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Molecular mechanisms of neurotoxin action on voltage-gated sodium channels
Abstract
Voltage-gated sodium channels are the molecular targets for a broad range of neurotoxins that act at six or more distinct receptor sites on the channel protein. These toxins fall into three groups. Both hydrophilic low molecular mass toxins and larger polypeptide toxins physically block the pore and prevent sodium conductance. Alkaloid toxins and related lipid-soluble toxins alter voltage-dependent gating of sodium channels via an allosteric mechanism through binding to intramembranous receptor sites. In contrast, polypeptide toxins alter channel gating by voltage sensor trapping through binding to extracellular receptor sites. The results of recent studies that define the receptor sites and mechanisms of action of these diverse toxins are reviewed here.
... Drugs that act on voltage-gated ion channels have for long been used as a treatment for many diseases, even before we knew such channels existed, in particular to control diseases of excitability (Brodie, 2010;Karagueuzian et al., 2017). Historically, natural compounds found in plants and toxins from animals have also been used to gain a better understanding of the structure and function of voltage-gated ion channels (Agnew et al., 1978;Cestèle and Catterall, 2000;MacKinnon, 1991;Stevens et al., 2011). From a clinical perspective, drugs that block NaV and CaV channels have classically been used to reduce the cellular excitability in diseases such as epilepsy, pain and cardiac arrhythmia (Bagal et al., 2015;Brodie, 2017a;Fozzard et al., 2011;Merritt and Putnam, 1938). ...
... The modulation of voltage-gated ion channels by different compounds, ions, toxins and lipids, is diverse in terms of binding site and mechanisms (Cestèle and Catterall, 2000;Elinder and Arhem, 2003;Liin et al., 2016b;Wulff et al., 2009). Metal ions affect the gating of voltage-gated ion channels, both electrostatically (e.g. ...
... binding to the channel), and indirectly from sites close to the channel, or directly by binding to the channel (Elinder and Arhem, 2003). Toxins also have several binding sites to voltage-gated ion channels, such as the pore domain, and the S3-S4 linker (voltage-sensor trapping toxins) (Cestèle and Catterall, 2000). In common with resin acids, the extracellular parts of S3 and S4 of the KV3.1 channel contribute to the sensitivity of the toxin gamberiol, presumably by modulating the accessibility to the binding pocket between the VSD and the pore domain, from which gamberiol stabilizes the KV3.1 channel in the closed state (Kopljar et al., 2016). ...
... Sea anemone, spider, and scorpion toxins bind to extracellular loops comprising receptor sites 3 or 4 in the voltage-sensing module of domains IV and II, respectively. These toxins modify channel gating by trapping the S4 segment voltage sensor in resting or activated states (reviewed by [22,[74][75][76][77][78][79]). Conotoxins target sites in each of these S3-S4 extracellular loops. ...
... This site includes residues of domain II S1-S2 and S3-S4 extracellular loops [212][213][214] and extends to the N-terminal pore loop in domain III, proximal to the DII VSD. β-scorpion and spider toxins act as gating modifiers by "trapping" the voltage sensor in domain II in the activated position (for reviews of voltage sensor trapping see [76,77]; Figure 1C,D). MrVIA inhibited NaV1.4 DII voltage sensor translocation [215], and a chimeric approach revealed its interaction with the N-terminal MrVIA blocked sodium current in molluscan (Aplysia) neurons and produced ataxia and/or coma when injected at nanomole doses (or less) into the central nervous system (intra-cranial injection) of mice [159]. ...
... This site includes residues of domain II S1-S2 and S3-S4 extracellular loops [212][213][214] and extends to the N-terminal pore loop in domain III, proximal to the DII VSD. β-scorpion and spider toxins act as gating modifiers by "trapping" the voltage sensor in domain II in the activated position (for reviews of voltage sensor trapping see [76,77]; Figure 1C,D). MrVIA inhibited Na V 1.4 DII voltage sensor translocation [215], and a chimeric approach revealed its interaction with the N-terminal pore loop in domain III [216]. ...
Marine toxins have potent actions on diverse sodium ion channels regulated by transmembrane voltage (voltage-gated ion channels) or by neurotransmitters (nicotinic acetylcholine receptor channels). Studies of these toxins have focused on varied aspects of venom peptides ranging from evolutionary relationships of predator and prey, biological actions on excitable tissues, potential application as pharmacological intervention in disease therapy, and as part of multiple experimental approaches towards an understanding of the atomistic characterization of ion channel structure. This review examines the historical perspective of the study of conotoxin peptides active on sodium channels gated by transmembrane voltage, which has led to recent advances in ion channel research made possible with the exploitation of the diversity of these marine toxins.
... They could be a new source of bioactive secondary metabolites, including toxins such as amphidinolides, amphidinol, azaspiracid, Among those toxic compounds, STXs, also known as paralytic shellfish toxins (PSTs), are neurotoxic alkaloids naturally produced by certain marine dinoflagellates [13]. PSTs block voltage-gated sodium channels of neurons in a reversible manner, inducing paralysis, and such STXs are 2000-times more lethal than sodium cyanide by weight [14]. They are classified into several subgroups based on their chemical structure, such as carbamoyl derivatives (STXs, neosaxitoxin (neoSTX), gonyautoxins1−4 (GTX1−4), N-sulphocarbamoyl derivatives (GTX5−6 and C1−4), dicarbamoyl derivatives [dicarbamoyl-STX (dcSTX), dicarbamoyl neosaxitoxin (dcneoSTX), and dicarbamoyl gonyautoxins 1−4 (dcGTX1−4)], and other less-frequent deoxy-decarbonylated, mono-hydroxy-benzoate, dihydroxy benzoate, and sulphated benzoate analogs [15]. ...
... Specifically, Alexandrium catenella, Alexandrium pacificum, and Alexandrium minutum are major species causing environmental Among those toxic compounds, STXs, also known as paralytic shellfish toxins (PSTs), are neurotoxic alkaloids naturally produced by certain marine dinoflagellates [13]. PSTs block voltage-gated sodium channels of neurons in a reversible manner, inducing paralysis, and such STXs are 2000-times more lethal than sodium cyanide by weight [14]. They are classified into several subgroups based on their chemical structure, such as carbamoyl derivatives (STXs, neosaxitoxin (neoSTX), gonyautoxins1-4 (GTX1-4), N-sulphocarbamoyl derivatives (GTX5-6 and C1-4), dicarbamoyl derivatives [dicarbamoyl-STX (dcSTX), dicarbamoyl neosaxitoxin (dcneoSTX), and dicarbamoyl gonyautoxins 1-4 (dcGTX1-4)], and other less-frequent deoxy-decarbonylated, mono-hydroxy-benzoate, di-hydroxy benzoate, and sulphated benzoate analogs [15]. ...
The marine dinoflagellate Alexandrium is known to form harmful algal blooms (HABs) and produces saxitoxin (STX) and its derivatives (STXs) that cause paralytic shellfish poisoning (PSP) in humans. Cell growth and cellular metabolism are affected by environmental conditions, including nutrients, temperature, light, and the salinity of aquatic systems. Abiotic factors not only engage in photosynthesis, but also modulate the production of toxic secondary metabolites, such as STXs, in dinoflagellates. STXs production is influenced by a variety of abiotic factors; however, the relationship between the regulation of these abiotic variables and STXs accumulation seems not to be consistent, and sometimes it is controversial. Few studies have suggested that abiotic factors may influence toxicity and STXs-biosynthesis gene (sxt) regulation in toxic Alexandrium, particularly in A. catenella, A. minutum, and A. pacificum. Hence, in this review, we focused on STXs production in toxic Alexandrium with respect to the major abiotic factors, such as temperature, salinity, nutrients, and light intensity. This review informs future research on more sxt genes involved in STXs production in relation to the abiotic factors in toxic dinoflagellates.
... Receptor mutation in the amino acid sequence of the sodium channel (IV S6) results in alteration of the site of action leading to local anesthetic resistance. 6,8 Adding an adjuvant like clonidine or fentanyl to the local anesthetic may reverse this action and help in achieving a successful block. 9 Furthermore, since the site of action of local anesthetics and the scorpion venom is the same, bupivacaine may offer partial protection against the inhibitory effects of the scorpion venom. ...
... β toxins bind to the receptor site 4 of the sodium channel and modify its activation leading to local anesthetic resistance causing delays or failures of spinal anesthesia. 8 The consequences of these failures are a waste ...
... NaTx binds to specific sites on sodium channels, disrupting their normal inactivation process and prolonging the channels' open state. This leads to a continuous influx of sodium ions into nerve cells, resulting in a cascade of electrophysiological abnormalities that trigger excessive neuronal excitation and intense pain [99]. The α-NaTx selectively binds to the S3-S4 loop of Domain IV (voltage-sensing domain IV, VSD4) in sodium channels, stabilizing the activated state of the voltage sensor to block fast inactivation and prolong channel opening. ...
Scorpion toxins significantly disrupt the normal function of ion channels, leading to abnormal nerve excitability and severe pain responses. Notably, α-type sodium channel toxins (α-NaTx) and β-type sodium channel toxins (β-NaTx) target sodium channels through distinct mechanisms: α-NaTx prolongs channel opening, while β-NaTx lowers the activation threshold, resulting in persistent nerve overexcitation and heightened pain. This review synthesizes current knowledge on pain-inducing venom peptides isolated from various scorpion species, elucidating the underlying molecular mechanisms involving ion channels. Furthermore, it explores the potential applications of these toxins in scientific research and drug development, highlighting their significance in advancing our understanding of pain mechanisms and facilitating the development of novel analgesic therapies.
... Interestingly, Na V channels are also the important targets for a wide range of neurotoxins such as saxitoxin, tetrodotoxin or peptide toxins from spiders, scorpions or sea anemones. [31] In human bodies, there are nine kinds of α-subunits in different Na V channels (from Na V 1.1 to Na V 1.9). And different subtype Na V channels showed tissue specific expression with distinct physiological functions. ...
Natural channel proteins allow the selective permeation of ions, water or other nutritious entities across bilayer membranes, facilitating various essential physiological functions in living systems. Inspired by nature, chemists endeavor to simulate the structural features and transport behaviors of channel proteins through biomimetic strategies. In this review, we start from introducing the inherent traits of channel proteins such as their crystal structures, functions and mechanisms. Subsequently, different kind of synthetic ion channels including their design principles, dynamic regulations and therapeutic applications were carefully reviewed. Finally, the potential challenges and opportunities in this research field were also carefully discussed. It is anticipated that this review could provide some inspiring ideas and future directions towards the construction of novel bionic ion channels with higher‐level structures, properties, functions and practical applications.
... [13] 2. Scorpion β-Toxins: The β-toxins are isolated from American scorpions, bind to receptor site 4 on vertebrate Na+ channels and producing a shift to a more negative membrane potential. [14] Several studies described the mode of action of these toxins that are related with the increment of sodium. The βscorpion toxin Css IV obtained from Centruroides suffussus scorpion venom is believed to specific bind in sodium channel. ...
... Experimental data show that the effect of these toxins facilitates the stimulation of VGSC and can increase the flow of sodium during each action potential. 3,4 The most important point in using these toxins is that they target specific molecules. They are able to find and interact with their own target, usually at low concentrations. ...
... The TTX treatment also diminished the internal Na + concentration by 50%, while the activation of the channels using veratridine showed no significant effect on an internal Na + concentration. Veratridine enhances sustained inward currents by blocking VGSC inactivation through allosteric mechanism (Cestèle and Catterall, 2000), with a concentration around 29 μM to induce 50% of the maximum effect (Catterall, 1977). The treatment with 0.5 μM TTX or 50 μM veratridine in H460 cells did not affect cell proliferation nor cell migration as determined by wound healing assay. ...
This review focuses on the expression and function of voltage-gated sodium channel subtype NaV1.7 in various cancers and explores its impact on the metastasis driving cell functions such as proliferation, migration, and invasiveness. An overview of its structural characteristics, drug binding sites, inhibitors and their likely mechanisms of action are presented. Despite the lack of clarity on the precise mechanism by which NaV1.7 contributes to cancer progression and metastasis; many studies have suggested a connection between NaV1.7 and proteins involved in multiple signaling pathways such as PKA and EGF/EGFR-ERK1/2. Moreover, the functional activity of NaV1.7 appears to elevate the expression levels of MACC1 and NHE-1, which are controlled by p38 MAPK activity, HGF/c-MET signaling and c-Jun activity. This cascade potentially enhances the secretion of extracellular matrix proteases, such as MMPs which play critical roles in cell migration and invasion activities. Furthermore, the NaV1.7 activity may indirectly upregulate Rho GTPases Rac activity, which is critical for cytoskeleton reorganization, cell adhesion, and actin polymerization. The relationship between NaV1.7 and cancer progression has prompted researchers to investigate the therapeutic potential of targeting NaV1.7 using inhibitors. The positive outcome of such studies resulted in the discovery of several inhibitors with the ability to reduce cancer cell migration, invasion, and tumor growth underscoring the significance of NaV1.7 as a promising pharmacological target for attenuating cancer cell proliferation and metastasis. The research findings summarized in this review suggest that the regulation of NaV1.7 expression and function by small molecules and/or by genetic engineering is a viable approach to discover novel therapeutics for the prevention and treatment of metastasis of cancers with elevated NaV1.7 expression.
... The most important peptides are those that recognize the voltage-gated sodium channels of excitable cells [13][14][15][16]. Their structures are similar in terms of the number of amino acids in the sequence and the three-dimensional structures [17]. ...
Alternative recombinant sources of antivenoms have been successfully generated. The application of such strategies requires the characterization of the venoms for the development of specific neutralizing molecules against the toxic components. Five toxic peptides to mammals from the Mexican scorpion Centruroides villegasi were isolated by chromatographic procedures by means of gel filtration on Sephadex G-50, followed by ion-exchange columns on carboxy-methyl-cellulose (CMC) resins and finally purified by high-performance chromatography (HPLC) columns. Their primary structures were determined by Edman degradation. They contain 66 amino acids and are maintained well packed by four disulfide bridges, with molecular mass from 7511.3 to 7750.1 Da. They are all relatively toxic and deadly to mice and show high sequence identity with known peptides that are specific modifiers of the gating mechanisms of Na⁺ ion channels of type beta-toxin (β-ScTx). They were named Cv1 to Cv5 and used to test their recognition by single-chain variable fragments (scFv) of antibodies, using surface plasmon resonance. Three different scFvs generated in our laboratory (10FG2, HV, LR) were tested for recognizing the various new peptides described here, paving the way for the development of a novel type of scorpion antivenom.
... They also can alter acetylcholine levels and cause dysfunction of the receptors. [81][82][83] This could explain some of the symptoms related to the nerves that some COVID 19 patients experience, such as reduced smell, taste, and signs of the Guillain-Barre syndrome. 72 Therefore, specific conotoxin peptides of C. flavidus may be able to inhibit the essential protein of life-threatening viruses COVID-19, potentially leading to their treatment or cure. ...
Background
Venomous marine cone snails produce unique neurotoxins called conopeptides or conotoxins, which are valuable for research and drug discovery. Characterizing Conus venom is important, especially for poorly studied species, as these tiny and steady molecules have considerable potential as research tools for detecting new pharmacological applications. In this study, a worm-hunting cone snail, Conus flavidus inhabiting the Red Sea coast were collected, dissected and the venom gland extraction was subjected to proteomic analysis to define the venom composition, and confirm the functional structure of conopeptides.
Results
Analysis of C. flavidus venom identified 117 peptide fragments and assorted them to conotoxin precursors and non-conotoxin proteins. In this procedure, 65 conotoxin precursors were classified and identified to 16 conotoxin precursors and hormone superfamilies. In the venom of C. flavidus, the four conotoxin superfamilies T, A, O2, and M were the most abundant peptides, accounting for 75.8% of the total conotoxin diversity. Additionally, 19 non-conotoxin proteins were specified in the venom, as well as several potentially biologically active peptides with putative applications.
Conclusion
Our research displayed that the structure of the C. flavidus-derived proteome is similar to other Conus species and includes toxins, ionic channel inhibitors, insulin-like peptides, and hyaluronidase. This study provides a foundation for discovering new conopeptides from C. flavidus venom for pharmaceutical use.
... These isoforms are expressed in different tissues and have different functions such as neurotransmitter release, secretion,muscle contraction and pain sensation (Papale et al. 2010;Stevens et al. 2011). Sodium channels consist of an α-subunit (260 kDa) and auxiliary β-subunits (30-40 kDa) (Cestèle and Catterall 2000;Gawali and Todt 2016). Due to the high binding specificity and affinity of neurotoxins to ion channels, neurotoxins can affect ion permeability or voltage-dependent gating properties. ...
Prior research has established that Jingzhaotoxin-III (JZTX-III) can selectively target Nav1.5 channels. It achieves this by anchoring the DII-S4 voltage sensor and attaching to the DII S3-S4 loop, classifying it within the receptor site 4 toxin family. Our investigation, however, unveiled that at elevated concentrations (1 or 5 μM), JZTX-III exhibits a dual functionality. It not only impedes channel activation but also decelerates the swift inactivation process of Nav1.5. A significant discovery from our study is the pivotal role of the positively charged residue at position 800 (R800) in mediating the interaction between Nav1.5 and JZTX-III. This was corroborated by the observation that the R800A mutation led to a 30-fold reduction in JZTX-III binding affinity. Intriguingly, at concentrations of 1 or 5 μM, JZTX-III was found to hinder the rapid inactivation in the Nav1.5 R800A mutant channel. While the toxin did not markedly inhibit activation, it notably delayed the fast inactivation of Nav1.5. These findings hint at a possible interaction of JZTX-III with DIV, given that DIVS4 is instrumental in fast inactivation. Further insights were gained through site-directed mutagenesis analysis, which showed that mutating two residues (Asn1612 and Lsy1613) in the DIV S3-S4 linker to alanine significantly diminished JZTX-III’s capacity to suppress Nav1.5’s peak current. For the double mutants R800A/Q1612A and R800A/K1613A, there was no inhibition of the peak current. Conversely, reverse mutations in the hNav1.7 genes E1589Q and T1590K notably heightened DIV’s sensitivity to JZTX-III. Hence, our findings suggest that JZTX-III’s binding is not confined to the DIVS3-S4 linker but extends to the DII S3-S4 linker of hNav1.5. This unveils a novel interaction mechanism between JZTX-III and Nav1.5, enriching our understanding of its intricate modulatory effects.
... 7 Scorpion toxins show a preference for specific sodium channel subtypes of mammals or insects. 8 Scorpion toxins (ScTxs) block or modify the function of voltage gated ion channels (VGIC). The long-chain scorpion toxins mainly act on voltage-gated sodium channels (VGSCs), while the short-chain scorpion toxins generally target potassium or chloride channels. ...
Background: Failure of neuraxial or regional anaesthesia can result from factors such as drug errors, technical inefficiencies, and poor patient positioning. While these causes are well-known, resistance to local anaesthetic action due to mutations in sodium channels or scorpion sting is a lesser-known contributor to block failure. In India, a tropical country with a significant number of patients presenting for surgical procedures, a history of scorpion bites is not uncommon. Case: We observed seven cases of failed regional anesthesia who had history of scorpion sting. All the patients received intrathecal bupivacaine by experienced anesthesiologists, of seven patients five patients did not develop sensory or motor block. One patient had delayed successful subarachnoid block after second attempt and one patient had successful block at first attempt. Conclusion: Our observations revealed instances of failed spinal blocks, despite adequate drug dosages and experienced anesthesiologists performing the procedures, in patients with a history of scorpion envenomation. Accordingly, our study concludes that obtaining a thorough scorpion sting history during pre-anesthesia check-ups, particularly in endemic areas, can effectively prevent unnecessary repeated pinpricks, escalating dosages, patient and surgeon discomfort, and skepticism towards the skills of anesthesiologists.
... Zapata, Jaramillo-Serna, & Avila-Reyes, 2007).Desde las ciencias básicas, hallazgos recientes definen los sitios receptores y mecanismos de acción de las toxinas hidrófilas y polipeptídicas, alteran los canales de sodio, potasio y calcio creando voltajes a manera de señalización para la generación del potencial acción y la conducción en una amplia variedad de neurotoxinas. Las toxinas polipeptídicas alteran el canal de apertura por la captura del sensor de voltaje a través de la unión a receptores extracelulares(Catterall WA, 2007) (Cestèle S, 2000). En el caso de los péptidos purificados del veneno del escorpión, demostraron la interacción con ciertos residuos de aminoácidos presentes en el vestíbulo exterior, diversos subtipos de canales de potasio, la pág. ...
Introduction: The Official Mexican Standard on scorpion stings (NOM-033-SSA2-2011) establishes official rules that nursing staff must know and comply with. Objective: analyze the knowledge and compliance of the NOM-033-SSA2-2011, of the nursing staff.
Methodology: descriptive and cross�sectional study carried out in a jurisdiction of the State of Colima, Mexico, where two instruments designed for its identification are applied, data that were analyzed through descriptive and inferential statistics. Results: the probability of occurrence of the phenomenon (knowledge/compliance) regarding the Standard in scorpion sting is 58.9% (OR 0.0589) and the risk of this occurring (RR 0.1064) this means that the occurrence of knowledge/ compliance is 10.64%, 95% CI (P≤ 0.001).
Conclusions: based on these results, it is concluded to reflect on the internal and external factors that influence the phenomenon of knowledge/compliance established by NOM-033-SSA2-2011.
... Based on the specific binding properties, scorpion toxins are divided into sodium channel, potassium channel, chloride channel, and calcium channel (Ca V ) toxins [3] and further clustered into 26 subfamilies according to structure, mechanism of action, and binding site on different channel subtypes [4,5]. The sodium channel-specific long-chain toxins (60-76 aa residues), subdivided into αand β-toxins targeting receptor sites 3 and 4 of vertebrate sodium channels, are the most numerous and well-studied scorpion toxins [6][7][8]. Calcium channel toxins are less studied, with the 63-aa kurtoxin from venom of the scorpion Parabuthus transvaalicus being the first and only scorpion toxin shown to selectively bind T-type Ca V s (Ca V 3.1 and Ca V 3.2) [9,10]. Low voltage-activated (LVA) T-type Ca V s are key players in signal amplification and synaptic integration, and a privileged gate for calcium influx that initiates many physiological events like secretion, neurotransmission, and cell proliferation. ...
Venom peptides have evolved to target a wide range of membrane proteins through diverse mechanisms of action and structures, providing promising therapeutic leads for diseases, including pain, epilepsy, and cancer, as well as unique probes of ion channel structure-function. In this work, a high-throughput FLIPR window current screening assay on T-type CaV3.2 guided the isolation of a novel peptide named ω-Buthitoxin-Hf1a from scorpion Hottentotta franzwerneri crude venom. At only 10 amino acid residues with one disulfide bond, it is not only the smallest venom peptide known to target T-type CaVs but also the smallest structured scorpion venom peptide yet discovered. Synthetic Hf1a peptides were prepared with C-terminal amidation (Hf1a-NH2) or a free C-terminus (Hf1a-OH). Electrophysiological characterization revealed Hf1a-NH2 to be a concentration-dependent partial inhibitor of CaV3.2 (IC50 = 1.18 μM) and CaV3.3 (IC50 = 0.49 μM) depolarized currents but was ineffective at CaV3.1. Hf1a-OH did not show activity against any of the three T-type subtypes. Additionally, neither form showed activity against N-type CaV2.2 or L-type calcium channels. The three-dimensional structure of Hf1a-NH2 was determined using NMR spectroscopy and used in docking studies to predict its binding site at CaV3.2 and CaV3.3. As both CaV3.2 and CaV3.3 have been implicated in peripheral pain signaling, the analgesic potential of Hf1a-NH2 was explored in vivo in a mouse model of incision-induced acute post-surgical pain. Consistent with this role, Hf1a-NH2 produced antiallodynia in both mechanical and thermal pain.
... Zapata, Jaramillo-Serna, & Avila-Reyes, 2007).Desde las ciencias básicas, hallazgos recientes definen los sitios receptores y mecanismos de acción de las toxinas hidrófilas y polipeptídicas, alteran los canales de sodio, potasio y calcio creando voltajes a manera de señalización para la generación del potencial acción y la conducción en una amplia variedad de neurotoxinas. Las toxinas polipeptídicas alteran el canal de apertura por la captura del sensor de voltaje a través de la unión a receptores extracelulares(Catterall WA, 2007) (Cestèle S, 2000). En el caso de los péptidos purificados del veneno del escorpión, demostraron la interacción con ciertos residuos de aminoácidos presentes en el vestíbulo exterior, diversos subtipos de canales de potasio, la pág. ...
Introducción: la Norma Oficial Mexicana en picadura de alacrán (NOM-033-SSA2-2011) establece reglas oficiales que se debe conocer y cumplir por parte del personal de enfermería. Objetivo: analizar el conocimiento y cumplimiento de la NOM-033-SSA2-2011, por parte del personal de enfermería. Metodología: estudio descriptivo y transversal realizado en una jurisdicción del Estado de Colima, México, donde se aplican dos instrumentos diseñados para su identificación, datos que fueron analizados mediante estadística descriptiva e inferencial. Resultados: la probabilidad de ocurrencia del fenómeno (conocimiento/cumplimiento) sobre la Norma en picadura Alacrán es 58,9%, (OR 0.0589) y el riesgo de que esto ocurra (RR 0.1064) esto quiere decir que la ocurrencia de conocimiento/cumplimiento es del 10.64%, IC 95% (P≤ 0.001). Conclusiones: con base en estos resultados se concluye reflexionar sobre los factores internos y externos que influyen en el fenómeno de conocimiento/cumplimiento establecido por la NOM-033-SSA2-2011.
... Harmful algal blooms (HAB) have made major negative influence on aquatic animals and potentially human health around the world (Glibert and Pitcher, 2001;Griffith and Gobler, 2020;Rolton et al., 2014). Of these harmful algae, the dinoflagellates Alexandrium and Gymnodinium, could produce a type of toxic alkaloid neurotoxin paralytic shellfish toxins (PSTs), which can poison marine animals and impact their survival by selectively blocking sodium channels and suppressing neural action potentials (Anderson et al., 2005;Cestèle and Catterall, 2000). Filter-feeding bivalves (e.g., scallops, clams, oysters, and mussels) filtered dinoflagellates or their cysts are key PST vectors in marine ecosystems, particularly during red tide outbreaks (Bricelj and Shumway, 1998;Yang et al., 2018). ...
... STXs are a water-soluble neurotoxins that bind to the voltage-dependent sodium channels in excitatory cells leading to hyperpolarization of the cell [8]. STXs are one of the most potent neurotoxins that were first reported in butter clam and mussel tissues in 1957 [9]. ...
Saxitoxins (STXs) are a highly marine neurotoxins derived from harmful algal blooms and cause paralytic shellfish poisoning (PSP) that pose a significant risk to public and environmental health. The study of STXs toxicity has been carried out but little and is known about the histopathological responses on mice. The purpose of this study was to evaluate immunotoxic and histological responses induced by STXs extracted from Acanthocardia tuberculatum. To this end, daily, mice were treated orally during 7 days with sublethal concentrations (10 mg /100 g mouse). Lymphocyte proliferation and brain histopathology were analysed after treatment. The results showed a significative increase of lymphocytes level and a decrease of polynuclears level. The histological study in brain mice showed an increase of the number of the nucleus as well as a hypercondensation of the chromatin brain, Also, we observed the presence of some multinucleated giant cells that indicate the inflammation in brain. We conclude that STXs induce inflammation and cells necrosis in brain mice and causes the importation of the immunizing cells and the development of the inflammatory reactions. This work is licensed under a Creative Commons Attribution Non-Commercial 4.0 International License.
... On the other hand, β-toxins bind to site four of the channel, immobilizing it and keeping it in the activated position [44,45]. The α and β-toxins, such as Ts1-5, Ts17, Ts18, Ts26-28, and Ts30, have a specific affinity for Na + channels, thereby modulating the activated channels. ...
Tityus serrulatus scorpion is responsible for a significant number of envenomings in Brazil, ranging from mild to severe, and in some cases, leading to fatalities. While supportive care is the primary treatment modality, moderate and severe cases require antivenom administration despite potential limitations and adverse effects. The remarkable proliferation of T. serrulatus scorpions, attributed to their biology and asexual reproduction, contributes to a high incidence of envenomation. T. serrulatus scorpion venom predominantly consists of short proteins acting as neurotoxins (α and β), that primarily target ion channels. Nevertheless, high molecular weight compounds, including metalloproteases, serine proteases, phospholipases, and hyaluronidases, are also present in the venom. These compounds play a crucial role in envenomation, influencing the severity of symptoms and the spread of venom. This review endeavors to comprehensively understand the T. serrulatus scorpion venom by elucidating the primary high molecular weight compounds and exploring their potential contributions to envenomation. Understanding these compounds' mechanisms of action can aid in developing more effective treatments and prevention strategies, ultimately mitigating the impact of scorpion envenomation on public health in Brazil.
... Saxitoxins are neurotoxic alkaloids that can cause paralysis by reversibly blocking voltage-gated Na + channels within the body (Kao 1966;Cestèle and Catterall 2000). The chemical structure of STX extracted from Alaskan butter clams was first reported in 1975 (Schantz et al. 1975) and more than 50 natural analogs have been found to date (Wiese et al. 2010). ...
The marine dinoflagellate Alexandrium catenella (Group I) produces saxitoxins (STXs) in marine environments, causing paralytic shellfish poisoning (PSP). The toxins are synthesized by STXs biosynthesis genes (sxt); of them, sxtA and sxtG are considered the most essential. The promoter region and 3'-untranslated region (3'-UTR) are known to regulate gene transcription; however, they have not been sufficiently elucidated in the sxt genes. Herein, we determined and characterized the genomic DNA sequence of the proximal regions of sxtA and sxtG in A. catenella. The promoter regions of sxtA and sxtG in A. catenella were found to contained distinct cis-regulatory elements (CREs), CpG islands, and TTTT-motif (instead of TATA-box), showing a typical eukaryotic promoter. Especially, a number of light-responsive CREs (G-box, SORLIP1AT/2AT, and Sp1) were found, and the gene expression level showed a significant relationship with photosynthesis. MiSeq sequencing of the long PCR amplicons revealed that sxtA was encoded as a single exonic structure that is discrete (> 5 kbp) from other core sxt. In addition, we reported for the first time two types of 3'-UTR in sxtG mRNA, and their lengths and sequences varied among strains and species. Such results suggest that sxt may be regulated by post-transcription. Our results provide insights into the toxin dynamics of toxic dinoflagellates from a molecular perspective.
... [1][2][3][4][5][6][7] The STX reversibly binds to specific amino acid residues near the sodium channels, blocking sodium permeableness of the excitatory membrane. 8,9 Blocking such voltagedependent sodium channels prevents the creation of appropriate action potentials in nerves and muscle fibers, resulting in neuromuscular paralysis and respiratory arrest. ...
Saxitoxin (STX) is a representative neurotoxin among paralytic shellfish poisons and poses a serious threat to human health. When ingested, it blocks sodium permeability of excitatory membranes and causes neuromuscular paralysis and respiratory arrest, leading to death. Therefore, technology capable of detecting STX in advance is required. In this study, to develop the bioreceptor that specifically binds to STX, a phage display was introduced. For this technique, the STX hapten was synthesized by reacting the amine group of STX with the carboxyl group of ovalbumin, a carrier protein, using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysulfosuccinimide sodium salt coupling reaction. The peptides discovered through this were selected according to frequency and similarity, and the 5′-terminal was modified with 5-carboxyfluorescein as the fluorophore. To construct a simple verification procedure for the discovered peptides, we developed a peptide-based fluorometric sensor using the graphene oxide (GO) quenching phenomenon. In this sensor, GO as a quencher can be strongly adsorbed through π–π interaction with a fluorophore and charge–charge interaction with STX at pH 7.4, and it showed a low detection limit of 1.5 ppb.
... Na V channel site 2 primarily comprises residues of the S6 helices of each channel domain which together line the hydrophobic central cavity of the channel pore (44). Binding of site 2 toxins causes (i) a characteristic large shift of the voltage-dependence of activation towards more negative potentials; (ii) slowing or inhibition of fast inactivation; and (iii) a reduction in Na + conductance and selectivity (45,46). Furthermore, the double-Boltzmann distribution of the voltage-dependence of activation observed for the ant venom Na V channel toxins is also shared by certain site two toxins (42,43,47), which in each case was interpreted as reflective of two separate populations of channels: toxin-modified and non-modified. ...
Harvester ants (genus Pogonomyrmex) are renowned for their stings which cause intense, long-lasting pain, and other neurotoxic symptoms in vertebrates. Here, we show that harvester ant venoms are relatively simple and composed largely of peptide toxins. One class of peptides is primarily responsible for the long-lasting local pain of envenomation via activation of peripheral sensory neurons. These hydrophobic, cysteine-free peptides potently modulate mammalian voltage-gated sodium (NaV) channels, reducing the voltage threshold for activation and inhibiting channel inactivation. These toxins appear to have evolved specifically to deter vertebrates.
... Seven binding sites for toxins and drugs were defined on the primary sequence of Na v channels based on rigorous functional characterizations [79][80][81][82][83][84][85]. For instance, receptor site 1 is targeted by the archetypal pore-blocking toxins tetrodotoxin (TTX) and saxitoxin (STX), and receptor sites 3 and 4 are for α and β-scorpion toxins, respectively ( Figure 2a). ...
Voltage-gated sodium (Nav) channels govern membrane excitability by initiating and propagating action potentials. Consistent with their physiological significance, dysfunction, or mutations in these channels are associated with various channelopathies. Nav channels are thereby major targets for various clinical and investigational drugs. In addition, a large number of natural toxins, both small molecules and peptides, can bind to Nav channels and modulate their functions. Technological breakthrough in cryo-electron microscopy (cryo-EM) has enabled the determination of high-resolution structures of eukaryotic and eventually human Nav channels, alone or in complex with auxiliary subunits, toxins, and drugs. These studies have not only advanced our comprehension of channel architecture and working mechanisms but also afforded unprecedented clarity to the molecular basis for the binding and mechanism of action (MOA) of prototypical drugs and toxins. In this review, we will provide an overview of the recent advances in structural pharmacology of Nav channels, encompassing the structural map for ligand binding on Nav channels. These findings have established a vital groundwork for future drug development.
... Saxitoxin is a paralytical neurotoxin, which also acts as a precursor to several other paralytic shellfish toxins (PSTs). Saxitoxin and its family members function largely by inhibition of the voltagegated sodium ion channel, though saxitoxin and some of its derivatives can also work to inhibit the potassium and calcium voltage-gated channels [10,11]. Anatoxins, sometimes referred to as the "very fast death factor," are another common form of cyanotoxin. ...
Cyanobacterial harmful algal blooms (CyanoHABs) are increasing in prevalence and severity globally and locally in the Great Lakes region. CyanoHABs have the potential to produce serious adverse human health effects due to the production of cyanotoxins from cyanobacteria. Common routes of exposure include recreational exposure (swimming, skiing, and boating), ingestion, and aerosolization of contaminated water sources. Cyanotoxins have been shown to adversely affect several major organ systems contributing to hepatotoxicity, gastrointestinal distress, and pulmonary inflammation. We present three pediatric case-reports that coincided with CyanoHABs exposure with a focus on presentation of illness, diagnostic work-up, and treatment of CyanoHAB-related illnesses. Potential cyanotoxin exposure occurred while swimming in the Maumee River and Maumee State Park in Northwest OH during the Summer months which coincide with peak CyanoHAB activity. Primary symptoms included generalized macular rash, fever, vomiting, diarrhea, and severe respiratory distress. Significant labs included leukocytosis and elevated C-reactive protein. All patients ultimately recovered with supportive care. Symptoms following potential cyanotoxin exposure coincide with multiple disease states representing an urgent need to develop specific diagnostic tests of exposure.
... Saxitoxin is a paralytical neurotoxin, which also acts as a precursor to several other paralytic shellfish toxins (PSTs). Saxitoxin and its family members function largely by inhibition of the voltage-gated sodium ion channel, though saxitoxin and some of its derivatives can also work to inhibit the potassium and calcium voltage-gated channels [10,11]. Anatoxins, sometimes referred to as the "very fast death factor", are another common form of cyanotoxin. ...
Cyanobacterial harmful algal blooms (CyanoHABs) are increasing in prevalence and severity in the Great Lakes region, as well as both globally and locally. CyanoHABs have the potential to cause adverse effects on human health due to the production of cyanotoxins from cyanobacteria. Common routes of exposure include recreational exposure (swimming, skiing, and boating), ingestion, and aerosolization of contaminated water sources. Cyanotoxins have been shown to adversely affect several major organ systems contributing to hepatotoxicity, gastrointestinal distress, and pulmonary inflammation. We present three pediatric case reports that coincided with CyanoHABs exposure with a focus on presentation of illness, diagnostic work-up, and treatment of CyanoHAB-related illnesses. Potential cyanotoxin exposure occurred while swimming in the Maumee River and Maumee Bay of Lake Erie in Ohio during the summer months with confirmed CyanoHAB activity. Primary symptoms included generalized macular rash, fever, vomiting, diarrhea, and severe respiratory distress. Significant labs included leukocytosis and elevated C-reactive protein. All patients ultimately recovered with supportive care. Symptoms following potential cyanotoxin exposure coincide with multiple disease states representing an urgent need to develop specific diagnostic tests of exposure.
... By regulating Na + flow across the cell membrane to initiate and propagate action potentials, NaV channels are responsible for the electrical excitability of cells and play crucial roles in ion regulation, neuromuscular communication, and digestion [54,55]. In addition, NaV channels are involved in regulating the cellular K + flux [56], which is required for the continuous functioning of the sodium-potassium pump (Na + /K + ATPase). ...
Harmful algal blooms (HABs) can produce a variety of noxious effects and, in some cases, the massive mortality of wild and farmed marine organisms. Some HAB species produce toxins that are released into seawater or transferred via food webs (particulate toxin fraction). The objective of the present study was to identify the toxicological effects of subacute exposure to saxitoxin (STX) during embryonic and early larval stages in Seriola rivoliana. Eggs were exposed to dissolved 19 STX (100 μg L⁻¹). The toxic effects of STX were evaluated via the hatching percentage, the activity of three enzymes (protein and alkaline phosphatases and peroxidase), and the expression of four genes (HSF2, Nav1.4b, PPRC1, and DUSP8). A low hatching percentage (less than 5%) was observed in 44 hpf (hours post fertilization) embryos exposed to STX compared to 71% in the unexposed control. At this STX concentration, no oxidative stress in the embryos was evident. However, STX induced the expression of the NaV1.4 channel α-subunit (NaV1.4b), which is the primary target of this toxin. Our results revealed the overexpression of all four candidate genes in STX-intoxicated lecithotrophic larvae, reflecting the activation of diverse cellular processes involved in stress responses (HSF2), lipid metabolism (PPRC1), and MAP kinase signaling pathways associated with cell proliferation and differentiation (DUSP8). The effects of STX were more pronounced in young larvae than in embryos, indicating a stage-specific sensitivity to the toxin.
... These blooms, often consisting of toxic dinoflagellates, can cause substantial damage to marine ecosystems, animal health, commercial industries such as mariculture, fisheries and tourism and human health (Murray et al., 2012;Vandersea et al., 2018;Wang, 2008). Among the many dinoflagellate species associated with HABs, Alexandrium minutum is notoriously known for its widespread distribution in coastal areas (Anderson et al., 2012) and its capacity to produce potent neurotoxins called saxitoxin (Abassi et al., 2023;Cestèle and Catterall, 2000;Murray et al., 2012). There has been a growing body of research in recent decades aimed at elucidating the factors influencing A. minutum abundance and saxitoxin production. ...
... Saxitoxin is a paralytical neurotoxin, which also acts as a precursor to several other paralytic shellfish toxins (PSTs). Saxitoxin and its family members function largely by inhibition of the voltagegated sodium ion channel, though saxitoxin and some of its derivatives can also work to inhibit the potassium and calcium voltage-gated channels [9,10]. Anatoxins, sometimes referred to as the "very fast death factor," are another common form of cyanotoxin. ...
Cyanobacterial harmful algal blooms (CyanoHABs) are increasing in prevalence and severity globally and locally in the Great Lakes region. CyanoHABs have the potential to produce serious adverse human health effects due to the production of cyanotoxins from cyanobacteria. Common routes of exposure include recreational exposure (swimming, skiing, and boating), ingestion, and aerosolization of contaminated water sources. Cyanotoxins have been shown to adversely affect several major organ systems contributing to hepatotoxicity, gastrointestinal distress, and pulmonary inflammation. We present three pediatric case-reports that coincided with CyanoHABs exposure with a focus on presentation of illness, diagnostic work-up, and treatment of CyanoHAB-related illnesses. Potential cyanotoxin exposure occurred while swimming in the Maumee River and Maumee State Park in Northwest OH during the Summer months which coincide with peak CyanoHAB activity. Primary symptoms included generalized macular rash, fever, vomiting, diarrhea, and severe respiratory distress. Significant labs included leukocytosis and elevated C-reactive protein. All patients ultimately recovered with supportive care. Symptoms following potential cyanotoxin exposure coincide with multiple disease states representing an urgent need to develop specific diagnostic tests of exposure.
... The neurotoxin known as saxitoxin (STX), which is soluble in water, has the ability to bind "to the voltage-dependent sodium channels that are present in excitatory cells". According to Cestelle and Catterall (2000), this interaction is responsible for cell hyperpolarization because it prevents sodium ions from entering the cell while enabling potassium ions to leave. Ions of sodium are permitted to exit the cell where they were produced. ...
The bioaccumulation of saxitoxins (STX) across the trophic chain is not well known, particularly in freshwater environments. This level of ignorance is quite concerning. In this work, the trophic bioassay was utilised to investigate "the effects that STX had on Hoplias malabaricus. The fish were given Astyanax sp. once every five days prior to receiving an intraperitoneal injection with the lysate of a Cylindrospermopsis raciborskii culture containing 97% STX, 3% neosaxitoxin, and 3% gonyautoxin for a period of 20 days. After this, the fish were given the inoculation via the intraperitoneal route. SOD, CAT, GST, GPx, GSH, LPO, and PCO were some of the biomarkers that were used in the evaluation of the animal's liver. The genotoxic and haematological effects of the drug were determined by examination of the blood. Both the hepatosomatic index and the relative condition factor failed to demonstrate a statistically significant difference between the groups that were exposed and those who served as controls. The STX group exhibited significantly greater mean corpuscular haemoglobin concentrations and values than the other groups. Both groups exhibited the typical patterns of hepatic tissue found in teleost fish. Both of these groups conformed to this trend". Increases in GPx activity, LPO concentrations, and GSH levels all point to an increase in the formation of reactive oxygen species. The SOD activity moved in the other direction. CAT and DNA damage were not affected in any way. Both PCO and CAT were unaffected by this. The neurotoxic cyanotoxin STX caused alterations in the biochemistry of liver tissue that may have important ecological repercussions.
... Symptoms include numbness and tingling sensations, headaches, nausea and paralysis, which are caused by saxitoxin (STX) and its analogues (STXs). The toxins reversibly bind the voltage-gated Na + channels of neurons (Etheridge, 2010;Cestèle and Catterall, 2000). STXs are produced by certain toxic dinoflagellates such as Alexandrium spp., Gymnodinium catenatum, and Pyrodinium bahamense (Wang, 2008). ...
The dinoflagellate Alexandrium pacificum (group IV) is of particular interest because of its involvement in harmful algal blooms and production of saxitoxin (STX), which causes paralytic shellfish poisoning. The toxicity from STX and its analogues (STXs) is suspected to be affected by nitrogen (N) availability. However, the toxicity-associated behavior and STX-biosynthesis gene responses of the toxic A. pacificum under N fluctuations have not been sufficiently investigated. In the present study, we identified the sxtI gene involved in sxt biosynthesis pathway and evaluated the effects of nitrate (NO3-) on STXs production and the expression of four sxt core genes (sxtA4, sxtG, sxtB, and sxtI). Quantification of total STXs levels in the cultures under different NO3- regimes showed that NO3- concentration influenced STXs production. In addition, the proportion and concentration of STXs varied depending on the NO3- concentration. Core sxt transcript abundance was also influenced by available NO3- in a time-dependent manner. Expressional levels and patterns of sxtI were correlated with those of sxtA and sxtB. The relationship between the toxins and sxt responses in A. pacificum under various NO3- regimes suggests the direct involvement of N in the STXs biosynthesis pathway. Understanding this link would provide a tool to understand the toxin dynamics of dinoflagellates following N shifts in marine environments.
... The first dinoflagellate associated with PSTs was Alexandrium catenella in 1927 near San Francisco, when it caused the death of 6 people and symptoms in more than 100 (Anderson, 2021;Cusick, 2013). PSTs bind on site 1 of the subunit voltage-gated sodium ion channels in a specific and reversible mode (Cestèle, 2000), blocking the Na + influx into the cell membrane and preventing the conduction of action potentials. Thus, PSTs affect nerve and muscle excitability (Cusick, 2013). ...
The chemical methodology to quantify paralytic shellfish toxins (PSTs) is ultrahigh-performance liquid chromatography with fluorescence detection and precolumn oxidation. It has been established as the European Union (EU) reference method replacing the mouse bioassay. There are some alternatives to this method for a better quantification of the whole range of PSTs; however, the detection of GC toxins in mollusks is still a challenge since they are retained when the C18 solid phase extraction (SPE) procedure is used to remove interferences. In addition to that they are not often included in the detection and quantification methods because of the lack of reference standards. Due to this in this work a new method is proposed to convert GC4 and GC5, for which analytical standards are not commercially available, into their decarbamoyl analogues dcGTX1 and dcGTX4, respectively. It was possible the conversion of these toxins, and the experimental conditions were tested and established regarding pH, temperature and time for the hydrolysis.
... As sodium channel receptor affinity is remarkably influenced by the membrane potential, the primary outcome of α-toxins is to extend the action potential of both neurons and muscular tissue (11). American scorpion-derived β-toxins alter the membrane potential to a more negative state by interacting with the fourth receptor site on vertebrate sodium channels (13). A sensor out of four voltage sensors in the Na+ channel is thought to be the sole sensor to which the -scorpion toxin binds (14). ...
Scorpion venom can lead to serious medical issues and even death. It comprises a complex of numerous toxins with diverse biological features and activities. The clinical manifestations of envenoming are thought to be caused by neurotoxins, major constituents in scorpion venom. They are extremely selective and powerful ligands for multiple ion channel types. Consequently, they present desirable molecules for the development of innovative medications, including those for the treatment of cardiovascular diseases, cancer, and neurological issues. Notwithstanding the uncertainty and complexity regarding the pathophysiology of envenomation, venom and its accompanying host immunological reactions are known to induce the synthesis of cytokines, critical molecules of inflammation. This review sheds light on scorpion-derived venom and its primary toxins, as well as their biological pathology and the treatment of scorpion stings.
... They are peptides containing 60 to 70 amino acid residues with four disulfide bonds [108,109], which bind to sodium channel receptor site 3, located in the extracellular segment S3-S4 of domain IV of the channel [100,110]. ...
The Buthidae family of scorpions consists of arthropods with significant medical relevance, as their venom contains a diverse range of biomolecules, including neurotoxins that selectively target ion channels in cell membranes. These ion channels play a crucial role in regulating physiological processes, and any disturbance in their activity can result in channelopathies, which can lead to various diseases such as autoimmune, cardiovascular, immunological, neurological, and neoplastic conditions. Given the importance of ion channels, scorpion peptides represent a valuable resource for developing drugs with targeted specificity for these channels. This review provides a comprehensive overview of the structure and classification of ion channels, the action of scorpion toxins on these channels, and potential avenues for future research. Overall, this review highlights the significance of scorpion venom as a promising source for discovering novel drugs with therapeutic potential for treating channelopathies.
P2Y receptors represent a class of purinergic receptors distributed across the human body, mainly involved in various physiological processes. These belong to the family of G protein-coupled receptors (GPCRs). Activated by nucleotides such as ATP and ADP, this family has eight distinct subtypes (P2Y1, P2Y2, P2Y4, P2Y6, P2Y11, P2Y12, P2Y13, and P2Y14). They play diverse functions in neurotransmission, immune modulation, and glial transmission by controlling neurotransmitters and neuronal excitability. Their implication in many neurodegenerative diseases like epilepsy, Alzheimer’s and Parkinson’s disease, as well as neuropathic pain, makes them an important therapeutic target.
Neuronal signaling depends on rapid changes in electrical fields across cell membranes mediated by ion channels. Three important properties of these transmembrane proteins facilitate the rapid changes in membrane potential: passive conduction of ions, ion selectivity, and open and closing (gating) in response to electrical, mechanical, or chemical signals.
Natural channel proteins allow the selective permeation of ions, water or other nutritious entities across bilayer membranes, facilitating various essential physiological functions in living systems. Inspired by nature, chemists endeavor to simulate the structural features and transport behaviors of channel proteins through biomimetic strategies. In this review, we start from introducing the inherent traits of channel proteins such as their crystal structures, functions and mechanisms. Subsequently, different kind of synthetic ion channels including their design principles, dynamic regulations and therapeutic applications were carefully reviewed. Finally, the potential challenges and opportunities in this research field were also carefully discussed. It is anticipated that this review could provide some inspiring ideas and future directions towards the construction of novel bionic ion channels with higher‐level structures, properties, functions and practical applications.
Neurotoxins present a substantial threat to human health and security as they disrupt and damage the nervous system. Their potent and structurally diverse nature poses challenges in developing effective countermeasures. In this study, a unique nanoparticle design that combines dual‐biomimicry mechanisms to enhance the detoxification efficacy of neurotoxins is introduced. Using saxitoxin (STX), one of the deadliest neurotoxins, and its natural binding protein saxiphilin (Sxph) as a model system, human neuronal membrane‐coated and Sxph‐loaded metal–organic framework (MOF) nanosponges (denoted “Neuron‐MOF/Sxph‐NS”) are successfully developed. The resulting Neuron‐MOF/Sxph‐NS exhibit a biomimetic design that not only emulates host neurons for function‐based detoxification through the neuronal membrane coating, but also mimics toxin‐resistant organisms by encapsulating the Sxph protein within the nanoparticle core. The comprehensive in vitro assays, including cell osmotic swelling, calcium flux, and cytotoxicity assays, demonstrate the improved detoxification efficacy of Neuron‐MOF/Sxph‐NS. Furthermore, in mouse models of STX intoxication, the application of Neuron‐MOF/Sxph‐NS shows significant survival benefits in both therapeutic and prophylactic regimens, without any apparent acute toxicity. Overall, the development of Neuron‐MOF/Sxph‐NS represents an important advancement in neurotoxin detoxification, offering promising potential for treating injuries and diseases caused by neurotoxins and addressing the current limitations in neurotoxin countermeasures.
The binding of the radioiodinated insect selective neurotoxin from the venom of the scorpion Androctonus australis (AaIT), to synaptic plasma membrane vesicles derived from osmotically shocked insect synaptosomes was studied under kinetic and equilibrium conditions. The integrity of these vesicles and the existence of membrane potential and its modifiability were demonstrated by assays of the uptake of the lipophilic cation tetraphenylphosphonium. It has been shown that 125I-labeled AaIT binds specifically and reversibly to a single class of noninteracting binding sites of high affinity () and low capacity (1.2–2.0 pmol/mg protein). The values of the rate association and dissociation constants k1 and k−1 are, respectively, 1.36 · 106 M−1 · s−1 and 1.9 · 10−3 s−1, and are in a good accordance with the equilibrium constant. The use of various ionophores and changes in external potassium concentration shown to modify the membrane potential of the present neuronal preparation, did not affect the binding of 125I-AaIT, thus indicating its voltage-independence. Veratridine, tetrodotoxin, sea anemone toxin and the α and β scorpion toxins specific for vertebrates did not affect the binding of 125I-AaIT. Furthermore, the above scorpion toxins were devoid of specific binding to the present insect neuronal preparation. Two additional insect toxins derived from the venom of the scorpion Buthotus judaicus, BjIT1 (spastic-excitatory toxin, homologus to the AaIT) and BjIT2 (flaccidity inducing-depressory toxin), were both shown to displace the 125I-AaIT with a high affinity (Kd = 2.2 and 1.3 nM, respectively). These data are compared and discussed in light of the information concerning the interaction of scorpion venom toxins affecting vertebrates with mammalian neuronal tissues.
The sodium channel is a voltage-gated ionic channel essential for the generation of action potentials. It has been reported that the sodium channels purified from the electric organ of Electrophorus electricus (electric eel) and from chick cardiac muscle consist of a single polypeptide of relative molecular mass (Mr) approximately 260,000 (260K), whereas those purified from rat brain and skeletal muscle contain, in addition to the large polypeptide, two or three smaller polypeptides of Mr 37-45K. Recently, we have elucidated the primary structure of the Electrophorus sodium channel by cloning and sequencing the DNA complementary to its messenger RNA. Despite the apparent homogeneity of the purified sodium channel preparations, several types of tetrodotoxin (or saxitoxin) binding sites or sodium currents have been observed in many excitable membranes. The occurrence of distinguishable populations of sodium channels may be attributable to different states of the same channel protein or to distinct channel proteins. We have now isolated complementary DNA clones derived from two distinct rat brain mRNAs encoding sodium channel large polypeptides and present here the complete amino-acid sequences of the two polypeptides (designated sodium channels I and II), as deduced from the cDNA sequences. A partial DNA sequence complementary to a third homologous mRNA from rat brain has also been cloned.
Toxin II isolated from the sea anemone Anemonia sulcata enhances activation of the action potential sodium ionophore of electrically excitable neuroblastoma cells by veratridine and batrachotoxin. This heterotropic cooperative effect is identical to that observed previously with scorpion toxin but occurs at a 110-fold higher concentration. Depolarization of the neuroblastoma cells inhibits the effect of sea anemone toxin as observed previously for scorpion toxin. Specific scorpion toxin binding is inhibited by sea anemone toxin with KD approximately equal to 90 nM. These results show that the polypeptides scorpion toxin and sea anemone toxin II share a common receptors site associated with action potential sodium ionophores.
Scorpion mono[125I]iodotoxin binds to a single class of receptor sites associated with voltage sensitive sodium channels in synaptic nerve ending particles (synaptosomes) with a K(D) of approximately 3 nM. Scorpion toxin binding is inhibited by depolarization of the synaptosomes with K+ or gramicidin or by lysis of the synaptosomes. Scorpion toxin binding is enhanced by batrachotoxin, veratridine, and aconitine if depolarization of the synaptosomes by these toxins is prevented by sodium free medium containing 1 μM tetrodotoxin. Batrachotoxin, veratridine, and aconitine act at a common receptor site in enhancing scorpion toxin binding. Batrachotoxin may be a full agonist at this receptor site, whereas veratridine and aconitine are partial agonists. The enhancement of scorpion toxin binding by batrachotoxin is accompanied by a large shift in the voltage dependence of scorpion toxin binding. These observations demonstrate allosteric interactions between the receptor sites for scorpion toxin and the alkaloid toxins. The results are considered in terms of a two-state allosteric model. In the presence of batrachotoxin, scorpion toxin binds to a single class of sites with a K(D) of 1.5 nM and a binding capacity of 1750 fmol/mg of protein. The voltage sensitivity of these sites suggests that they are all associated with synaptosomes. Therefore, synaptosomes contain 63 scorpion toxin sites/individual nerve ending or 35 sites/μm2 of surface membrane. This density of sodium channels is similar to that observed in unmyelinated nerves.
Purified scorpion toxin (Leiurus quinquestriatus) slows inactivation of sodium channels in frog muscle at concentrations in the range of 17-170 nM. Mono[125I]iodo scorpion toxin binds to a single class of sites in frog sartorius muscle with a dissociation constant of 14 nM and a binding capacity of 13 fmol/mg wet weight. Specific binding is inhibited more than 90% by 3 microM sea anemone toxin II and by depolarization with 165 mM K+. Half-maximal inhibition of binding is observed on depolarization to -41 mV. The voltage dependence of scorpion toxin binding is correlated with the voltage dependence of activation of sodium channels. Removal of calcium from the bathing medium shifts both activation and inhibition of scorpion toxin binding to more negative membrane potentials. The results are considered in terms of the hypothesis that activation of sodium channels causes a conformational change in the scorpion toxin receptor site resulting in reduced affinity for scorpion toxin.
The inward Na+ current underlying the action potential in nerve is terminated by inactivation. The preceding report shows that deletions within the intracellular linker between domains III and IV remove inactivation, but mutation of conserved basic and paired acidic amino acids has little effect. Here we show that substitution of glutamine for three clustered hydrophobic amino acids, Ile-1488, Phe-1489, and Met-1490, completely removes fast inactivation. Substitution of Met-1490 alone slows inactivation significantly, substitution of Ile-1488 alone both slows inactivation and makes it incomplete, and substitution of Phe-1489 alone removes inactivation nearly completely. These results demonstrate an essential role of Phe-1489 in Na(+)-channel inactivation. It is proposed that the hydrophobic cluster of Ile-1488, Phe-1489, and Met-1490 serves as a hydrophobic latch that stabilizes the inactivated state in a hinged-lid mechanism of Na(+)-channel inactivation.
Voltage-sensitive sodium channels are responsible for the initiation and propagation of the action potential and therefore are important for neuronal excitability. Complementary DNA clones encoding the beta 1 subunit of the rat brain sodium channel were isolated by a combination of polymerase chain reaction and library screening techniques. The deduced primary structure indicates that the beta 1 subunit is a 22,851-dalton protein that contains a single putative transmembrane domain and four potential extracellular N-linked glycosylation sites, consistent with biochemical data. Northern blot analysis reveals a 1,400-nucleotide messenger RNA in rat brain, heart, skeletal muscle, and spinal cord. Coexpression of beta 1 subunits with alpha subunits increases the size of the peak sodium current, accelerates its inactivation, and shifts the voltage dependence of inactivation to more negative membrane potentials. These results indicate that the beta 1 subunit is crucial in the assembly, expression, and functional modulation of the heterotrimeric complex of the rat brain sodium channel.
We describe the properties of a family of 22-amino acid peptides, the mu-conotoxins, which are useful probes for investigating voltage-dependent sodium channels of excitable tissues. The mu-conotoxins are present in the venom of the piscivorous marine snail, Conus geographus L. We have purified seven homologs of the mu-conotoxin set and determined their amino acid sequences, as follows, where Hyp = trans-4-hydroxyproline. GIIIA R.D.C.C.T.Hyp.Hyp.K.K.C.K.D.R.Q.C.K.Hyp.Q.R.C.C.A-NH2 [Pro6]GIIIA R.D.C.C. T.P.Hyp.K.K.C.K.D.R.Q.C.K.Hyp.Q.R.C.C.A-NH2 [Pro7]GIIIA R.D.C.C.T.Hyp.P.K.K.C.K.D.R.Q.C.R.Hyp.Q.R.C.C.A-NH2 GIIIB R.D.C.C.T.Hyp.Hyp.R.K.C.K.D.R.R.C.K.Hyp.M.K.C.C.A-NH2 [Pro6]GIIIB R.D.C.C.T.P.Hyp.R.K.C.K.D.R.R. C.K.Hyp.M.K.C.C.A-NH2 [Pro7]GIIIB R.D.C.C.T.Hyp.P.R.K.C.K.D.R.R.C.K.Hyp.M.K.C.C.A-NH2 GIIIC R.D.C.C.T.Hyp.Hyp.K.K.C.K.D.R.R.C.K.Hyp.L.K.C.C.A-NH2. Using the major peptide (GIIIA) in electrophysiological studies on nerve-muscle preparations and in single channel studies using planar lipid bilayers, we have established that the toxin blocks muscle sodium channels, while having no discernible effect on nerve or brain sodium channels. In bilayers the blocking kinetics of GIIIA were derived by statistical analysis of discrete transitions between blocked and unblocked states of batrachotoxin-activated sodium channels from rat muscle. The kinetics conform to a single-site, reversible binding equilibrium with a voltage-dependent binding constant. The measured value of the equilibrium KD for GIIIA is 100 nM at OmV, decreasing e-fold/34 mV of hyperpolarization. This voltage dependence of blocking is similar to that of tetrodotoxin and saxitoxin as measured by the same technique. The tissue specificity and kinetic characteristics suggest that the mu-conotoxins may serve as useful ligands to distinguish sodium channel subtypes in different tissues.
Two insect selective toxins were purified by gel-permeation and ion-exchange chromatographies from the venom of the scorpion, Leiurus quinquestriatus quinquestriatus, and their chemical and pharmacological properties were studied. The first toxin (LqqIT1) induces a fast excitatory contraction paralysis of fly larvae and is about 40 times more toxic than the crude venom. It is a polypeptide composed of 71 amino acids, including 8 half-cystines and devoid of methionine and tryptophan, with an estimated molecular weight of 8189 and a pI value of 8.5. The second toxin (LqqIT2) induces a slow depressant, flaccid paralysis of fly larvae. It is composed of 72 amino acids, including 8 half-cystines, is devoid of proline methionine and histidine, and has an estimated molecular weight of 7990 and a pI value of 8.3. The contrasting symptomatology of these toxins is interpreted in terms of their effects on an isolated axonal preparation of the cockroach in current and voltage clamp conditions. LqqIT1 (0.5-4 microM) induced repetitive firing of the axon which was attributable to two changes in the sodium conductance, a small increase in the peak conductance and a slowing of its turning off. LqqIT2 (1-8 microM) caused a blockage of the evoked action potentials, attributable to both a strong depolarization of the axonal membrane and a progressive suppression of the sodium current. Neither toxin affected potassium conductance. The two toxins differ mainly in their opposite effects on the activatable sodium permeability. In binding assays to a preparation of insect synaptosomal membrane vesicles, the two toxins were shown to competitively displace the radioiodinated excitatory insect toxin derived from the venom of the scorpion, Androctonus australis [( 125I]AaIT), which strongly resembles, in its chemistry and action, the LqqIT1 toxin. The present two toxins have demonstrated a strong affinity closely resembling the AaIT, with KD values of 0.4, 1.9, and 1.0 nM for LqqIT1, LqqIT2, and AaIT, respectively. These data suggest the possibility that the excitatory and depressant insect toxins share a common binding site associated with sodium channels in insect neuronal membranes.
Geographutoxin II (GTX II), a peptide toxin isolated from Conus geographus, inhibited [3H]saxitoxin binding to receptor sites associated with voltage-sensitive Na channels in rat skeletal muscle homogenates and rabbit T-tubular membranes with K0.5 values of 60 nM for homogenates and 35 nM for T-tubular membranes in close agreement with concentrations that block muscle contraction. Scatchard analysis of [3H]saxitoxin binding to T-tubular membranes gave values of KD = 9.3 nM and Bmax = 300 fmol/mg of protein and revealed a primarily competitive mode of inhibition of saxitoxin binding by GTX II. The calculated KD values for GTX II were 24 nM for T-tubules and 35 nM for homogenates, respectively. In rat brain synaptosomes, GTX II caused a similar inhibitory effect on [3H]saxitoxin binding at substantially higher concentrations (K0.5 = 2 microM). In contrast, binding of [3H]batrachotoxin A 20-alpha-benzoate and 125I-labeled scorpion toxin to receptor sites associated with Na channels in synaptosomes was not affected by GTX II at concentrations up to 10 microM. Furthermore, [3H]saxitoxin binding to membranes of rat superior cervical ganglion was only blocked 10% by GTX II at 10 microM. These results indicate that GTX II interacts competitively with saxitoxin in binding at neurotoxin receptor site 1 on the sodium channel in a highly tissue-specific manner. GTX II is the first polypeptide ligand for this receptor site and the first to discriminate between this site on nerve and adult muscle sodium channels.
The effect of two mu-conotoxin peptides on the specific binding of [3H]saxitoxin was examined in isolated plasma membranes of various excitable tissues. mu-Conotoxins GIIIA and GIIIB inhibit [3H]saxitoxin binding in Electrophorus electric organ membranes with similar KdS of approximately equal to 50 X 10(-9) M in a manner consistent with direct competition for a common binding site. GIIIA and GIIIB similarly compete with the majority (80-95%) of [3H]saxitoxin binding sites in rat skeletal muscle with KdS of approximately 25 and approximately 140 X 10(-9) M, respectively. However, the high-affinity saxitoxin sites in lobster axons, rat brain, and rat heart are virtually insensitive to GIIIA concentrations up to 10 microM. These results and previously published data suggest that three Na-channel subtypes can be distinguished on the basis of toxin pharmacology: Na channels of skeletal muscle and Electrophorus electroplax have high affinity for mu-conotoxins and tetrodotoxin, neuronal Na channels have low affinity for mu-conotoxins and high affinity for tetrodotoxin, while heart Na channels and a similar subtype also found in denervated muscle have low affinity for both mu-conotoxin and tetrodotoxin.
Structure-function relationships of the sodium channel expressed in Xenopus oocytes have been investigated by the combined use of site-directed mutagenesis and patch-clamp recording. This study provides evidence that the positive charges in segment S4 are involved in the voltage-sensing mechanism for activation of the channel and that the region between repeats III and IV is important for its inactivation.
1.The effects of the major neurotoxic fraction isolated from scorpion venom ofTityus serrulatus, TiTx?, on peripheral nerve membrane ofXenopus laevis were studied under current- and voltage-clamp conditions.2.700 nmol/l TiTx? depolarized the membrane and induced spontaneous activity (150s-1, maximum value), which ceased within a few minutes. It reduced the amplitude of the action potentials from 109 mV to 52 mV and increased their duration from 1.25 ms to 4.5 ms.3.440 nmol/l TiTx? induced inward Na current flow at resting potential. The descending branch of the Na current-voltage curve was flattened and shifted approximately 10 mV to more negative potentials. Maximum Na permeability was reduced to about 20%.4.Both development of and recovery from inactivation of Na permeability were slowed. The steepness of the steady-state inactivation curve was decreased, but the mid-potential changed only insignificantly.5.No prepulse was necessary to elicit either a shift of activation or an inward current at resting potential.6.Expressing the toxin effect either in terms of the decrease of Na peak current or of the slowing of inactivation, half-maximum effects were found with 0.3±0.1 and 3.7±0.7 µmol/l TiTx?, respectively.
1.
The effect of highly purified toxin γ from the venom of the scorpionTityus serrulatus (TiTxγ) on nerve membrane ionic channels have been investigated using the suction electrodes voltage clamp technique on neuroblastoma
cells.
2.
The amplitude of the normally voltage-dependent Na current is reversible reduced by approximately 50% after 15–105 nM TiTxγ,
whereas even the highest toxin concentrations have no significant effect on the outward K current in the presence of tetrodotoxin.
3.
TiTxγ causes a transient inward current to appear at membrane potentials between −70 and −40 mV, a potential region in which
no significant inward current is observed in control experiments.
4.
Tetrodotoxin (300 nM) rapidly blocks both the TiTxγ-induced inward current and the remaining normally voltagedependent Na
current. The binding of radiolabelled TiTxγ to the Na channels in the neuroblastoma cell membrane is prevented by native TiTxγ
with aK
0.5=0.75 nM.
5.
Both activation and inactivation of the TiTxγ-induced Na current are shifted 30–40 mV towards more negative potential values
as compared to normally voltage-dependent Na current. The TiTxγ-induced Na current exhibits sigmoidal activation kinetics
and relatively slow, exponential inactivation kinetics.
6.
The local anesthetic procaine at an external concentration of 1 mM blocks more effectively the remaining normally voltage-dependent
Na current than the TiTxγ-induced Na current. Both Na current components are equally blocked by 1 mM of the local anesthetic
propoxycaine.
7.
The relation between the effects of TiTxγ on Na+ channels and those of other known neurotoxins specific of this channel is discussed. It is concluded that the characteristic
effects of TiTxγ differ from those of all other known toxins including other scorpion toxins that bind to the same site on
the Na channel.
This article concludes a series of papers concerned with the flow of electric current through the surface membrane of a giant
nerve fibre (Hodgkinet al., 1952,J. Physiol.
116, 424–448; Hodgkin and Huxley, 1952,J. Physiol.
116, 449–566). Its general object is to discuss the results of the preceding papers (Section 1), to put them into mathematical
form (Section 2) and to show that they will account for conduction and excitation in quantitative terms (Sections 3–6).
125I-iododerivatives of the insect toxin (AaH IT1) from Androctonus australis Hector and of one toxin (Ts VII) from Tityus serrulatus were purified by high performance liquid chromatography (HPLC) and used in binding experiments on a nerve cord synaptosomal fraction of cockroach (P. americana). [125I]AaH IT1-b binds specifically and reversibly to a single class of non-interacting binding sites of high affinity ( nM, n = 5) and low capacity (1.7 ± 0.6 pmol/mg protein). The association (k1) and dissociation (k−1) constants are 3 × 106 M−1 s−1 (n = 2) and 7 × 10−4 s−1 (n = 2), respectively, and are in good agreement with the equilibrium constant. The interaction of [125I]Ts VII-d was irreversible and the association kinetics constant (k) was 2–5 × 107 M−1 s−1 (n = 4). As already shown Ts VII and AaH IT1 compete for the same binding site. The binding site capacity of [125I]Ts VII-d (1.6 ± 0.12 pmol/mg of protein, n = 2) was similar to that obtained with [125I]AaH IT1-b in the present experiments and with [125I]AaH IT1 on an insect synaptosomal fraction of central nervous system, and to that of a similar toxin (toxin γ) on rat brain synaptosomes. The injection of Ts VII in the sixth abdominal ganglion of the cockroach induces a progressive decrease in excitatory postsynaptic potentials and action potential.
A single point mutation of the rat sodium channel II reduces its sensitivity to tetrodotoxin and saxitoxin by more than three orders of magnitude. The mutation replaces glutamic acid 387 with a glutamine and has only slight effects on the macroscopic current properties, as measured under voltage-clamp in Xenopus oocytes injected with the corresponding cDNA-derived mRNA.
The protein neurotoxin II from the venom of the scorpion Hector was labeled with 125I by the lactoperoxidase method to a specific radioactivity of about 100 μCi/μg without loss of biological activity. The labeled neurotoxin binds specifically to a single class of non intereacting binding sites of high affinity (KD = 0.3 – 0.6 nM) and low capacity (4000 – 8000 sites/cell) to electrically excitable neuroblastoma cells. Relation of these sites to the action potential Na+ channel is derived from identical concentration dependence of scorpion toxin binding and increase in duration and amplitude of action potential. The protein neurotoxin II from the sea anemone also affects the closing of the action potential Na+ ionophore in nerve axons. The unlabelled sea anemone toxin modifies 125I-labeled scorpion toxin II binding to neuroblastoma cells by increasing the apparent KD for labeled scorpion toxin without modification of the number of binding sites. It is concluded that both scorpion toxin II and sea anemone toxin II interact competitively with a regulatory component of the action potential Na+ channel.
Chemical modification of some trifunctional amino acid residues in toxins I, II, and III of the scorpion Androctonus australis Hector have been performed. The results indicate: (1) Reduction and methylation of one disulfide bridge destroy toxic activity of toxin II. (2) The only tryptophan residue of toxin II (position 38) is not included in the active site of the molecule. (3) Modification of five carboxylates out of the seven contained in toxin II suppresses the toxic activity. (4) Acetylation of the lysine and tyrosine residues in toxin II leads to the loss of both toxic and antigenic activity. Treatment of the acetylated toxin by hydroxylamine restores partially the antigenic activity. In the case of toxin I, total acetylation abolishes only the toxic activity. It is concluded that at least one tyrosine residue must be involved in an antigenic site of toxin II. (5) Citraconylation of toxins II and III leads to complete loss of toxicity; decitraconylation restores full activity. (6) Guanidination of toxin II does not affect its toxicity significantly. (7) Alkylation of toxin II by iodoacetic acid affects both amino groups and histidine residues. The loss of toxicity is mainly due to the modification of the lysine residues. In the case of toxin I, the kinetics of toxicity loss closely parallel the covalent modification of one lysine residue.
Depolarization of neuroblastoma cells causes a 70-fold increase in the apparent dissociation constant KD for scorpion toxin enhancement of activation of the action potential Na+ ionophore by veratridine and a large increase in the rate of reversal of scorpion toxin action. Depolarization also inhibits binding of 125I-labeled scorpion toxin to a small number of saturable binding sites on electrically excitable neuroblastoma cells and increases the rate of dissociation of scorpion toxin from these sites. The results suggest that scorpion toxin binds to a regulatory component of the action potential Na+ ionophore whose conformation changes on depolarization.
1. The effect of Centruroides sculpturatus scorpion venom on single frog myelinated nerve fibres was studied. Sodium currents through the nodal membrane were measured under voltage-clamp conditions before and after exposure to venom in Ringer solution 1-5 mug/ml. for 1-3 min. 2. Centruroides venom brings about repetitive firing and increased membrane potential noise. Spontaneous firing was also observed. Eventually the nodal membrane becomes inexcitable following venom treatment. 3. Under voltage clamp with a step depolarization of the membrane potential, activation and inactivation of sodium currents turns on, reaches a peak within about 25 msec, and then declines over several hundred milliseconds. As the amplitude and duration of the depolarizing pulse are increased, the size of the venom-induced current that follows also increases. 4. The venom-induced current turns on exponentially with a time constant near the value of the time constant for recovery from inactivation, tau-h, at the resting membrane potential. A depolarizing pulse inactivates this new current component, while a hyperpolarizing pulse leads to a larger venom-induced current immediately after the hyperpolarization. Its time course and membrane potential dependence indicate that the venom-induced current is modulated by the sodium inactivation process. 5. The membrane potential dependence of sodium activation in some channels is shifted by 40-50 mV in the hyperpolarizing direction. Depolarization increases the proportion of channels with shifted activation gating by first-older kinetics. Following a depolarizing pulse the activation parameter, m-3, remains elevated for hundreds of milliseconds, allowing channels to reopen as recovery from inactivation occurs. 6. A kinetic model with normal inactivation gating and shifted activation gating in some channels accounts for the observed voltage-clamp currents and for the repetitive firing evoked by Centruroides venom. In the model normal channels are converted to channels with shifted activation gating by a voltage dependent reaction. 7. The results suggest limits to possible coupling between sodium channel activation and inactivation. Transitions of the inactivation parameter, h, can occur normally in channels with a shifted membrane potential dependence for activation.
The effects of two toxins extracted from the tentacles of Anemonia sulcata on ionic currents have been tested on the nodal membrane of myelinated nerve fibres from Rana esculenta. While external application of Toxin I at 100 muM leaves both specific ionic currents unmodified, Toxin II at 10 muM reacts with a receptor site associated with the sodium conductance inactivation gating. Since internal application by diffusion of Toxin II at a concentration of 700 muM leaves the ionic currents unchanged, the receptor site is most likely located on the external side of the nodal membrane. An equilibrium dissociation constant for the effects of Toxin II was estimated as 20 muM. The on-reaction is fast (rate constant for the on-reaction roughly equal to 3.103 M-1) suggesting a readily accesible receptor site for the toxin. The kinetics characteristics of the sodium currents recorded in the presence of Toxin II suggest that there are at least two steps in the reaction leading to Na+ -channels with the inactivation gate completely immobilized. The relatively fast reversibility of the intermediate stage of the reaction and the rather slow but, in the end, complete reversal of the toxin effects suggest that the toxin acts by modifying the energy profile for the transition "inactivation gate in the open configuration to inactivation gate in the closed configuration." Toxin II at higher concentrations (greater than 100 muM) also inhibits the potassium currents but these effects were not studied in any detail.
The sodium channel, one of the family of structurally homologous voltage-gated ion channels, differs from other members, such as the calcium and the potassium channels, in its high selectivity for Na+. This selectivity presumably reflects a distinct structure of its ion-conducting pore. We have recently identified two clusters of predominantly negatively charged amino-acid residues, located at equivalent positions in the four internal repeats of the sodium channel as the main determinants of sensitivity to the blockers tetrodotoxin and saxitoxin. All site-directed mutations reducing net negative charge at these positions also caused a marked decrease in single-channel conductance. Thus these two amino-acid clusters probably form part of the extracellular mouth and/or the pore wall of the sodium channel. We report here the effects on ion selectivity of replacing lysine at position 1,422 in repeat III and/or alanine at position 1,714 in repeat IV of rat sodium channel II (ref. 3), each located in one of the two clusters, by glutamic acid, which occurs at the equivalent positions in calcium channels. These amino-acid substitutions, unlike other substitutions in the adjacent regions, alter ion-selection properties of the sodium channel to resemble those of calcium channels. This result indicates that lysine 1,422 and alanine 1,714 are critical in determining the ion selectivity of the sodium channel, suggesting that these residues constitute part of the selectivity filter of the channel.
Sodium channels are the major proteins that underlie excitability in nerve, heart, and skeletal muscle. Chemical reaction
rate theory was used to analyze the blockage of single wild-type and mutant sodium channels by cadmium ions. The affinity
of cadmium for the native tetrodotoxin (TTX)-resistant cardiac channel was much higher than its affinity for the TTX-sensitive
skeletal muscle isoform of the channel (microliters). Mutation of Tyr401 to Cys, the corresponding residue in the cardiac
sequence, rendered microliters highly susceptible to cadmium blockage but resistant to TTX. The binding site was localized
approximately 20% of the distance down the electrical field, thus defining the position of a critical residue within the sodium
channel pore.
Site-directed antibodies corresponding to conserved putative extracellular segments of sodium channels, coupled with binding studies of radiolabeled insect-selective scorpion neurotoxins, were employed to clarify the relationship between the toxins' receptor sites and the insect sodium channel. (1) The depressant insect toxin LqhIT2 was shown to possess two noninteracting binding sites in locust neuronal membranes: a high-affinity (KD1 = 0.9 +/- 0.6 nM) and low-capacity (Bmax1 = 0.1 +/- 0.07 pmol/mg) binding site as well as a low-affinity (KD2 = 185 +/- 13 nM) and high-capacity (Bmax2 = 10.0 +/- 0.6 pmol/mg) binding site. (2) The high-affinity site serves as a target for binding competition by the excitatory insect toxin AaIT. (3) The binding of LqhIT2 was significantly inhibited in a dose-dependent manner by each of four site-directed antibodies. The binding inhibition resulted from reduction in the number of binding sites. (4) The antibody-mediated inhibition of [125I]AaIT binding differs from that of LqhIT2: three out of the four antibodies which inhibited LqhIT2 binding only partially affected AaIT binding. Two antibodies, one corresponding to extracellular and one to intracellular segments of the channel, did not affect the binding of either toxin. These data suggest that the receptors to the depressant and excitatory insect toxins (a) comprise an integral part of the insect sodium channel, (b) are formed by segments of external loops in domains I, III, and IV of the sodium channel, and (c) are localized in close proximity but are not identical in spite of the competitive interaction between these toxins.
Sodium channels from brain and heart, whose primary structures are known, differ in their sensitivity to block by the guadinium toxins tetrodotoxin and saxitoxin and to block by external Zn2+ and Cd2+. Studies using site-directed mutagenesis have identified the SS2 and adjacent regions of all four repeats as critical determinants for toxin sensitivity. Within and in the immediate vicinities of the SS2 segments, there are only two amino-acid differences between rat brain sodium channel II and rat heart I sodium channel, both located in repeat I. Here we show that replacement of phenylalanine 385 of brain sodium channel by cysteine that is present at the equivalent position in heart channel (F385C) not only reduces sensitivity to the guadinium toxins but also increases sensitivity to Zn2+ and Cd2+, thus conferring properties of heart sodium channel on brain sodium channel. Replacement of asparagine at the second non-conserved position by arginine (N388R) only marginally affects sensitivity to the toxins, Zn2+ or Cd2+, but this mutation markedly reduces sensitivity to block by Ca2+ and Co2+. The double mutant channel (F385C.N388R) shows combined properties of the two mutant channels. These results give a structural insight into the different properties of the two channel proteins.
The cardiac sodium channel alpha subunit (RHI) is less sensitive to tetrodotoxin (TTX) and saxitoxin (STX) and more sensitive to cadmium than brain and skeletal muscle (microliter) isoforms. An RHI mutant, with Tyr substituted for Cys at position 374 (as in microliter) confers three properties of TTX-sensitive channels: (i) greater sensitivity to TTX (730-fold); (ii) lower sensitivity to cadmium (28-fold); and (iii) altered additional block by toxin upon repetitive stimulation. Thus, the primary determinant of high-affinity TTX-STX binding is a critical aromatic residue at position 374, and the interaction may take place possibly through an ionized hydrogen bond. This finding requires revision of the sodium channel pore structure that has been previously suggested by homology with the potassium channel.
The SS2 and adjacent regions of the 4 internal repeats of sodium channel II were subjected to single mutations involving, mainly, charged amino acid residues. These sodium channel mutants, expressed in Xenopus oocytes by microinjection of cDNA-derived mRNAs, were tested for sensitivity to tetrodotoxin and saxitoxin and for single-channel conductance. The results obtained show that mutations involving 2 clusters of predominantly negatively charged residues, located at equivalent positions in the SS2 segment of the 4 repeats, strongly reduce toxin sensitivity, whereas mutations of adjacent residues exert much smaller or no effects. This suggests that the 2 clusters of residues, probably forming ring structures, take part in the extracellular mouth and/or the pore wall of the sodium channel. This view is further supported by our finding that all mutations reducing net negative charge in these amino acid clusters cause a marked decrease in single-channel conductance.
Brevetoxin, a neurotoxin isolated from the marine dinoflagellate Ptychodiscus brevis, has been derivatized into a photoaffinity probe by carbodiimide linkage to p-azidobenzoic acid. Rosenthal analysis of a tritiated p-azidobenzoate brevetoxin derivative indicates that specific binding of the toxin occurs at two distinct and separate sites, with Kd and Bmax values of 0.21 nM and 2.12 pmol/mg of protein for the high affinity site and 50.7 nM and 91.5 pmol/mg of protein for the low affinity site, respectively. Binding of tritiated photoaffinity probe to the high affinity/low capacity site can be displaced in a competitive manner by native brevetoxin (Kd = 1.9 nM), demonstrating a specific competitive interaction with the receptor site. Rat brain synaptosomes, covalently labeled with the brevetoxin photoaffinity probe, were subjected to detergent solubilization. The covalently labeled membrane protein was estimated to have a Stokes radius of 55 +/- 3 A. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed specific labeling of a 260-kDa protein. Treatment with 2-mercaptoethanol and neuraminidase resulted in retention of brevetoxin binding to this high molecular weight protein. The affinity-purified membrane protein-brevetoxin photoaffinity probe complex was specifically recognized by a sodium channel antibody directed against the intracellular side of transmembrane segment IS6. The sodium channel alpha subunit is implicated as the specific site of brevetoxin interaction.
The organs of 195 specimens of 4 species of the gastropod Naticidae, collected from fish markets in Taiwan, were assayed for toxicity. The calf moon shell Natica vitellus contained weak toxicity (10-99 MU/g) in the muscle and digestive gland. The bladder moon shell Polinices didyma contained moderate (100-999 MU/g) and weak toxicity in the muscle and digestive gland, respectively. The digestive gland in 1 out of 20 specimens of the pear-shaped moon shell P. tumidus was toxic (4 MU/g). All tissues of the butterfly moon shell N. alapapilionis were non-toxic. The toxins were partially purified from the toxic specimens of the calf moon shell and the bladder moon shell, and identified to be tetrodotoxin and anhydrotetrodotoxin.
Depressant insect-selective neurotoxins derived from scorpion venoms (a) induce in blowfly larvae a short, transient phase of contraction similar to that induced by excitatory neurotoxins followed by a prolonged flaccid paralysis and (b) displace excitatory toxins from their binding sites on insect neuronal membranes. The present study was undertaken in order to examine the basis of these similarities by comparing the primary structures and neuromuscular effects of depressant and excitatory toxins. A new depressant toxin (LqhIT2) was purified from the venom of the Israeli yellow scorpion. The effects of this toxin on a prepupal housefly neuromuscular preparation mimic the effects on the intact animal; i.e., a brief period of repetitive bursts of junction potentials is followed by suppression of their amplitude and finally by a block of neuromuscular transmission. Loose patch clamp recordings indicate that the repetitive activity has a presynaptic origin in the motor nerve and closely resembles the effect of the excitatory toxin AaIT. The final synaptic block is attributed to neuronal membrane depolarization, which results in an increase in spontaneous transmitter release; this effect is not induced by excitatory toxin. The amino acid sequences of three depressant toxins were determined by automatic Edman degradation. The depressant toxins comprise a well-defined family of polypeptides with a high degree of sequence conservation. This group differs considerably in primary structure from the excitatory toxin, with which it shares identical or related binding sites, and from the two groups of scorpion toxins that affect sodium conductance in mammals. The two opposing pharmacological effects of depressant toxins are discussed in light of the above data.
The amino acid sequences of insect-selective scorpion toxins, purified from the venom of Leiurus quinquestriatus quinquestriatus, have been determined by automatic phenyl isothiocyanate degradation of the S-carboxymethylated proteins and derived proteolytic peptides. The excitatory toxin Lqq IT1 and Lqq IT1' (70 residues) show the shift of one half-cystine from an external position, which is characteristic of anti-mammal toxins, to an internal sequence position. Lqq IT2 (61 residues) displays the half-cystine residue in position 12, common to the sequence of all known anti-mammal toxins; it induces flaccid paralysis on insects but is non-toxic for the mouse. Lqq IT2 structurally defines a new type of anti-insect toxins from scorpion venoms. CD spectra and immunological data are in agreement with this finding.
A new toxin, Lqh alpha IT, which caused a unique mode of paralysis of blowfly larvae, was purified from the venom of the scorpion Leiurus quinquestriatus hebraeus, and its structural and pharmacological properties were compared to those of three other groups of neurotoxins found in Buthinae scorpion venoms. Like the excitatory and depressant insect-selective neurotoxins, Lqh alpha IT was highly toxic to insects, but it differed from these toxins in two important characteristics: (a) Lqh alpha IT lacked strict selectivity for insects; it was highly toxic to crustaceans and had a measurable but low toxicity to mice. (b) It did not displace an excitatory insect toxin, 125I-AaIT, from its binding sites in the insect neuronal membrane; this indicates that the binding sites for Lqh alpha IT are different from those shared by the excitatory and depressant toxins. However, in its primary structure and its effect on excitable tissues, Lqh alpha IT strongly resembled the well-characterized alpha scorpion toxins, which affect mammals. The amino acid sequence was identical with alpha toxin sequences in 55%-75% of positions. This degree of similarity is comparable to that seen among the alpha toxins themselves. Voltage- and current-clamp studies showed that Lqh alpha IT caused an extreme prolongation of the action potential in both cockroach giant axon and rat skeletal muscle preparations as a result of the slowing and incomplete inactivation of the sodium currents. These observations indicate that Lqh alpha IT is an alpha toxin which acts on insect sodium channels.(ABSTRACT TRUNCATED AT 250 WORDS)
Receptor sites for some of the most important toxins known to be specific for voltage-sensitive Na+ channel in the mammalian nervous system have been identified in a purified membrane preparation of house fly brain. Very high affinities have been found for the association of tetrodotoxin or tetrodotoxin derivatives with the insect Na+ channel (Kd = 0.03 - 0.08 nM). The gamma toxin from the Brazilian scorpion Tityus serrulatus forms a complex with the Na+ channel having a Kd of 6.1 pM. The Kd value for toxin II from the sea anemone Anemonia sulcata is 0.12 microM. These results show a high degree of conservation of the pharmacological properties of the brain Na+ channels between insects and mammals.
Conotoxin GIII from Conus geographus inhibited the binding of [3H]lysine-tetrodotoxin (4 nM) to electroplax membranes from Electrophorus electricus and to the rat brain P2 fraction with IC50 values of 13 nM and 7.9 microM, respectively. This inhibition observed with electroplax membranes was irreversible. These and physiological findings (Life Sci., 21 (1977) 1759-1770 suggest that conotoxin GIII inhibits Na channel activation by its interaction with the tetrodotoxin binding site of the Na channel. The differences in structures related to the activation of Na channels between the eel electroplax and the rat brain are indicated.
The effects of 0.25 X 10(-9) and 1.25 X 10(-9) g/ml of purified ciguatoxin (CgTX) upon the node of Ranvier of frog isolated nerve fibres were investigated under current and voltage clamp conditions. When added to the external solution, CgTX induced spontaneous action potentials at a frequency of about 100 Hz, which were reversible upon removal of the toxin. Under voltage clamp conditions, CgTX modified neither linear leakage and capacity currents nor K current, but reversibly induced a maintained (late) inward current (IL) during long lasting depolarizations. IL, as well as the peak Na current, was suppressed by tetrodotoxin (300 nM). The steady-state inactivation curve of the Na current showed that a fraction of the current (corresponding to IL) did not inactivate. IL activated and reversed at voltages about 30 mV more negative than the peak Na current (recorded under control conditions or in the presence of CgTX). During a given depolarizing pulse, the amplitude of IL depended on the holding potential. IL was about three times greater when the holding potential was -70 mV rather than -120 mV. We conclude that CgTX specifically interacts with and modifies Na channels. We also conclude that the effects of CgTX depend on membrane potential.
The effects of the major neurotoxic fraction isolated from scorpion venom of Tityus serrulatus, TiTx gamma, on peripheral nerve membrane of Xenopus laevis were studied under current- and voltage-clamp conditions. 700 nmol/l TiTx gamma depolarized the membrane and induced spontaneous activity (150 s-1, maximum value), which ceased within a few minutes. It reduced the amplitude of the action potentials from 109 mV to 52 mV and increased their duration from 1.25 ms to 4.5 ms. 440 nmol/l TiTx gamma induced inward Na current flow at resting potential. The descending branch of the Na current-voltage curve was flattened and shifted approximately 10 mV to more negative potentials. Maximum Na permeability was reduced to about 20%. Both development of and recovery from inactivation of Na permeability were slowed. The steepness of the steady-state inactivation curve was decreased, but the mid-potential changed only insignificantly. No prepulse was necessary to elicit either a shift of activation or an inward current at resting potential. Expressing the toxin effect either in terms of the decrease of Na peak current or of the slowing of inactivation, half-maximum effects were found with 0.3 +/- 0.1 and 3.7 +/- 0.7 mumol/l TiTx gamma, respectively.
The polyether lipid-soluble toxins isolated from the marine dinoflagellate Ptychodiscus brevis (formerly Gymnodinium breve) have been determined to bind to a unique site associated with rat brain synaptosomes. Using [3H]brevetoxin PbTx-3 as a specific probe, binding was determined at 4 degrees in rat brain synaptosomes using a rapid centrifugation technique. Rosenthal analysis yields a KD of 2.9 nM and a Bmax of 6.8 pmol of toxin/mg of protein. Labeled probe can be displaced by unlabeled PbTx-3, PbTx-2, or synthetic PbTx-3 (reduced PbTx-2) but not by a nontoxic, synthetic oxidized derivative of PbTx-2. Competition experiments using natural toxin probes specific for sites 1-4 of the voltage-dependent sodium channel have illustrated that PbTx-3 does not bind to any of the previously described sites associated with the channel. A fifth site is proposed. In addition, because of the varied nomenclature associated with the brevetoxins, a new classification system is proposed.
The neurotoxins that modify Na channels have actions that are characterized by different degrees of specificity (Table 2). These specificities can be correlated with their chemical properties. For example, guanidinium toxins, which are small charged ligands, appear only to "block" Na channels by binding to a site on the external surface. Peptide toxins, which are also positively charged and relatively small, also act from the external solution to modify channel activation and inactivation processes but do not alter ion selectivity. The lipophilic toxins, hydrophobic, neutral drugs, act from either side of the membrane and modify all the functions of Na channels. From such differences, and from the independence of toxin binding as well as toxin action, separate binding sites for these agents have been classified (Catterall 1980). Recent findings reviewed here suggest that all these toxins share certain features: They differentiate between various states of the channel. Effects of lipophilic activators, polypeptide toxins, and, indeed, even STX and TTX are enhanced or reversed in fractions of seconds under voltage clamp by patterns of membrane potential that selectively populate the channel open state, or the slow or fast inactivated states. Other assays--such as the binding of radiolabeled ligands or the changes of steady-state Na flux that require seconds to minutes of toxin-channel interaction--reveal interactions of the toxins with states of the channel not detected in the usual voltage-clamp analysis. Pharmacological probes may thus reveal channel states or transitions previously unrecognized. The bound toxins appear to interact with one another. The well-documented synergism at equilibrium of alpha-toxins with lipophilic activators provided a model for allosteric interactions between two separate binding sites (Catterall 1979, 1980). The other toxin interactions are more ephemeral and are characterized by kinetic variations that reflect the availability of reactive channel states. For example, the appearance of beta-toxin induced modifications of Na currents is accelerated in the presence of alpha-toxin (Wang & Strichartz 1983), whereas the modifications of inactivation by alpha-toxins are prevented by concurrent incubation with tetrodotoxin, although such modifications, once effected, are not reversed by the subsequent addition of TTX. Modifications of gating by lipophilic toxins confer a selective voltage-dependence on STX and TTX inhibition of open channels that is not observed in drug-free channels.(ABSTRACT TRUNCATED AT 400 WORDS)
Binding studies indicate that ciguatoxin and brevetoxin allosterically enhance in a very similar way the binding of [3H]batrachotoxinin A 20-alpha-benzoate to the neuronal Na+ channel protein. Moreover ciguatoxin competitively inhibits the binding of [3H]brevetoxin-3 to rat brain membranes. The affinity of ciguatoxin for the Na+ channel is at least 20-50-times higher than that of brevetoxin. These results indicate that ciguatoxin and brevetoxins act at the same binding site on the sodium channel.
Site-directed and monoclonal antibodies recognizing different extracellular regions of the RII sodium channel alpha subunit have been used to determine the sequences that comprise the receptor for alpha-scorpion toxins by evaluating the effect of antibody on voltage-dependent binding of radio-labeled toxin isolated from Leiurus quinquestriatus to both reconstituted rat brain sodium channel and rat brain synaptosomes. Of six antibodies tested, two recognizing amino acid residues 355-371 and 382-400 located on an extracellular loop between transmembrane segments S5 and S6 of domain I and one recognizing residues 1686-1703 of a similar loop of domain IV inhibit binding by 30-55%. Inhibition is concentration-(EC50 = 0.4-2 microM) and time- (t1/2 = 40-80 min) dependent. Five different monoclonal antibodies recognizing the same extracellular loop in domain I inhibit binding completely with similar EC50 values as observed for site-directed antibodies. Kinetic studies of the antibody effect are consistent with a slowly reversible competition for the toxin receptor site. Our results suggest that the extracellular loops between segments S5 and S6 of domains I and IV comprise at least part of the alpha-scorpion toxin receptor site and support the membrane topology models in which domains I and IV are adjacent in the tertiary structure of the channel protein and six transmembrane sequences are contained in each of the four homologous domains.
Chemical modifications of tyrosine and tryptophan residues of scorpion alpha-neurotoxins II and III from Androctonus australis Hector were performed as well as modification of the two arginines and the alpha-amino group of toxin I. The pharmacological potencies of each derivative were assessed in vivo by LD50 measurement and in vitro by competition experiments with 125I-toxin for synaptosomal receptors. Arginine residues in positions 2 and 60 and the alpha-amino group of Androctonus toxin I were derivatized by p-hydroxyphenylglyoxal; the corresponding modified toxins exhibit low pharmacological potencies. Tryptophan 38 of toxin II and tryptophan 45 of toxin III were modified by nitrophenylsulfenyl chloride, leading respectively to a poorly and a fully active derivative. The tetranitromethane modification of tyrosine residues in positions 60, 5 and 14 of toxin III induced respectively 60%, 40% and 30% of loss of biological activity. Circular dichroic analysis indicated that for every derivative, except the nitrophenylsulfenyl derivative of Trp-45 of AaH III, the conformation of the toxin was not altered by derivatization. Conformational integrity was also confirmed by full activity of the derivatives in radioimmunoassays. Taken together, the results suggest that aromatic residues belonging to the conserved hydrophobic surface, to the C-terminal and to the loop region 37-44 are involved in the molecular mechanisms by which scorpion alpha-toxins act. Charged residues in the N-terminal and C-terminal also contribute to the high efficacy of the binding process. It appears that all important residues are clustered on one face of the toxin, suggesting a multipoint interaction with the proteins of the sodium channel.