTx-1 might be useful in developing anti-arrhythmic drugs given its high affinity, high selectivity and unique action mode in interacting with the KV4.2/4.3 channels.

The insecticidal effects of ω-hexatoxin-Hv1a, κ-hexatoxin-Hv1c and ω/κ-hexatoxin-Hv1h are currently attributed to action at calcium and potassium channels. By characterizing the binding of these toxins to neuronal membranes, we show that they have more potent effects as positive allosteric modulators (PAMs) of insect nicotinic acetylcholine receptors (nAChRs), consistent with their neuroexcitatory toxicology. Alanine scanning analysis of ω-hexatoxin-Hv1a reveals a structure-activity relationship for binding that mirrors that for insecticidal activity. Spinosyn A does not compete with ω-hexatoxin-Hv-1a for binding, and we show that these two PAMs have distinct pharmacology of binding indicating that they act at different receptor populations. These toxins represent valuable tools for the characterization of insect nAChRs and for the development of more selective agrochemicals.

Ligand-gated ion channels (LGIC) are membranous proteins that allow ionic flow across plasma membrane upon ligand binding. The channels are implicated in many physiological and pathological process...

Calcium-permeable AMPA-type glutamate receptors (CP-AMPARs) contribute to many forms of synaptic plasticity and pathology. They can be distinguished from GluA2-containing calcium-impermeable AMPARs by the inward rectification of their currents, which reflects voltage-dependent block by intracellular spermine. However, the efficacy of this weakly permeant blocker is differentially altered by the presence of AMPAR auxiliary subunits – including transmembrane AMPAR regulatory proteins, cornichons and GSG1L – that are widely expressed in neurons and glia. This complicates the interpretation of rectification as a measure of CP-AMPAR expression. Here we show that inclusion of the spider toxin analogue 1-naphthylacetyl spermine (NASPM) in the intracellular recording solution results in complete block of GluA1-mediated outward currents irrespective of the type of associated auxiliary subunit. In neurons from GluA2-knockout mice expressing only CP-AMPARs, intracellular NASPM, unlike spermine, blocks all outward synaptic current. Thus, our results identify an unambiguous functional measure, sensitive solely to changes in CP-AMPAR prevalence.

Spider venoms include various peptide toxins that modify the ion currents, mainly of excitable insect cells. Consequently, scientific research on spider venoms has revealed a broad range of peptide toxins with different pharmacological properties, even for mammal species. In this work, thirty animal venoms were screened against hKv1.5, a potential target for atrial fibrillation therapy. The whole venom of the spider Oculicosa supermirabilis, which is also insecticidal to house crickets, caused voltage-gated potassium ion channel modulation in hKv1.5. Therefore, a peptide from the spider O. supermirabilis venom, named Osu1, was identified through HPLC reverse-phase fractionation. Osu1 displayed similar biological properties as the whole venom; so, the primary sequence of Osu1 was elucidated by both of N-terminal degradation and endoproteolytic cleavage. Based on its primary structure, a gene that codifies for Osu1 was constructed de novo from protein to DNA by reverse translation. A recombinant Osu1 was expressed using a pQE30 vector inside the E. coli SHuffle expression system. recombinant Osu1 had voltage-gated potassium ion channel modulation of human hKv1.5, and it was also as insecticidal as the native toxin. Due to its novel primary structure, and hypothesized disulfide pairing motif, Osu1 may represent a new family of spider toxins.

Peptide toxins of arthropods are one of the potential sources of bioactive substances. Toxins are able to bind to calcium channels and block them. Ca2+ ions play an important role in many cell processes, in particular, in apoptosis. In this work, we study the effect of some arthropod toxins on intracellular processes associated with the induction of apoptosis. Synthetic analogs of U5 -scytotoxin-Sth1a, ω-hexatoxin-Hv1a, ω-theraphotoxin-Hhn2a, and μ-agatoxin-Aa1a toxins-inhibitors of calcium L, P, and Q channels and sodium channels were used in the study. Apoptosis was induced by AC-1001 H3 peptide. We study the effect of toxins on the level of apoptosis, ROS, mitochondrial potential, GSH, and ATP in CHO-K1 cells. We show that all the tested toxins are able to dose dependently block the induction of apoptosis triggered by AC-1001 H3 and reduce the level of natural apoptosis in CHO-K1 cells. Cell incubation with apoptosis inducer AC-1001 H3 in the presence and absence of toxins causes an increase in the intracellular concentrations of ROS, ATP, and mitochondrial potential and decreases the GSH concentration. The present study reveals the antiapoptotic effect of a number of arthropod peptide toxins. The toxins studied can represent a novel approach used in the treatment of pathologies associated with the activation of apoptotic mechanisms.

Background: Phoneutria nigriventer spider venom contains several cysteine-rich peptide toxins that act on different ion channels. Despite extensive studies on its venom and description of cDNA sequences of several of its toxin precursors, the gene structure of these toxins remains unknown. Methods: Genomic regions encoding the precursors of three previously characterized P. nigriventer toxins - PnTx1, PnTx2-5 and PnTx4(5-5) - were amplified by PCR using specific primers. PCR fragments were cloned and sequenced. Obtained sequences were compared with their corresponding cDNA sequences. Results: The size of PCR fragments obtained and sequences corresponding to genomic regions encoding for the toxin precursors matched their cDNA sequences. Conclusions: Despite a few nucleotide substitutions in the genomic regions encoding for the toxin precursors when compared with cDNA sequences, the results of the present work indicate that P. nigriventer toxins do not contain introns in their genes sequences.

Peptide toxins that adopt the inhibitory cystine knot (ICK) scaffold have very stable three-dimensional structures as a result of the conformational constraints imposed by the configuration of the three disulfide bonds that are the hallmark of this fold. Understanding the oxidative folding pathways of these complex peptides, many of which are important therapeutic leads, is important in order to devise reliable synthetic routes to correctly folded, biologically active peptides. Previous research on the ICK peptide ProTx-II has shown that in the absence of an equilibrating redox buffer, misfolded intermediates form that prevent the formation of the native disulfide bond configuration. In this paper, we used tandem mass spectrometry to examine these misfolded peptides, and identified two non-native singly bridged peptides, one with a Cys(III)-Cys(IV) linkage and one with a Cys(V)-Cys(VI) linkage. Based on these results, we propose that the C-terminus of ProTx-II has an important role in initiating the folding of this peptide. To test this hypothesis, we have also studied the folding pathways of analogues of ProTx-II containing the disulfide-bond directing group penicillamine (Pen) under the same conditions. We find that placing Pen residues at the C-terminus of the ProTx-II analogues directs the folding pathway away from the singly bridged misfolded intermediates that represent a kinetic trap for the native sequence, and allows a fully oxidized final product to be formed with three disulfide bridges. However, multiple two-disulfide peptides were also produced, indicating that further study is required to fully control the folding pathways of this modified scaffold.

SThe human voltage-gated sodium channel subtype 1.7 (hNaV1.7) is an attractive target for the development of potent and selective novel analgesics. HwTx-IV, a spider derived peptide toxin with 35-residue, inhibits hNaV1.7 with high potency by influencing the kinetics and gating behaviors of the channel, kinetics refers to the control of ion channels on and off by binding to a voltage sensor domains, so it is classified as gating modifier toxins (GMTs). In this study, we study how HwTx-IV and its variant exert its inhibitory potency on hNav1.7 using a range of biophysical techniques including homology modelling, molecular docking, molecular dynamics simulation, and umbrella sampling. The results show that the binding free energy of HwTx-IV and m3-HwTx-IV to hNaV1.7 is -15.00 kJ/mol and -16.2 kJ/mol, respectively, which are consistent with the experiential results；hydrophobic and electrostatic interaction both are important concerns about toxin blocking ion channels：the interactions of m3-HwTx-IV-hNaV1.7 are enhanced by mutating several residues in HwTx-IV. In comparison with the other peptide toxins of NaSpTx-F1, it is found that NaSpTx-F1 also had a similar binding characteristic. Combined above results, it was concluded that K32, W30 and F6, K7 and A8 in N-groove were critical for the interaction strength；G1, L3, G4, I5 in the N terminus and W33, I35 in the C terminus together can determine the peptide binding orientation relative to the channel and ultimately altered the inhibitory effect. This conclusion would be useful for designing high potency peptide inhibitor for hNav1.7.Communicated by Ramaswamy H. Sarma.

Human genetic and pharmacological studies have demonstrated that voltage-gated sodium channels (VGSCs) are promising therapeutic targets for the treatment of pain. Spider venom contains many toxins that modulate the activity of VGSCs. To date, only 0.01% of such spider toxins has been explored, and thus there is a great potential for discovery of novel VGSC modulators as useful pharmacological tools or potential therapeutics. In the current study, we identified a novel peptide, µ-TRTX-Ca1a (Ca1a), in the venom of the tarantula Cyriopagopus albostriatus. This peptide consisted of 38 residues, including 6 cysteines, i.e. IFECSISCEIEKEGNGKKCKPKKCKGGWKCKFNICVKV. In HEK293T or ND7/23 cells expressing mammalian VGSCs, this peptide exhibited the strongest inhibitory activity on Na(v)1.7 (IC(50) 378 nM), followed by Na(v)1.6 (IC(50) 547 nM), Na(v)1.2 (IC(50) 728 nM), Na(v)1.3 (IC(50) 2.2 µM) and Na(v)1.4 (IC(50) 3.2 µM), and produced negligible inhibitory effect on Na(v)1.5, Na(v)1.8, and Na(v)1.9, even at high concentrations of up to 10 µM. Furthermore, this peptide did not significantly affect the activation and inactivation of Na(v)1.7. Using site-directed mutagenesis of Na(v)1.7 and Na(v)1.4, we revealed that its binding site was localized to the DIIS3-S4 linker region involving the D816 and E818 residues. In three different mouse models of pain, pretreatment with Cala (100, 200, 500 µg/kg) dose-dependently suppressed the nociceptive responses induced by formalin, acetic acid or heat. These results suggest that Ca1a is a novel neurotoxin against VGSCs and has a potential to be developed as a novel analgesic.

Spider toxins are molecularly diverse and some display not only a strong antibacterial effect but also exhibit significant inhibition of tumor growth and promote tumor cell apoptosis. The aim of the present investigation was to explore different antitumor effects of the spider peptide toxin lycosin-I through different pathways at different concentrations. It was found that by inactivating STAT3 pathway, high concentrations of lycosin-I induce apoptosis in prostate cancer cells and low concentrations of lycosin-I inhibit the migration of prostate cancer cells. This finding provides favorable evidence for further study of the molecular diversity of spider toxins. Impact statement The spider peptide toxin has become an important research topic. These toxins are molecularly diverse and some display not only a strong antibacterial effect but also exhibit significant inhibition of tumor growth and promote tumor cell apoptosis. Inspired by previous studies, the present study aims to investigate the effects of different concentrations of lycosin-I on the invasiveness and apoptosis of human prostate cancer cells. The findings provide favorable evidence for further study of the molecular diversity of spider toxins.

Significance Voltage-gated ion channels contain domains that have discrete functionalities. The central pore domain allows current flow and provides ion selectivity, whereas peripherally located voltage-sensing domains (VSDs) are needed for voltage-dependent gating. Certain mutations trigger a leak current through VSDs, known as gating pore current. Hypokalemic periodic paralysis (HypoPP) type 2 is caused by mutations in the skeletal muscle voltage-gated sodium channel NaV1.4 that neutralize positive charges in S4 voltage-sensing segments of VSDs. We show that Hm-3 toxin from the crab spider Heriaeus melloteei inhibits gating pore currents through such mutant channels. We propose that Hm-3 and similar toxins may constitute useful hits in developing gating pore current inhibitors and HypoPP therapy., Gating pore currents through the voltage-sensing domains (VSDs) of the skeletal muscle voltage-gated sodium channel NaV1.4 underlie hypokalemic periodic paralysis (HypoPP) type 2. Gating modifier toxins target ion channels by modifying the function of the VSDs. We tested the hypothesis that these toxins could function as blockers of the pathogenic gating pore currents. We report that a crab spider toxin Hm-3 from Heriaeus melloteei can inhibit gating pore currents due to mutations affecting the second arginine residue in the S4 helix of VSD-I that we have found in patients with HypoPP and describe here. NMR studies show that Hm-3 partitions into micelles through a hydrophobic cluster formed by aromatic residues and reveal complex formation with VSD-I through electrostatic and hydrophobic interactions with the S3b helix and the S3–S4 extracellular loop. Our data identify VSD-I as a specific binding site for neurotoxins on sodium channels. Gating modifier toxins may constitute useful hits for the treatment of HypoPP.

Voltage-gated sodium (NaV ) channel gating is a complex phenomenon which involves a distinct contribution of four integral voltage-sensing domains (VSDI, VSDII, VSDIII and VSDIV). Utilizing accrued pharmacological and structural insights, we build on an established chimera approach to introduce animal toxin sensitivity in each VSD of an acceptor channel by transferring in portable S3b-S4 motifs from the four VSDs of a toxin-susceptible donor channel (NaV 1.2). By doing so, we observe that in NaV 1.8, a relatively unexplored channel subtype with distinctly slow gating kinetics, VSDI-III participate in channel opening whereas VSDIV can regulate opening as well as fast inactivation. These results illustrate the effectiveness of a pharmacological approach to investigate the mechanism underlying gating of a mammalian NaV channel complex.

The need for molecules with high specificity against noxious insects leads the search towards spider venoms that have evolved highly selective toxins for insect preys. In this respect, spiders as a highly diversified group of almost exclusive insect predators appear to possess infinite potential for the discovery of novel insect-selective toxins. In 2003, a group of toxins was isolated from the spider Macrothele gigas and the amino acid sequence was reported. We obtained, by molecular biology techniques in a heterologous system, one of these toxins. Purification process was optimized by chromatographic methods to determine the three-dimensional structure by nuclear magnetic resonance in solution, and, finally, their biological activity was tested. rMagi3 resulted to be a specific insect toxin with no effect on mice.

Spider peptide toxins have attracted attention because of their ability to target voltage-gated ion channels, which are involved in several pathologies including chronic pain and some cardiovascular conditions. A class of these peptides acts by modulating the gating mechanism of voltage-gated ion channels and are thus called gating modifier toxins (GMTs). In addition to their interactions with voltage-gated ion channels, some GMTs have affinity for lipid bilayers. This review discusses the potential importance of the cell membrane on the mode of action of GMTs. We propose that peptide-membrane interactions can anchor GMTs at the cell surface, thereby increasing GMT concentration in the vicinity of the channel binding site. We also propose that modulating peptide-membrane interactions might be useful for increasing the therapeutic potential of spider toxins. Furthermore, we explore the advantages and limitations of the methodologies currently used to examine peptide-membrane interactions. Although GMT-lipid membrane binding does not appear to be a requirement for the activity of all GMTs, it is an important feature, and future studies with GMTs should consider the trimolecular peptide-lipid membrane-channel complex. This article is part of the Special Issue entitled 'Venom-derived Peptides as Pharmacological Tools.'

Voltage-gated potassium channels play a key role in human physiology and pathology. Reflecting their importance, numerous channelopathies have been characterised that arise from mutations in these channels or from autoimmune attack on the channels. Voltage-gated potassium channels are also the target of a broad range of peptide toxins from venomous organisms, including sea anemones, scorpions, spiders, snakes and cone snails; many of these peptides bind to the channels with high potency and selectivity. In this review we describe the various classes of peptide toxins that block these channels and illustrate the broad range of three-dimensional structures that support channel blockade. The therapeutic opportunities afforded by these peptides are also highlighted. This article is part of the Special Issue entitled 'Venom-derived Peptides as Pharmacological Tools.'

Background and purpose Naturally occurring dysfunction in NaV channels results in complex disorders such as chronic pain, making these channels an attractive target for new therapies. In the pursuit of novel NaV modulators, we investigated spider venoms for new inhibitors of NaV channels. Experimental Approach We used high-throughput screens to identify a NaV modulator in venom of the spider Davus fasciatus. Further characterization of this venom peptide was undertaken using fluorescent and electrophysiological assays, molecular modeling and a rodent pain model. Key Results We identified a potent NaV inhibitor named μ-TRTX-Df1a. This 34-residue peptide fully inhibited responses mediated by NaV1.7 endogenously expressed in SH-SY5Y cells. Df1a also inhibited CaV3 currents but had no activity against KV2. The modelled structure of Df1a, which contains an inhibitor cystine knot motif, is reminiscent of the NaV channel toxin ProTx-I. Electrophysiology revealed that Df1a inhibits all NaV subtypes tested (hNaV1.1–1.7). Df1a also slowed fast inactivation of NaV1.1, NaV1.3 and NaV1.5, and modified the voltage-dependence of activation and inactivation of most NaV subtypes. Df1a preferentially binds to the domain II voltage-sensor and has additional interactions with the voltage sensors domains III and IV, which likely explains its modulatory features. Df1a was analgesic in vivo, reversing the spontaneous pain behaviors induced by the NaV activator OD1. Conclusion and Implication μ-TRTX-Df1a shows potential as a new molecule for the development of therapies to treat voltage-gated ion channels mediated pain disorders.

We have recently demonstrated that a common phenomenon in evolution of spider venom composition is the emergence of so-called modular toxins consisting of two domains, each corresponding to a “usual” single-domain toxin. In this paper, we describe the structure of two domains that build up a modular toxin named spiderine or OtTx1a from the venom of Oxyopes takobius. Both domains were investigated by solution NMR in water and detergent micelles used to mimic membrane environment. The N-terminal spiderine domain OtTx1a-AMP (41 amino acid residues) contains no cysteines. It is disordered in aqueous solution but in micelles it assumes a stable amphiphilic structure consisting of two α-helices separated by a flexible linker. On the contrary, the C-terminal domain OtTx1a-ICK (59 residues) is a disulfide-rich polypeptide reticulated by five S-S bridges. It presents a stable structure in water and its core is the inhibitor cystine knot (ICK) or knottin motif that is common among single-domain neurotoxins. OtTx1a-ICK structure is the first knottin with five disulfide bridges and it represents a good reference for the whole oxytoxin family. The affinity of both domains to membranes was measured with NMR using titration by liposome suspensions. In agreement with biological tests, OtTx1a-AMP was found to show high membrane affinity explaining its potent antimicrobial properties. This article is protected by copyright. All rights reserved.

Snake and spider venom is a complex mixture that contains proteins, peptides, and small organic and inorganic compounds. In contrast to spider venom, snake venom proteins are well known both functionally and structurally. This work describes methods for purification and crystallization of snake and spider venom toxins and their three-dimensional structure determination by X-ray crystallography.

Voltage-gated sodium (NaV) channels are essential for the normal functioning of cardiovascular, muscular, and nervous systems. These channels have modular organization; the central pore domain allows current flow and provides ion selectivity, whereas four peripherally located voltage-sensing domains (VSDs-I/IV) are needed for voltage-dependent gating. Mutations in the S4 voltage-sensing segments of VSDs in the skeletal muscle channel NaV1.4 trigger leak (gating pore) currents and cause hypokalemic and normokalemic periodic paralyses. Previously, we have shown that the gating modifier toxin Hm-3 from the crab spider Heriaeus melloteei binds to the S3-S4 extracellular loop in VSD-I of NaV1.4 channel and inhibits gating pore currents through the channel with mutations in VSD-I. Here, we report that Hm-3 also inhibits gating pore currents through the same channel with the R675G mutation in VSD-II. To investigate the molecular basis of Hm-3 interaction with VSD-II, we produced the corresponding 554-696 fragment of NaV1.4 in a continuous exchange cell-free expression system based on the Escherichia coli S30 extract. We then performed a combined nuclear magnetic resonance (NMR) and electron paramagnetic resonance spectroscopy study of isolated VSD-II in zwitterionic dodecylphosphocholine/lauryldimethylamine-N-oxide or dodecylphosphocholine micelles. To speed up the assignment of backbone resonances, five selectively 13C,15N-labeled VSD-II samples were produced in accordance with specially calculated combinatorial scheme. This labeling approach provides assignment for ∼50% of the backbone. Obtained NMR and electron paramagnetic resonance data revealed correct secondary structure, quasi-native VSD-II fold, and enhanced ps–ns timescale dynamics in the micelle-solubilized domain. We modeled the structure of the VSD-II/Hm-3 complex by protein–protein docking involving binding surfaces mapped by NMR. Hm-3 binds to VSDs I and II using different modes. In VSD-II, the protruding ß-hairpin of Hm-3 interacts with the S1-S2 extracellular loop, and the complex is stabilized by ionic interactions between the positively charged toxin residue K24 and the negatively charged channel residues E604 or D607. We suggest that Hm-3 binding to these charged groups inhibits voltage sensor transition to the activated state and blocks the depolarization-activated gating pore currents. Our results indicate that spider toxins represent a useful hit for periodic paralyses therapy development and may have multiple structurally different binding sites within one NaV molecule.

The incoming signals from injured sensory neurons upon peripheral inflammation are processed in the dorsal horn of spinal cord, where glial cells accumulate and play a critical role in initiating allodynia (increased pain in response to light-touch). However, how painful stimuli in the periphery engage glial reactivity in the spinal cord remains unclear. Here, we found that a hind paw inflammation induced by CFA produces robust morphological changes in spinal astrocytes and microglia compatible with the reactive phenotype. Strikingly, we discovered that a single intrathecal injection with venom peptides that inhibit calcium channels reversed all the glial pathological features of the peripheral inflammation. These effects were more apparent in rats treated with the Phα1β spider toxin (non-specific calcium channel antagonist) than ω-MVIIA cone snail toxin (selective N-type calcium channel antagonist). These data reveal for the first time a venom peptide acting on glial structural remodeling in vivo. We, therefore, suggest that calcium-dependent plasticity is an essential trigger for glial cells to initiate reactivity, which may represent a new target for the antinociceptive effects of Phα1β and ω-MVIIA toxins in inflammatory pain conditions.

Background: The venom of Phoneutria nigriventer spider is a source of numerous bioactive substances, including some toxins active in insects. An example is PnTx4(5-5) that shows a high insecticidal activity and no apparent toxicity to mice, although it inhibited NMDA-evoked currents in rat hippocampal neurons. In this work the analgesic activity of PnTx4(5-5) (renamed Γ-ctenitoxin-Pn1a) was investigated. Methods: The antinociceptive activity was evaluated using the paw pressure test in rats, after hyperalgesia induction with intraplantar injection of carrageenan or prostaglandin E2 (PGE2). Results: PnTx4(5-5), subcutaneously injected, was able to reduce the hyperalgesia induced by PGE2 in rat paw, demonstrating a systemic effect. PnTx4(5-5) administered in the plantar surface of the paw caused a peripheral and dose-dependent antinociceptive effect on hyperalgesia induced by carrageenan or PGE2. The hyperalgesic effect observed in these two pain models was completely reversed with 5 µg of PnTx4(5-5). Intraplantar administration of L-glutamate induced hyperalgesic effect that was significantly reverted by 5 μg of PnTx4(5-5) injection in rat paw. Conclusion: The antinociceptive effect for PnTx4(5-5) was demonstrated against different rat pain models, i.e. induced by PGE2, carrageenan or glutamate. We suggest that the antinociceptive effect of PnTx4(5-5) may be related to an inhibitory activity on the glutamatergic system.

Gating modifier toxins (GMTs) from spider venom can inhibit voltage gated sodium channels (NaVs) involved in pain signal transmission, including the NaV1.7 subtype. GMTs have a conserved amphipathic structure that allow them to interact with membranes and also with charged residues in regions of NaV that are exposed at the cell surface. ProTx-II and GpTx-1 are GMTs able to inhibit NaV1.7 with high potency, but they differ in their ability to bind to membranes and in their selectivity over other NaV subtypes. To explore these differences and gain detailed information on their membrane-binding ability and how this relates to potency and selectivity, we examined previously described NaV1.7 potent/selective GpTx-1 analogues, and new ProTx-II analogues designed to reduce membrane binding and improve selectivity for NaV1.7. Our studies reveal that the number and type of hydrophobic residues as well as how they are presented at the surface determine the affinity of ProTx-II and GpTx-1 for membranes, and that altering these residues can have dramatic effects on NaV inhibitory activity. We demonstrate that strong peptide-membrane interactions are not essential for inhibiting NaV1.7, and propose that hydrophobic interactions instead play an important role in positioning the GMT at the membrane surface proximal to exposed NaV residues, thereby affecting peptide-channel interactions. Our detailed structure activity relationship study highlights the challenges of designing GMT-based molecules that simultaneously achieve high potency and selectivity for NaV1.7, as single mutations can induce local changes in GMT structure that can have a major impact on NaV-inhibitory activity.

Epilepsy is considered as one of the major disabling neuropathologies. Almost one third of adult patients with temporal lobe epilepsy (TLE) do not respond to current antiepileptic drugs (AEDs). Additionally, most AEDs do not have neuroprotective effects against the inherent neurodegenerative process underlying the hippocampal sclerosis on TLE. Dysfunctions in the GABAergic neurotransmission may contribute not only to the onset of epileptic activity but also constitute an important system for therapeutic approaches. Therefore, molecules that enhance GABA inhibitory effects could open novel avenues for the understanding of epileptic plasticity and for drug development. Parawixin2, a compound isolated from Parawixia bistriata spider venom, inhibits both GABA and glycine uptake and has an anticonvulsant effect against a wide range of chemoconvulsants. The neuroprotective potential of Parawixin2 was analyzed in a model of TLE induced by a long-lasting Status Epilepticus (SE), and its efficiency was compared to well-known neuroprotective drugs, such as riluzole and nipecotic acid. Neuroprotection was assessed through histological markers for cell density (Nissl), astrocytic reactivity (GFAP) and cell death labeling (TUNEL), which were performed 24 h and 72 h after SE. Parawixin2 treatment resulted in neuroprotective effects in a dose dependent manner at 24 h and 72 h after SE, as well as reduced reactive astrocytes and apoptotic cell death. Based on these findings, Parawixin2 has a great potential to be used as a tool for neuroscience research and as a probe to the development of novel GABAergic neuroprotective agents.

Voltage-gated sodium (Na(V)) channels play crucial roles in a range of (patho)physiological processes. Much interest has arisen within the pharmaceutical industry to pursue these channels as analgesic targets following overwhelming evidence that Na(V) channel subtypes Na(V)1.7–Na(V)1.9 are involved in nociception. More recently, Na(V)1.1, Na(V)1.3 and Na(V)1.6 have also been identified to be involved in pain pathways. Venom-derived disulfide-rich peptide toxins, isolated from spiders and cone snails, have been used extensively as probes to investigate these channels and have attracted much interest as drug leads. However, few peptide-based leads have made it as drugs due to unfavourable physiochemical attributes including poor in vivo pharmacokinetics and limited oral bioavailability. The present work aims to bridge the gap in the development pipeline between drug leads and drug candidates by downsizing these larger venom-derived Na(V) inhibitors into smaller, more “drug-like” molecules. Here, we use molecular engineering of small cyclic peptides to aid in the determination of what drives subtype selectivity and molecular interactions of these downsized inhibitors across Na(V) subtypes. We designed a series of small, stable and novel Na(V) probes displaying Na(V) subtype selectivity and potency in vitro coupled with potent in vivo analgesic activity, involving yet to be elucidated analgesic pathways in addition to Na(V) subtype modulation.

Chinese tarantula Ornithoctonus huwena is one of the most venomous spiders distributing in the hilly areas of southern China. In this study, using whole-cell patch-clamp technique we investigated electrophysiological and pharmacological properties of ion channels from tarantula subesophageal ganglion neurons. It was found that the neurons express multiple kinds of ion channels at least including voltage-gated calcium channels, TTX-sensitive sodium channels and two types of potassium channels. They exhibit pharmacological properties similar to mammalian subtypes. Spider calcium channels were sensitive to ω-conotoxin GVIA and diltiazem, two well-known inhibitors of mammalian neuronal high-voltage-activated (HVA) subtypes. 4-Aminopyridine and tetraethylammonium could inhibit spider outward transient and delayed-rectifier potassium channels, respectively. Huwentoxin-I and huwentoxin-IV are two abundant toxic components in the venom of Ornithoctonus huwena. Interestingly, although in our previous work they inhibit HVA calcium channels and TTX-sensitive sodium channels from mammalian sensory neurons, respectively, they fail to affect the subtypes from spider neurons. Moreover, the crude venom has no effect on delayed-rectifier potassium channels and only slightly reduces transient outward potassium channels with an IC50 value of ∼51.3 mg/L. Therefore, our findings provide important evidence for ion channels from spiders having an evolution as self-defense and prey mechanism.

Voltage-gated sodium (Na-v) channels initiate action potentials in most neurons, including primary afferent nerve fibres of the pain pathway. Local anaesthetics block pain through non-specific actions at all Na-v channels, but the discovery of selective modulators would facilitate the analysis of individual subtypes of these channels and their contributions to chemical, mechanical, or thermal pain. Here we identify and characterize spider (Heteroscodra maculata) toxins that selectively activate the Na(v)1.1 subtype, the role of which in nociception and pain has not been elucidated. We use these probes to show that Na(v)1.1-expressing fibres are modality-specific nociceptors: their activation elicits robust pain behaviours without neurogenic inflammation and produces profound hypersensitivity to mechanical, but not thermal, stimuli. In the gut, high-threshold mechanosensitive fibres also express Na(v)1.1 and show enhanced toxin sensitivity in a mouse model of irritable bowel syndrome. Together, these findings establish an unexpected role for Na(v)1.1 channels in regulating the excitability of sensory nerve fibres that mediate mechanical pain.

ω-Agatoxin IV A, a peptidyl toxin from Agelenopsis aperta venom, selectively binds to voltage-gated P/Q-type calcium channels. ω-Agatoxin IV A has been used as a selective tool in pharmacological and electrophysiological studies. Visualization of P/Q-type calcium channels has previously been accomplished using biotin-conjugated ω-Agatoxin IV A in freshly prepared mouse cerebellar and hippocampal slices (Nakanishi et al, J. Neurosci. Res., 41: , 532, 1995). Here biotinylated ω-agatoxin IV A was applied to transcardially fixed brain slices prepared with various fixatives. ω-Agatoxin IV A did not bind to fixed tissues from P/Q-type calcium channel knockout mice, confirming that binding to normal, fixed tissues was not an artifact. Using transmission electron microscopy, locations of biotinylated ω-agatoxin IV A binding sites visualized with gold-conjugated streptavidin showed a similar pattern to those visualized with antibody. The ability of biotinylated ω-agatoxin IV A to bind to fixed tissue provides a new cytochemical technique to study molecular architecture of synapses.

Peptides that bind to ion channels have attracted much interest as potential lead molecules for the development of new drugs and insecticides. However, the structure determination of large peptide-channel complexes using experimental methods is challenging. Thus structural models are often derived from combining experimental information with restraint-driven docking approaches. Using the complex formed by the venom peptide PcTx1 and the acid sensing ion channel (ASIC) 1a as a case study, we have examined the effect of different combinations of restraints and input structures on the statistical likelihood of (a) correctly predicting the structure of the binding interface and (b) the ability to predict which residues are involved in specific pairwise peptide-channel interactions. For this, we have analyzed over 200 000 water-refined docked structures obtained with various amounts and types of restraints of the peptide-channel complex predicted using the docking program HADDOCK. We found that increasing the ...

Summary ArachnoServer is a manually curated database that consolidates information on the sequence, structure, function and pharmacology of spider-venom toxins. Although spider venoms are complex chemical arsenals, the primary constituents are small disulfide-bridged peptides that target neuronal ion channels and receptors. Due to their high potency and selectivity, these peptides have been developed as pharmacological tools, bioinsecticides and drug leads. A new version of ArachnoServer (v3.0) has been developed that includes a bioinformatics pipeline for automated detection and analysis of peptide toxin transcripts in assembled venom-gland transcriptomes. ArachnoServer v3.0 was updated with the latest sequence, structure and functional data, the search-by-mass feature has been enhanced, and toxin cards provide additional information about each mature toxin. Availability and implementation http://arachnoserver.org Supplementary information Supplementary data are available at Bioinformatics online.

Capsaicin, an agonist of TRPV1, evokes intracellular [Ca2+] transients and glutamate release from perfused trigeminal ganglion. The spider toxin PnTx3-5, native or recombinant is more potent than the selective TRPV1 blocker SB-366791 with IC50 of 47 ± 0.18 nM, 45 ± 1.18 nM and 390 ± 5.1 nM in the same experimental conditions. PnTx3-5 is thus more potent than the selective TRPV1 blocker SB-366791. PnTx3-5 (40 nM) and SB-366791 (3 μM) also inhibited the capsaicin-induced increase in intracellular Ca2+ in HEK293 cells transfected with TRPV1 by 75 ± 16% and 84 ± 3.2%, respectively. In HEK293 cells transfected with TRPA1, cinnamaldehyde (30 μM) generated an increase in intracellular Ca2+ that was blocked by the TRPA1 antagonist HC-030031 (10 μM, 89% inhibition), but not by PnTx3-5 (40 nM), indicating selectivity of the toxin for TRPV1. In whole-cell patch-clamp experiments on HEK293 cells transfected with TRPV1, capsaicin (10 μM) generated inward currents that were blocked by SB-366791 and by both native and recombinant PnTx3-5 by 47 ± 1.4%; 54 ± 7.8% and 56 ± 9.0%, respectively. Intradermal injection of capsaicin into the rat left vibrissa induced nociceptive behavior that was blocked by pre-injection with either SB-366791 (3 nmol/site i.d., 83.3 ± 7.2% inhibition) or PnTx3-5 (100 fmol/site, 89 ± 8.4% inhibition). We conclude that both native and recombinant PnTx3-5 are potent TRPV1 receptor antagonists with antinociceptive action on pain behavior evoked by capsaicin.

Pain is a medical condition that interferes with normal human life and work and reduces human well-being worldwide. The voltage-gated sodium channel (VGSC) human NaV1.7 (hNaV1.7) is a compelling target that plays a key role in human pain signaling. The 33-residue peptide &micro, TRTX-Hhn2b (HNTX-I), a member of NaV-targeting spider toxin (NaSpTx) family 1, has shown negligible activity on mammalian VGSCs, including the hNaV1.7 channel. We engineered analogues of HNTX-I based on sequence conservation in NaSpTx family 1. Substitution of Asn for Ser at position 23 or Asp for His at position 26 conferred potent activity against hNaV1.7. Moreover, multiple site mutations combined together afforded improvements in potency. Ultimately, we generated an analogue E1G&ndash, N23S&ndash, D26H&ndash, L32W with &gt, 300-fold improved potency compared with wild-type HNTX-1 on hNaV1.7 (IC50 0.036 &plusmn, 0.007 &micro, M). Structural simulation suggested that the charged surface and the hydrophobic surface of the modified peptide are responsible for binding affinity to the hNaV1.7 channel, while variable residues may determine pharmacological specificity. Therefore, this study provides a profile for drug design targeting the hNaV1.7 channel.

Spider venoms are complex mixtures of biologically active components with potentially interesting applications for drug discovery or for agricultural purposes. The spider Phoneutria nigriventer is responsible for a number of envenomations with sometimes severe clinical manifestations in humans. A more efficient treatment requires a comprehensive knowledge of the venom composition and of the action mechanism of the constituting components. PnTx2-1 (also called &delta, ctenitoxin-Pn1a) is a 53-amino-acid-residue peptide isolated from the venom fraction PhTx2. Although PnTx2-1 is classified as a neurotoxin, its molecular target has remained unknown. This study describes the electrophysiological characterization of PnTx2-1 as a modulator of voltage-gated sodium channels. PnTx2-1 is investigated for its activity on seven mammalian NaV-channel isoforms, one insect NaV channel and one arachnid NaV channel. Furthermore, comparison of the activity of both PnTx2-1 and PnTx2-6 on NaV1.5 channels reveals that this family of Phoneutria toxins modulates the cardiac NaV channel in a bifunctional manner, resulting in an alteration of the inactivation process and a reduction of the sodium peak current.

The synthetic peptide PnPP-19 comprehends 19 amino acid residues and it represents part of the primary structure of the toxin δ-CNTX-Pn1c (PnTx2-6), isolated from the venom of the spider Phoneutria nigriventer. Behavioural tests suggest that PnPP-19 induces antinociception by activation of CB1, μ and δ opioid receptors. Since the peripheral and central antinociception induced by PnPP-19 involves opioid activation, the aim of this work was to identify whether this synthetic peptide could directly activate opioid receptors and investigate the subtype selectivity for μ-, δ- and/or κ-opioid receptors. Furthermore, we also studied the modulation of calcium influx driven by PnPP-19 in dorsal root ganglion neurons, and analyzed whether this modulation was opioid-mediated. PnPP-19 selectively activates μ-opioid receptors inducing indirectly inhibition of calcium channels and hereby impairing calcium influx in dorsal root ganglion (DRG) neurons. Interestingly, notwithstanding the activation of opioid receptors, PnPP-19 does not induce β-arrestin2 recruitment. PnPP-19 is the first spider toxin derivative that, among opioid receptors, selectively activates μ-opioid receptors. The lack of β-arrestin2 recruitment highlights its potential for the design of new improved opioid agonists. ispartof: TOXINS vol:10 issue:1 ispartof: location:Switzerland status: published

The transport of ions across biological membranes, via ion channels, is a critical event that regulates signal transduction, ionic homeostasis, and other cellular physiological and biochemical processes like the osmotic health of cells, providing nutritional requirements, and maintaining electrogenic gradients. Ion channels are integral pore-forming membrane proteins that are activated by changes in membrane voltage or by binding of specific ligands. Specific binding sites on ion channels are also modulatory targets for a variety of toxic compounds, which affect ion channel function resulting in deleterious toxicological effects. Several toxins that target one specific subtype of ion channels have been isolated from a variety of plants, invertebrates, and vertebrates. Voltage-gated sodium channels are ubiquitously expressed in several cell types. The classification of toxins is based on toxin-specific binding sites. Sodium channel physiology and toxicology have been extensively researched, and the major types of toxins that modulate these channels include tetrodotoxin, saxitoxin, scorpion toxins, spider toxins, sea anemone toxins, and pyrethroids. The potassium channels show great variation in terms of their genetic diversity and channel properties. The major toxins that modulate potassium channels include scorpion toxins and dendrotoxins. The voltage-gated calcium channels are classified based upon their voltage threshold for activation and channel properties and are affected by toxins such as ω-conotoxins, ω-agatoxins, huwenotoxins, and glycerotoxin. Chloride channels are inhibited by Georgia anion toxin and are different from the ligand-gated γ-aminobutyric acid (GABA) and glycine receptors. GABA receptors are well characterized in terms of toxicology and are prominent targets of certain insecticides. The ionotropic glutamate receptors are one class of ligand-gated receptors that are affected by heavy metal ions and toxins, both synthetic and natural. The other major types of receptors include acetylcholine receptors and serotonin receptors; the former are targets of peptide toxins from venoms and the latter are targets of abused drugs of plant origin. The ion channel toxins, especially the ones with high binding affinities, have served as valuable tools for elucidating the structure and function of various ion channels. In addition, the knowledge of ion channel modulations by toxins serves as a basis for the future development of effective therapies. This chapter provides an updated systematic review of ion channel toxicology, particularly of the voltage-gated ion channels.

Background: The great diversity of molecules found in spider venoms include amino acids, polyamines, proteins and peptides, among others. Some of these compounds can interact with different neuronal receptors and ion channels including those present in the ocular system. To study potential toxicity and safety of intravitreal injection in rabbits of LyeTx I b, a synthetic peptide derived from the toxin LyeTx I found in venom from the spider Lycosa eritrognatha and to evaluate the angiogenic activity on a CAM model. Methods: ARPE-19 cells were treated with LyeTx I b (0.36; 0.54; 0.72; 2.89; 4.34 or 9.06 μM). In this study, New Zealand rabbits were used. LyeTx I b (2.89 μM) labeled with FITC dissolved in PBS, or only PBS, were injected into vitreous humor. Electroretinogram (ERG) was recorded 1 day before injection and at 7,14 and 28 days post-injection. Clinical examination of the retina was conducted through tonometer and eye fundus after ERG. Eyes were enucleated and retinas were prepared for histology in order to assess retinal structure. CAMs were exposed to LyeTx I b (0.54; 0.72; 2.17 or 2.89 μM). Results: ARPE-19 cells exposed to LyeTx I b showed cell viability at the same levels of the control. The fluorescence of LyeTx I b labeled with FITC indicated its retinal localization. Our findings indicate ERG responses from rats injected in the eye with LyeTx I b were very similar to the corresponding responses of those animals injected only with vehicle. Clinical examination found no alterations of intraocular pressure or retinal integrity. No histological damage in retinal layers was observed. CAM presented reduced neovascularization when exposed to LyeTx I b. Conclusions: Intravitreal injection of LyeTx I b is safe for use in the rabbit eye and prevents neovascularization in the CAM model, at Bevacizumab levels. These findings support intravitreal LyeTx l b as a good candidate to develop future alternative treatment for the retina in neovascularization diseases.

The inhibitor cystine knot (ICK) is an unusual three-disulfide architecture in which one of the disulfide bonds bisects a loop formed by the two other disulfide bridges and the intervening sections of the protein backbone. Peptides containing an ICK motif are frequently considered to have high levels of thermal, chemical and enzymatic stability due to cross-bracing provided by the disulfide bonds. Experimental studies supporting this contention are rare, in particular for spider-venom toxins, which represent the largest diversity of ICK peptides. We used ω-hexatoxin-Hv1a (Hv1a), an insecticidal toxin from the deadly Australian funnel-web spider, as a model system to examine the contribution of the cystine knot to the stability of ICK peptides. We show that Hv1a is highly stable when subjected to temperatures up to 75 °C, pH values as low as 1, and various organic solvents. Moreover, Hv1a was highly resistant to digestion by proteinase K and when incubated in insect hemolymph and human plasma. We demonstrate that the ICK motif is essential for the remarkable stability of Hv1a, with the peptide’s stability being dramatically reduced when the disulfide bonds are eliminated. Thus, this study demonstrates that the ICK motif significantly enhances the chemical and thermal stability of spider-venom peptides and provides them with a high level of protease resistance. This study also provides guidance to the conditions under which Hv1a could be stored and deployed as a bioinsecticide.

A recently introduced new SPS resin, possessing a 2-(ortho-nitrophenyl)ethanal linker, was used for the regioselective on-resin synthesis of N-mono-hydroxylated and N-mono-methylated polyamine spider toxins of Agelenopsis aperta and Larinioides folium. The polyamine backbones of the target compounds were efficiently constructed from the center by reductive amination of the aldehyde linker, followed by stepwise alkylation and acylation on solid support. Depending on the cleavage conditions, employing either oxidation/Cope elimination or methylation/Hofmann elimination, regioselectively the respective N-hydroxyl or N-methyl products were obtained. Employing this methodology, a number of acylpolyamine spider toxins were synthesized and identified as venom components by UHPLC and ESI-MS/MS.

Specific peptide toxins interact with voltage-gated sodium channels by regulating the activation or inactivation of targeted channels. However, few toxins possessing dual effects have been identified. In the present study, we showed that jingzhaotoxin-XI/κ-theraphotoxin-Cj1a (JZTX-XI), a 34-residue peptide from the venom of the Chinese spider Chilobrachys jingzhao, inhibits the sodium conductance (IC50 = 124 ± 26 nM) and slows the fast inactivation (EC50 = 1.18 ± 0.2 μM) of Nav1.5 expressed in Chinese hamster ovary (CHO-K1) cells. JZTX-XI significantly shifted the activation to more depolarized voltages and decreased the deactivation of Nav1.5 currents upon extreme depolarization, but only slightly affected voltage-dependence of steady-state inactivation. In addition, JZTX-XI caused an approximately five-fold decrease in the rate of recovery from inactivation and an approximately 1.9-fold reduction in the closed-state inactivation rate. Our data suggest that JZTX-XI integrates the functions of site 3 toxins (α-scorpion toxins) with site 4 toxins (β-scorpion and spider toxins) by targeting multiple sites on Nav1.5. The unique properties displayed by JZTX-XI in its inhibitory activity on Nav1.5 suggest that its mechanism of action is distinct from those of site 3 and site 4 toxins, making JZTX-XI a useful probe for investigating the gating mechanism of Nav1.5 and toxin-channel interactions.

Spider venoms constitute incredibly diverse libraries of compounds, many of which are involved in prey capture and defence. Polyamines are often prevalent in the venom and target ionotropic glutamate receptors. Here we show that a novel spider polyamine, PA366, containing a hydroxyphenyl-based structure is present in the venom of several species of tarantula, and has selective toxicity against MCF-7 breast cancer cells. By contrast, a polyamine from an Australian funnel-web spider venom, which contains an identical polyamine tail to PA366 but an indole-based head-group, is only cytotoxic at high concentrations. Our results suggest that the ring structure plays a role in the cytotoxicity and that modification to the polyamine head group might lead to more potent and selective compounds with potential as novel cancer treatments.

Beyond providing evolutionary advantages, venoms offer unique research tools, as they were developed to target functionally important proteins and pathways. As a key pain receptor in the nociceptive pathway, transient receptor potential vanilloid 1 (TRPV1) of the TRP superfamily has been shown to be a target for several toxins, as a way of producing pain to deter predators. Importantly, TRPV1 is involved in thermoregulation, inflammation, and acute nociception. As such, toxins provide tools to understand TRPV1 activation and modulation, a critical step in advancing pain research and the development of novel analgesics. Indeed, the phytotoxin capsaicin, which is the spicy chemical in chili peppers, was invaluable in the original cloning and characterization of TRPV1. The unique properties of each subsequently characterized toxin have continued to advance our understanding of functional, structural, and biophysical characteristics of TRPV1. By building on previous reviews, this work aims to provide a comprehensive summary of the advancements made in TRPV1 research in recent years by employing animal toxins, in particular DkTx, RhTx, BmP01, Echis coloratus toxins, APHCs and HCRG21. We examine each toxin’s functional aspects, behavioral effects, and structural features, all of which have contributed to our current knowledge of TRPV1. We additionally discuss the key features of TRPV1’s outer pore domain, which proves to be the target of the currently discussed toxins.