Channel Toxins Research Articles

LCTX-F2, a Novel Potentiator of Coagulation Factors From the Spider Venom of Lycosa singoriensis.

Spider venoms contain many functional proteins/peptides such as proteinases, serine/cysteine proteinase inhibitors, insecticidal toxins, and ion channel toxins. However, to date, no peptide toxin with procoagulant activities has been identified from spider venom. In this study, a novel toxin LCTX-F2 with coagulation-promoting activity was identified and characterized in the venom of the spider Lycosa singoriensis (L. singoriensis). LCTX-F2 significantly shortened activated partial thromboplastin time (APTT), clotting time, and plasma recalcification time. This toxin directly interacted with several coagulation factors such as FXIIa, kallikrein, thrombin, and FXa and increased their protease activities. In liver bleeding and tail bleeding mouse models, LCTX-F2 significantly decreased the number of blood cells and bleeding time in a dose-dependent manner. At the same dosage, LCTX-F2 exhibited a more significant procoagulant effect than epsilon aminocaproic acid (EACA). Moreover, LCTX-F2 showed no cytotoxic or hemolytic activity against either normal cells or red blood cells. Our results suggested that LCTX-F2 is a potentiator of coagulation factors with the potential for use in the development of procoagulant drugs.

Cloning and expression of OdTx12, a β excitatory toxin from Odontobuthus doriae, in Escherichia coli and evaluation of its bioactivity in Locusta migratoria

The venom of Odontobuthus doriae contains several peptide toxins that interfere with the sodium channel function of cell membranes, some of which specifically act on the insect's sodium channel without affecting mammalian cells. In this study sodium channel toxins of Odontobuthus doriae were aligned to other closely related toxins by BLAST and ClustalW servers. Among these toxins, NaTx12 (OdTx12) showed more than 90% similarity to the most known beta excitatory toxin, AaHIT1; furthermore, our modeling studies confirmed high tertiary structure similarity of these proteins. OdTx12 was cloned and expressed in E.coli, using pET26-b and pET28-a expression vectors. Tris-tricine SDS-PAGE and Western blot analysis showed OdTx12 expression by pET28-a, only. After purification, bioactivity of the purified protein was analyzed by injection and oral administration to Locusta migratoria larvae, and toxicity to mammals was tested on mice. Injection of OdTx12 resulted in the killing of larvae with LD50 of 0.4 and 0.2 after 48 and 72 h respectively, but oral administration of OdTx12 had no significant effect on Locusta migratoria, nor did the injection to mice show any signs of toxicity. These results showed that OdTx12, as a novel β excitatory toxin can be considered as a candidate for insect control purposes.

Correlation of Venom Toxinome Composition of Indian Red Scorpion (Mesobuthus tamulus) with Clinical Manifestations of Scorpion Stings: Failure of Commercial Antivenom to Immune-Recognize the Abundance of Low Molecular Mass Toxins of This Venom.

The Indian red scorpion (Mesobuthus tamulus), with its life-threatening sting, is the world's most dangerous species of scorpion. The toxinome composition of M. tamulus venom was determined by tandem mass spectrometry (MS) analysis of venom protein bands separated by SDS-PAGE. A total of 110 venom toxins were identified from searching the MS data against the Buthidae family (taxid: 6855) of toxin entries in nonredundant protein databases. The Na+ and K+ ion channel toxins taken together are the most abundant toxins (76.7%) giving rise to the neurotoxic nature of this venom. The other minor toxin classes in the M. tamulus venom proteome are serine protease-like protein (2.9%), serine protease inhibitor (2.2%), antimicrobial peptide (2.3%), hyaluronidase (2.2%), makatoxin (2.1%), lipolysis potentiating peptides (1.2%), neurotoxin affecting Cl- channel (1%), parabutoporin (0.6%), Ca2+ channel toxins (0.8%), bradykinin potentiating peptides (0.2%), HMG CoA reductase inhibitor (0.1%), and other toxins with unknown pharmacological activity (7.7%). Several of these toxins have been shown to be promising drug candidates. M. tamulus venom does not show enzymatic activity (phospholipase A2, l-amino acid oxidase, adenosine tri-, di-, and monophosphatase, hyaluronidase, metalloproteinase, and fibrinogenolytic), in vitro hemolytic activity, interference with blood coagulation, or platelet modulation properties. The clinical manifestations post M. tamulus sting have been described in the literature and are well correlated with its venom proteome composition. An abundance of low molecular mass toxins (3-15 kDa) are responsible for exerting the major pharmacological effects of M. tamulus venom, though they are poorly immune-recognized by commercial scorpion antivenom. This is a major concern for the development of effective antivenom therapy against scorpion stings.

Transcriptomic and proteomic analyses of the venom and venom glands of Centruroides hirsutipalpus, a dangerous scorpion from Mexico

Centruroides hirsutipalpus (Scorpiones: Buthidae) is related to the “striped scorpion” group inhabiting the western Pacific region of Mexico. Human accidents caused by this species are medically important due to the great number of people stung and the severity of the resulting intoxication. This communication reports an extensive venom characterization using high-throughput proteomic and Illumina transcriptomic sequencing performed with RNA purified from its venom glands. 2,553,529 reads were assembled into 44,579 transcripts. From these transcripts, 23,880 were successfully annoted using Trinotate. Using specialized databases and by performing bioinformatic searches, it was possible to identify 147 putative venom protein transcripts. These include α- and β–type sodium channel toxins (NaScTx), potassium channel toxins (KScTx) (α-, β-, δ-, γ- and λ–types), enzymes (metalloproteases, hyaluronidases, phospholipases, serine proteases, and monooxygenases), protease inhibitors, host defense peptides (HDPs) such as defensins, non-disulfide bridge peptides (NDBPs), anionic peptides, superfamily CAP proteins, insulin growth factor-binding proteins (IGFBPs), orphan peptides, and other venom components (La1 peptides). De novo tandem mass spectrometric sequencing of digested venom identificatied 50 peptides. The venom of C. hirsutipalpus contains the highest reported number (77) of transcripts encoding NaScTxs, which are the components responsible for human fatalities.

Small Ion Transport Properties of the Anthrax Toxin Channels

Anthrax toxin channel, protective antigen (PA63), is known to be strongly cation selective, the property that has determined the development of positively charged inhibitors as channel blockers. Successful interpretation of small ion transport through PA63 can also contribute to the description of the translocase activity of the channel that transports the enzymatic factors of anthrax toxin, lethal factor (LF) and edema factor (EF), into the cytosol. It was recently suggested by Kalu et al (FEBS Letters, 2012) that PA63 conductance is determined by the two contributing factors: ionic strength-dependent electrical potential and access resistance, with PA63 dependence on KCl concentration showing two distinct patterns at pH 6.5 and 4.5. The model also offered an explanation for the fact that the protonation of the cavity residues does not change PA63 small ion selectivity. The authors suggested that the channel conductance is determined by the narrowest φ clamp part of the channel and the ionizable residues in proximity to it. In contrast, selectivity is governed by the concerted action of many residues in the channel lumen. To validate this, so far putative model, here we investigate PA63 small ion transport properties using different electrolyte solutions, including LiCl, NaCl, KCl, RbCl, CsCl, NH4Cl, and CaCl2. We demonstrate that PA63 conductance shows an unexpectedly strong dependence on the nature of the electrolyte solution cation, compared to the bulk conductivity change. At the same time, the channel's selectivity remains essentially unchanged for monovalent cation solutions. In CaCl2, the selectivity is reversed. We discuss the influence of PA63 lumen residues, cation hydration shells, and access resistance on small ion transport properties of the channel.

Weaponisation ‘on the fly’: Convergent recruitment of knottin and defensin peptide scaffolds into the venom of predatory assassin flies

Many arthropod venom peptides have potential as bioinsecticides, drug leads, and pharmacological tools due to their specific neuromodulatory functions. Assassin flies (Asilidae) are a family of predaceous dipterans that produce a unique and complex peptide-rich venom for killing insect prey and deterring predators. However, very little is known about the structure and function of their venom peptides. We therefore used an E. coli periplasmic expression system to express four disulfide-rich peptides that we previously reported to exist in venom of the giant assassin fly Dolopus genitalis. After purification, each recombinant peptide eluted from a C18 column at a position closely matching its natural counterpart, strongly suggesting adoption of the native tertiary fold. Injection of purified recombinant peptides into blowflies (Lucilia cuprina) and crickets (Acheta domestica) revealed that two of the four recombinant peptides, named rDg3b and rDg12, inhibited escape behaviour in a manner that was rapid in onset (<1 min) and reversible. Homonuclear NMR solution structures revealed that rDg3b and rDg12 adopt cystine-stabilised α/ß defensin and inhibitor cystine knot folds, respectively. Although the closest known homologues of rDg3b at the level of primary structure are dipteran antimicrobial peptides such as sapecin and lucifensin, a DALI search showed that the tertiary structure of rDg3b most closely resembles the KV11.1-specific α-potassium channel toxin CnErg1 from venom of the scorpion Centruroides noxius. This is mainly due to the deletion of a large, unstructured loop between the first and second cysteine residues present in Dg3b homologues from non-asiloid, but not existing in asiloid, species. Patch-clamp electrophysiology experiments revealed that rDg3b shifts the voltage-dependence of KV11.1 channel activation to more depolarised potentials, but has no effect on KV1.3, KV2.1, KV10.1, KCa1.1, or the Drosophila Shaker channel. Although rDg12 shares the inhibitor cystine knot structure of many gating modifier toxins, rDg12 did not affect any of these KV channel subtypes. Our results demonstrate that multiple disulfide-rich peptide scaffolds have been convergently recruited into asilid and other animal venoms, and they provide insight into the molecular evolution accompanying their weaponisation.

Characterization, molecular modeling and phylogenetic analysis of a long mammalian neurotoxin from the venom of the Iranian scorpion Androctonus crassicauda

Scorpion venoms are highly complex polypeptides with a wide spectrum of bioactivities. A long chain peptide with eight cysteine residues and four disulfide bonds isolated from the venom of the Iranian scorpion Androctonus crassicauda, a dangerously venomous species belongs to the Buthidae family. Toxin was purified using size exclusion and reverse phase chromatography and was characterized by molecular weight determination and amino acid sequence. The primary structure analysis shows that AnCra1 is a long peptide with 64 amino acid residues and 7255.23 Da. as molecular weight. This toxic peptide, causing severe paralysis and leading to dead. It shows highest homology with voltage-gated sodium channel alpha toxins isolated from Middle-East and North-Africa scorpions. Phylogenetic relationship of AnCra1 and other ion channel toxins from Buthidae family suggested that, separation between these families of scorpions caused by the geographical conditions led to evolutionary alterations in these venoms polypeptide sequences. Homology modeling and three-dimensional structure of AnCra1 indicated that binding residues on exposed part of the peptide have high affinity for receptor residues in Na+V ion channel, site-3. The contact surfaces characteristics and charges may be eventuate to interaction between toxin peptide and targeted channel. Molecular homology between AnCra1 with structure of Na+V neurotoxic peptides illustrated that, AnCra1 can act as novel functional putative Alpha-mammal voltage-gated sodium channel toxin.

Immunosuppressive effects of a novel potassium channel toxin Ktx-Sp2 from Scorpiops Pocoki

BackgroundThe cDNA Library of venomous animals could provide abundant bioactive peptides coding information and is an important resource for screening bioactive peptides that target and regulate disease-related ion channels. To further explore the potential medicinal usage of the transcriptome database of Scorpiops Pocoki’s venom gland, this research identified the function of a new potassium channel toxin Ktx-Sp2, whose gene was screened from the database by sequence alignment.ResultsThe mature peptide of Ktx-Sp2 was obtained by genetic engineering. Whole-cell patch-clamp experiment showed that Ktx-Sp2 peptide could effectively block three types of exogenous voltage-gated potassium channels—Kv1.1, Kv1.2 and Kv1.3, among which, the blocking activity for Kv1.3 was relatively high, showing selectivity to some extent. Taking Jurkat T cells as the cell model, this study found that Ktx-Sp2 peptide could also effectively block endogenous Kv1.3, significantly reduce the free calcium concentration in Jurkat T cells, inhibit the activation of Jurkat T cells and reduce the release of inflammatory cytokines IL-2, showing a strong immunosuppressant effect.ConclusionsThis study further proves that the transcriptome database of the Scorpiops Pocoki venom gland is an important resource for discovery of novel bioactive polypeptide coding genes. The newly screened Kv1.3 channel blocker Ktx-Sp2 expanded the range of leading compounds for the treatment of autoimmune diseases and promoted the development and application of scorpion toxin peptides in the field of biomedicine.

Hadrurid Scorpion Toxins: Evolutionary Conservation and Selective Pressures.

Scorpion toxins are thought to have originated from ancestral housekeeping genes that underwent diversification and neofunctionalization, as a result of positive selection. Our understanding of the evolutionary origin of these peptides is hindered by the patchiness of existing taxonomic sampling. While recent studies have shown phylogenetic inertia in some scorpion toxins at higher systematic levels, evolutionary dynamics of toxins among closely related taxa remain unexplored. In this study, we used new and previously published transcriptomic resources to assess evolutionary relationships of closely related scorpions from the family Hadruridae and their toxins. In addition, we surveyed the incidence of scorpine-like peptides (SLP, a type of potassium channel toxin), which were previously known from 21 scorpion species. We demonstrate that scorpine-like peptides exhibit gene duplications. Our molecular analyses demonstrate that only eight sites of two SLP copies found in scorpions are evolving under positive selection, with more sites evolving under negative selection, in contrast to previous findings. These results show evolutionary conservation in toxin diversity at shallow taxonomic scale.

ImKTx96, a peptide blocker of the Kv1.2 ion channel from the venom of the scorpion Isometrus maculates

Scorpion venom contains diverse bioactive peptides that can recognize and interact with membrane proteins such as ion channels. These natural toxins are believed to be useful tools for exploring the structure and function of ion channels. In this study, we characterized a K+-channel toxin gene, ImKTx96, from the venom gland cDNA library of the scorpion Isometrus maculates. The peptide deduced from the ImKTx96 precursor nucleotide sequence contains a signal peptide of 27 amino acid residues and a mature peptide of 29 residues with three disulfide bridges. Multiple sequence alignment indicated that ImKTx96 is similar with the scorpion toxins that typically target K+-channels. The recombined ImKTx96 peptide (rImKTx96) was expressed in the Escherichia coli system, and purified by GST-affinity chromatography and RP-HPLC. Results from whole-cell patch-clamp experiments revealed that rImKTx96 can inhibit the current of the Kv1.2 ion channel expressed in HEK293 cells. The 3D structure of ImKTx96 was constructed by molecular modeling, and the complex formed by ImKTx96 interacting with the Kv1.2 ion channel was obtained by molecular docking. Based on its structural features and pharmacological functions, ImKTx96 was identified as one member of K+-channel scorpion toxin α-KTx10 group and may be useful as a molecular probe for investigating the structure and function of the Kv1.2 ion channel.

Exploring the Nature of Cationic Blocker Recognition by the Anthrax Toxin Channel

Obstructing conductive pathways of the channel-forming toxins with targeted blockers is a promising drug design approach. Nearly all tested positively charged ligands have been shown to reversibly block the cation-selective channel-forming protective antigen (PA63) component of the binary anthrax toxin. The cationic ligands with more hydrophobic surfaces, particularly those carrying aromatic moieties, inhibited PA63 more effectively. To understand the physical basis of PA63 selectivity for a particular ligand, detailed information is required on how the blocker structural elements (e.g., positively charged and aromatic groups) influence the molecular kinetics of the blocker/channel binding reactions. In this study, we address this problem using the high-resolution single-channel planar lipid bilayer technique. Several structurally distinct cationic blockers, namely per-6-S-(3-amino) propyl-β-cyclodextrin, per-6-S-(3-aminomethyl) benzyl-α-cyclodextrin, per-6-S-(3-aminomethyl) benzyl-β-cyclodextrin, per-6-S-(3-aminomethyl) benzyl-γ-cyclodextrin, methyltriphenylphosphonium ion, and G0 polyamidoamine dendrimer are tested for their ability to inhibit the heptameric and octameric PA63 variants and PA63F427A mutant. The F427 residues form a hydrophobic constriction region inside the channel, known as the “ϕ-clamp.” We show that the cationic blockers interact with PA63 through a combination of forces. Analysis of the binding reaction kinetics suggests the involvement of cation-π, Coulomb, and salt-concentration-independent π-π or hydrophobic interactions in the cationic cyclodextrin binding. It is possible that these blockers bind to the ϕ-clamp and are also stabilized by the Coulomb interactions of their terminal amino groups with the water-exposed negatively charged channel residues. In PA63F427A, only the suggested Coulomb component of the cyclodextrin interaction remains. Methyltriphenylphosphonium ion and G0 polyamidoamine dendrimer, despite being positively charged, interact primarily with the ϕ-clamp. We also show that seven- and eightfold symmetric cyclodextrins effectively block the heptameric and octameric forms of PA63 interchangeably, adding flexibility to the earlier formulated blocker/target symmetry match requirement.

Wild Honey Poisoning: A Case Report

Wild honey is consumed in many parts of the world as an alternative source of medicine with the belief of reducing cardiovascular, gastrointestinal and many other ailments. However, intoxication secondary to consumption of wild honey produced from the nectar of a few species rhododendrons has been encountered due to a toxin known as grayanotoxin. It is a sodium channel toxin causing symptoms like bradycardia, arrhythmias, hypotension, sweating, dizziness and altered sensorium. We report a case of a 58 year old man who presented to the emergency room following ingestion of wild honey with bradycardia, hypotension and altered mental status.&#x0D; Keywords: bradycardia; grayanotoxin; hypotension; wild honey poisoning.

The Dual α-Amidation System in Scorpion Venom Glands.

Many peptides in scorpion venoms are amidated at their C-termini. This post-translational modification is paramount for the correct biological function of ion channel toxins and antimicrobial peptides, among others. The discovery of canonical amidation sequences in transcriptome-derived scorpion proproteins suggests that a conserved enzymatic α-amidation system must be responsible for this modification of scorpion peptides. A transcriptomic approach was employed to identify sequences putatively encoding enzymes of the α-amidation pathway. A dual enzymatic α-amidation system was found, consisting of the membrane-anchored, bifunctional, peptidylglycine α-amidating monooxygenase (PAM) and its paralogs, soluble monofunctional peptidylglycine α-hydroxylating monooxygenase (PHMm) and peptidyl-α-hydroxyglycine α-amidating lyase (PALm). Independent genes encode these three enzymes. Amino acid residues responsible for ion coordination and enzymatic activity are conserved in these sequences, suggesting that the enzymes are functional. Potential endoproteolytic recognition sites for proprotein convertases in the PAM sequence indicate that PAM-derived soluble isoforms may also be expressed. Sequences potentially encoding proprotein convertases (PC1 and PC2), carboxypeptidase E (CPE), and other enzymes of the α-amidation pathway, were also found, confirming the presence of this pathway in scorpions.

Purification and biochemical characterisation of a putative sodium channel agonist secreted from the South African Knobbly sea anemone Bunodosoma capense

Voltage gated ion channels have become a subject of investigation as possible pharmaceutical targets. Research has linked the activity of ion channels directly to anti-inflammatory pathways, energy homeostasis, cancer proliferation and painful diabetic neuropathy. Sea anemones secrete a diverse array of bioactive compounds including potassium and sodium channel toxins. A putative novel sodium channel agonist (molecular mass of 4619.7 Da) with a predicted sequence: CLCNSDGPSV RGNTLSGILW LAGCPSGWHN CKKHKPTIGW CCK was isolated from Bunodosoma capense using a modified stimulation technique to induce the secretion of the neurotoxin rich mucus confirmed by an Artemia nauplii bio-assay. The peptide purification combined size-exclusion and reverse-phase high performance liquid chromatography. A thallium-based ion flux assay confirmed the presence of a sodium channel agonist/inhibitor and purity was determined using a modified tricine SDS-PAGE system. The peptide isolated indicated the presence of multiple disulfide bonds in a tight β-defensin cystine conformation. An IC50 value of 26 nM was determined for total channel inhibition on MCF-7 cells. The unique putative sodium channel agonist initiating with a cystine bond indicates a divergent evolution to those previously isolated from Bunodosoma species.

Interaction of PEGs with the Anthrax Toxin Channel and their Role in Altering the 1/F Current Noise

Interaction of PEGs with the Anthrax Toxin Channel and their Role in Altering the 1/F Current Noise

Effect of Late Endosomal Dobmp Lipid and Traditional Model Lipids of Electrophysiology on the Anthrax Toxin Channel Activity

Abstract Anthrax toxin action requires triggering of natural endocytic transport mechanisms whereby the binding component of the toxin forms channels (PA63) within endosomal limiting and intraluminal vesicle membranes to deliver the toxin's enzymatic components into the cytosol. Membrane lipid composition varies at different stages of anthrax toxin internalization, with intraluminal vesicle membranes containing ~70% of anionic bis(monoacylglycero)phosphate lipid. Using model bilayer measurements, we show that membrane lipids can have a strong effect on the anthrax toxin channel properties, including the channel-forming activity, voltage-gating, conductance, selectivity, and enzymatic factor binding. Interestingly, the highest PA63 insertion rate was observed in bis(monoacylglycero)phosphate membranes. The molecular dynamics simulation data show that the conformational properties of the channel are different in bis(monoacylglycero)phosphate compared to PC, PE, and PS lipids. The anthrax toxin protein/lipid bilayer system can be advanced as a novel robust model to directly investigate lipid influence on membrane protein properties and protein/protein interactions.

New Kv, NAv, and ASIC channel toxins from the sea anemone Heteractis crispa

New Kv, NAv, and ASIC channel toxins from the sea anemone Heteractis crispa

Three New Scorpion Chloride Channel Toxins as Potential Anti-Cancer Drugs: Computational Prediction of The Interactions With Hmmp-2 by Docking and Steered Molecular Dynamics Simulations.

Scorpion venom is a rich source of toxins which have great potential to develop new therapeutic agents. Scorpion chloride channel toxins (ClTxs), such as Chlorotoxin selectively inhibit human Matrix Methaloproteinase-2 (hMMP-2). The inhibitors of hMMP-2 have potential use in cancer therapy. Three new ClTxs, meuCl14, meuCl15 and meuCl16, derived from the venom transcriptome of Iranian scorpion, M. eupeus (Buthidea family), show high sequence identity (71.4%) with Chlorotoxin. Here, 3-D homology model of new ClTxs were constructed. The models were optimized by Molecular Dynamics simulation based on MDFF (molecular dynamics flexible fitting) method. New ClTxs indicate the presence of CSαβ folding of other scorpion toxins. A docking followed by steered molecular dynamics (SMD) simulations to investigate the interactions of meuCl14, meuCl15, and meuCl16 with hMMP-2 was applied. The current study creates a correlation between the unbinding force and the inhibition activities of meuCl14, meuCl15 and meuCl16 to shed some insights as to which toxin may be used as a drug deliverer. To this aim, SMD simulations using Constant Force Pulling method were carried out. The SMD provided useful details related to the changes of electrostatic, van de Waals (vdW), and hydrogen-bonding (H-bonding) interactions between ligands and receptor during the pathway of unbinding. According to SMD results, the interaction of hMMP-2 with meuCl14 is more stable. In addition, Arginine residue was found to contribute significantly in interaction of ClTxs with hMMP-2. All in all, the present study is a dynamical approach whose results are capable of being implemented in structure-based drug design.

Batrachotoxin acts as a stent to hold open homotetrameric prokaryotic voltage-gated sodium channels.

Batrachotoxin (BTX), an alkaloid from skin secretions of dendrobatid frogs, causes paralysis and death by facilitating activation and inhibiting deactivation of eukaryotic voltage-gated sodium (Nav) channels, which underlie action potentials in nerve, muscle, and heart. A full understanding of the mechanism by which BTX modifies eukaryotic Nav gating awaits determination of high-resolution structures of functional toxin-channel complexes. Here, we investigate the action of BTX on the homotetrameric prokaryotic Nav channels NaChBac and NavSp1. By combining mutational analysis and whole-cell patch clamp with molecular and kinetic modeling, we show that BTX hinders deactivation and facilitates activation in a use-dependent fashion. Our molecular model shows the horseshoe-shaped BTX molecule bound within the open pore, forming hydrophobic H-bonds and cation-π contacts with the pore-lining helices, leaving space for partially dehydrated sodium ions to permeate through the hydrophilic inner surface of the horseshoe. We infer that bulky BTX, bound at the level of the gating-hinge residues, prevents the S6 rearrangements that are necessary for closure of the activation gate. Our results reveal general similarities to, and differences from, BTX actions on eukaryotic Nav channels, whose major subunit is a single polypeptide formed by four concatenated, homologous, nonidentical domains that form a pseudosymmetric pore. Our determination of the mechanism by which BTX activates homotetrameric voltage-gated channels reveals further similarities between eukaryotic and prokaryotic Nav channels and emphasizes the tractability of bacterial Nav channels as models of voltage-dependent ion channel gating. The results contribute toward a deeper, atomic-level understanding of use-dependent natural and synthetic Nav channel agonists and antagonists, despite their overlapping binding motifs on the channel proteins.

Scorpion Toxins: Positive Selection at a Distal Site Modulates Functional Evolution at a Bioactive Site.

The bioactive sites of proteins are those that directly interact with their targets. In many immunity- and predation-related proteins, they frequently experience positive selection for dealing with the changes of their targets from competitors. However, some sites that are far away from the interface between proteins and their targets are also identified to evolve under positive selection. Here, we explore the evolutionary implication of such a site in scorpion α-type toxins affecting sodium (Na+) channels (abbreviated as α-ScNaTxs) using a combination of experimental and computational approaches. We found that despite no direct involvement in interaction with Na+ channels, mutations at this site by different types of amino acids led to toxicity change on both rats and insects in three α-ScNaTxs, accompanying differential effects on their structures. Molecular dynamics simulations indicated that the mutations changed the conformational dynamics of the positively selected bioactive site-containing functional regions by allosteric communication, suggesting a potential evolutionary correlation between these bioactive sites and the distant nonbioactive site. Our results reveal for the first time the cause of fast evolution at nonbioactive sites of scorpion neurotoxins, which is presumably to adapt to the change of their bioactive sites through coevolution to maintain an active conformation for channel binding. This might aid rational design of scorpion Na+ channel toxins with improved phyletic selectivity via modification of a distant nonbioactive site.