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The hypothesis that glutamate is the afferent transmitter in the acoustico-lateralis receptors was examined in Plotosus electroreceptors. JSTX, a spider toxin known to specifically block glutamate receptors, irreversibly abolished afferent impulse discharges induced by iontophoretically applied glutamate, whereas those induced synaptically by focal stimulation of receptor cells were little affected. Such differential blocking effects by JSTX, complementary to other biochemical data, further provide pharmacological evidence against the glutamate hypothesis.

M. Yoshioka , N. Narai , H. Pan-Hou , K. Shimazaki , A. Miwa and N. Kawai . Color development upon reaction of ferric ion with the toxin JSTX, a glutamate receptor blocker present in the venom gland of the spider Nephila clavata (Joro spider). Toxicon26, 414 – 416, 1988. — A spider toxin, JSTX, derived from Nephila clavata, which blocks glutamate receptor was found to react with Fe3+. Mechanism of the coloration may be chelate formation since the green color completely faded upon the addition of EDTA. The colored JSTX significantly lost its neurophysiological activity. This unique coloration may be useful for not only detecting specific blockers of the glutamate receptor in spider venom but also for characterizing the glutamate receptor.

Effects of a spider toxin (JSTX) — a specific blocker of glutamate receptors — on single pyramidal neurons of the hippocampus were studied using tissue slices in vitro. JSTX blocked the synaptic response in CA1 pyramidal cells evoked by Schaffer collateral stimulation without affecting the antidromic spike potential. The toxin suppressed glutamate-induced cell firings whereas it had little effect on aspartate-induced responses. The results suggest that glutamate is a neurotransmitter of the Schaffer collateral input to CA1 pyramidal neurons.

The effect of a toxin (JSTX) obtained from Nephila clavata (Joro spider) on the CA1 pyramidal neurons of the hippocampus was studied using slice preparations. JSTX blocked the excitatory postsynaptic potentials (EPSPs) in the pyramidal neuron evoked by Schaffer collateral stimulation but was without effect on the antidromic action potentials or on the resting conductance. Depolarization induced by ionophoretic application of glutamate was readily suppressed by JSTX but aspartate-induced depolarization was much less sensitive to the toxin. Among preferential agonists activating 3 receptor subtypes for excitatory amino acids, quisqualate responses were most effectively suppressed by JSTX. Kainate responses were similarly suppressed but in some cells higher concentration of the toxin was needed to block the responses. N-methyl- D -aspartate (NMDA) responses were the least sensitive to JSTX but they were suppressed by ± 2-amino-5-phonovaleric acid (APV). Long term potentiation (LTP) once it had taken place was not completely inhibited by APV. In the presence of JSTX, however, LTP was blocked and tetanic stimuli produced only a short-lived potentiation. In Mg2+ free solution, an orthodromic stimulation evoked repetitive spike responses which were superimposed on the depolarization following the initial spike. APV suppressed the depolarization and associated spikes leaving an orthodromic response which was sensitive to JSTX. The results suggest that JSTX blocks EPSPs in CA1 pyramidal neurons which are mediated by non-NMDA type receptors.

Joro spider toxin (JSTX), a specific blocker of glutamate receptors, was found to exert a prominent suppressive action on the Na+-dependent binding of L-glutamate to synaptic membranes and on glutamate uptake by synaptosomes in a dose-dependent manner. In contrast, the synthesized 2,4-dihydroxyphenylacetylasparagine (2,4-DHPA-ASN), a common moiety of spider toxins, which has been shown to exhibit almost the same activity as intact JSTX with respect to the inhibition of Na+-independent glutamate binding to its synaptic membrane receptors, shows lower potency in inhibiting Na+-dependent binding and uptake of L-glutamate. From these findings, it is clear that JSTX has the ability to inhibit not only L-glutamate binding to its synaptic membrane receptors but also L-glutamate uptake by synaptosomes, and that polyamines linked to 2,4-DHPA-ASN in the molecule of spider toxins may participate in the inhibition of L-glutamate uptake.

The blocking action ofNephila clavata spider neurotoxin, or JSTX, on ionic currents activated by L-glutamate and its agonists when applied to the membrane of neurons isolated from the rat hippocampus was investigated using a concentration clamp technique. Crude JSTX venom was found to block L-glutamate-, quisqualate, and kainate-activated ionic currents induced by activating non-N-methyl-D-aspartate (non-NMDA) membrane receptors. Following the effects of JSTX, ionic currents activated by L-glutamate and its agonists declined to 34–36% of their initial value with no recovery during JSTX washout. An active fraction of JSTX at concentrations of 10−4–10−5 produced almost total but partially reversible blockade of ionic currents. The action of JSTX became less effective during depolarization. The concentration dependence of JSTX-induced blockade of kainate-activated ionic currents was investigated and the velocity constants of interaction between the toxin and glutamate receptors obtained. It is postulated that JSTX interacts with chemically-operated non-NMDA ionic channels, blocking their transition into a number of their possible open states.

Recombinant fusion protein technology allows specific insecticidal protein and peptide toxins to display activity in orally-delivered biopesticides. The spider venom peptide δ-amaurobitoxin-PI1a, which targets insect voltage-gated sodium channels, was fused to the “carrier” snowdrop lectin (GNA) to confer oral toxicity. The toxin itself (PI1a) and an amaurobitoxin/GNA fusion protein (PI1a/GNA) were produced using the yeast Pichia pastoris as expression host. Although both proteins caused mortality when injected into cabbage moth (Mamestra brassicae) larvae, the PI1a/GNA fusion was approximately 6 times as effective as recombinant PI1a on a molar basis. PI1a alone was not orally active against cabbage moth larvae, but a single 30 μg dose of the PI1a/GNA fusion protein caused 100% larval mortality within 6 days when fed to 3rd instar larvae, and caused significant reductions in survival, growth and feeding in 4th – 6th instar larvae. Transport of fusion protein from gut contents to the haemolymph of cabbage moth larvae, and binding to the nerve chord, was shown by Western blotting. The PI1a/GNA fusion protein also caused mortality when delivered orally to dipteran (Musca domestica; housefly) and hemipteran (Acyrthosiphon pisum; pea aphid) insects, making it a promising candidate for development as a biopesticide.

N8-Coumaroyl spermidine (N8-CS) is an example of hydroxycinnamic acid - polyamine conjugates found in certain plants. Because of its structural similarity to some spider toxins, N8-CS was tested for its ability to block arthropod neuromuscular synapses. It inhibited chemical synaptic transmission at crayfish (Procambarus clarkii) neuromuscular junctions, the IC50 being approximately 200 µM. Its effect was at least partially reversed by washing with physiological saline. Joro spider toxin, a structurally similar compound, also blocked crayfish neuromuscular synapses but its effect was irreversible. These results suggest that plant-derived cinnamoyl spermidines might have paralytic properties similar to those of spider toxins.

Although necessary for human survival, pain may sometimes become pathologic if long-lasting and associated with alterations in its signaling pathway. Opioid painkillers are officially used to treat moderate to severe, and even mild, pain. However, the consequent strong and not so rare complications that occur, including addiction and overdose, combined with pain management costs, remain an important societal and economic concern. In this context, animal venom toxins represent an original source of antinociceptive peptides that mainly target ion channels (such as ASICs as well as TRP, CaV, KV and NaV channels) involved in pain transmission. The present review aims to highlight the NaV1.7 channel subtype as an antinociceptive target for spider toxins in adult dorsal root ganglia neurons. It will detail (i) the characteristics of these primary sensory neurons, the first ones in contact with pain stimulus and conveying the nociceptive message, (ii) the electrophysiological properties of the different NaV channel subtypes expressed in these neurons, with a particular attention on the NaV1.7 subtype, an antinociceptive target of choice that has been validated by human genetic evidence, and (iii) the features of spider venom toxins, shaped of inhibitory cysteine knot motif, that present high affinity for the NaV1.7 subtype associated with evidenced analgesic efficacy in animal models.

Phlotoxin-1 (PhlTx1) is a peptide previously identified in tarantula venom (Phlogius species) that belongs to the inhibitory cysteine-knot (ICK) toxin family. Like many ICK-based spider toxins, the synthesis of PhlTx1 appears particularly challenging, mostly for obtaining appropriate folding and concomitant suitable disulfide bridge formation. Herein, we describe a procedure for the chemical synthesis and the directed sequential disulfide bridge formation of PhlTx1 that allows for a straightforward production of this challenging peptide. We also performed extensive functional testing of PhlTx1 on 31 ion channel types and identified the voltage-gated sodium (Nav) channel Nav1.7 as the main target of this toxin. Moreover, we compared PhlTx1 activity to 10 other spider toxin activities on an automated patch-clamp system with Chinese Hamster Ovary (CHO) cells expressing human Nav1.7. Performing these analyses in reproducible conditions allowed for classification according to the potency of the best natural Nav1.7 peptide blockers. Finally, subsequent in vivo testing revealed that intrathecal injection of PhlTx1 reduces the response of mice to formalin in both the acute pain and inflammation phase without signs of neurotoxicity. PhlTx1 is thus an interesting toxin to investigate Nav1.7 involvement in cellular excitability and pain.

The suitability of a newly developed cell-based functional assay was tested for the detection of the activity of a range of neurotoxins and neuroactive pharmaceuticals which act by stimulation or inhibition of calcium-dependent neurotransmitter release. In this functional assay, a reporter enzyme is released concomitantly with the neurotransmitter from neurosecretory vesicles. The current study showed that the release of a luciferase from a differentiated human neuroblastoma-based reporter cell line (SIMA-hPOMC1-26-GLuc cells) can be stimulated by a carbachol-mediated activation of the Gq-coupled muscarinic-acetylcholine receptor and by the Ca2+-channel forming spider toxin α-latrotoxin. Carbachol-stimulated luciferase release was completely inhibited by the muscarinic acetylcholine receptor antagonist atropine and α-latrotoxin-mediated release by the Ca2+-chelator EGTA, demonstrating the specificity of luciferase-release stimulation. SIMA-hPOMC1-26-GLuc cells express mainly L- and N-type and to a lesser extent T-type VGCC on the mRNA and protein level. In accordance with the expression profile a depolarization-stimulated luciferase release by a high K+-buffer was effectively and dose-dependently inhibited by L-type VGCC inhibitors and to a lesser extent by N-type and T-type inhibitors. P/Q- and R-type inhibitors did not affect the K+-stimulated luciferase release. In summary, the newly established cell-based assay may represent a versatile tool to analyze the biological efficiency of a range of neurotoxins and neuroactive pharmaceuticals which mediate their activity by the modulation of calcium-dependent neurotransmitter release.

In contrast to the reported effects of polyamines on the high affinity neurotransmitter uptake, two polyamine-like spider toxins significantly increase the high affinity uptake of glutamate as demonstrated with high resolution autoradiography. The effects of both spider toxins were compared to those of a polyamine toxin from the wasp Philanthus triangulum, which is known to inhibit the high affinity glutamate uptake.

A new spider toxin NSTX-3 was chemically synthesized for confirmation of the proposed structure and use in the investigation of neuroscience.

The NMDA-type of ionotropic glutamate receptors (iGluRs) are involved in excitatory transmission in the mammalian brain and in a range of neurological and psychiatric diseases for which NMDA receptors are considered potential therapeutic drug targets. The majority of NMDA receptor subtypes are comprised of two glycine-binding GluN1 subunits and two glutamate-binding GluN2 subunits (NR2A-D); leading to the existence of four major receptor subtypes with distinct expression patterns and functional properties: GluN1/2A, GluN1/2B, GluN1/2C, and GluN1/2D. While the GluN1/2B subtype can be selectively inhibited by the highly subtype-selective non-competitive antagonist ifenprodile, the lack of inhibitors with similar subtype selectivity for the GluN1/2A, GluN1/2C, and GluN1/2D receptors is a limitation in studies exploring the role of these subtypes in many aspects of brain function and disease.Polyamine toxins isolated from the venom of spiders are open-channel blockers of ion channels, in particular iGluRs. Argiotoxin-636 (ArgTX-636) isolated from the venom of the orb weaver spider Argiope lobata consists of an aromatic amino acid head-group coupled to a polyamine tail. ArgTX-636 is a potent inhibitor of mammalian iGluRs; presumably by binding to the ion channel region in a use- and voltage-dependent manner that prevents ion conduction. We have recently shown1 that modifications within the polyamine tail of ArgTX-636 can control selectivity between the NMDA- and the AMPA-type of iGluRs (1). Further exploration of the analog lead-structures have recently developed a series of analogs of the spider toxin Argiotoxin-636 (ArgTX-636), which display robust selectivity towards GluN1/2A and GluN1/2B over GluN1/2C, and GluN1/2D subtypes of NMDA receptors. In this study we have explorer the molecular determinants for subtype-selectivity of the novel analogs ArgTX-48 and ArgTX-75 within the NMDA receptor ion channel using electrophysiological characterization of chimeric and mutant GluN1/2A and GluN1/2D NMDA receptors.

Background Venomous animals incapacitate their prey using complex venoms that can contain hundreds of unique protein toxins. The realisation that many of these toxins may have pharmaceutical and insecticidal potential due to their remarkable potency and selectivity against target receptors has led to an explosion in the number of new toxins being discovered and characterised. From an evolutionary perspective, spiders are the most successful venomous animals and they maintain by far the largest pool of toxic peptides. However, at present, there are no databases dedicated to spider toxins and hence it is difficult to realise their full potential as drugs, insecticides, and pharmacological probes. Description We have developed ArachnoServer, a manually curated database that provides detailed information about proteinaceous toxins from spiders. Key features of ArachnoServer include a new molecular target ontology designed especially for venom toxins, the most up-to-date taxonomic information available, and a powerful advanced search interface. Toxin information can be browsed through dynamic trees, and each toxin has a dedicated page summarising all available information about its sequence, structure, and biological activity. ArachnoServer currently manages 567 protein sequences, 334 nucleic acid sequences, and 51 protein structures. Conclusion ArachnoServer provides a single source of high-quality information about proteinaceous spider toxins that will be an invaluable resource for pharmacologists, neuroscientists, toxinologists, medicinal chemists, ion channel scientists, clinicians, and structural biologists. ArachnoServer is available online at http://www.arachnoserver.org.