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Abstract The Tityus serrulatus scorpion species represents a serious human health threat to in Brazil because it is among the animals that produces the most dangerous venoms for mammals in South America. Its venom has provided several highly selective ligands that specifically interact with sodium and potassium channels. During the past decades, several international groups published an increasing amount of data on the isolation and the chemical, pharmacological and immunological characterisation of its main β-toxin, Ts1. In this review, we compiled the best available past and recent knowledge on Ts1. Aside from its intricate purification, the state-of-the-art understanding concerning its pharmacological activities is presented. Its solved three-dimensional structure is shown, as well as the possible surface areas of contact between Ts1 and its diverse voltage-gated Na+ channel targets. Organisations of the gene and the precursor encoding Ts1 are also tackled based on available cDNA clones or on information obtained from polymerase chain reactions of stretches of scorpion DNA. At last, the immunological studies complete with Ts1 to set up an efficient immunotherapy against the Tityus serrulatus venom are summarized. Graphical abstract Image 1 Highlights • An updated review on the current knowledge about the Tityus serrulatus toxin Ts1. • Comparison between the diverse purification procedures of Ts1. • Ts1 cDNA cloning and genomic organisations. • Ts1 3D-structure and structure-function relationships with its targets. • Special emphasis given to Ts1 pharmacology on voltage-gated Na+ channels. [ABSTRACT FROM AUTHOR]

Potassium (K + ) channels are trans-membrane proteins, which play a key role in cellular excitability and signal transduction pathways. Scorpion toxins blocking the ion-conducting pore from the external side have been invaluable probes to elucidate the structural, functional, and physio-pathological characteristics of these ion channels. This review will focus on the interaction between K + channels and their peptide blockers isolated from the venom of the scorpion Tityus serrulatus , which is considered as the most dangerous scorpion in Brazil, in particular in Minas-Gerais State, where many casualties are described each year. The primary mechanisms of action of these K + blockers will be discussed in correlation with their structure, very often non-canonical compared to those of other well known K + channels blockers purified from other scorpion venoms. Also, special attention will be brought to the most recent data obtained by proteomic and transcriptomic analyses on Tityus serrulatus venoms and venom glands. [ABSTRACT FROM AUTHOR]

Androctonus australis hector scorpion venom is well known by its high toxicity, it induces massive release of neurotransmitters that lead to pathophysiological disorders in cardiovascular, neuro-hormonal and immune systems. Previous studies have shown the relationship between the severity of scorpion envenoming and immune system activation. This study was assessed to investigate the involvement of complement system and inflammatory mediators after sublethal injection of Aah venom, its toxic fraction (FtoxG50) and its main toxins (AahI and AahII) into NMRI mice. The Activation complement system by the venom is also compared to that induced of lipoplysaccharides (LPS). Obtained results showed that seric complement system (CS) is activated by the venom and by its toxic components; this activation is more pronounced into liver tissue when toxic components (FtoxG50, AahI or AahII) are used. Increase of cytokine levels (IL1β, TNFα and ICAM) into hepatic tissue induced by AahI or AahII neurotoxins is correlated with tissue alterations. Aprotinin, a non specific inhibitor of complement system seems to be able to reduce CS consumption and to restore partially the induced tissue damage by venom. The mechanisms by which toxic fraction or LPS induced the activation of complement system seem to be different. Sensitivity of hepatic tissue is more pronounced after FtoxG50 injection; however lung tissue is more sensible to LPS than FoxG50. [ABSTRACT FROM AUTHOR]

Abstract: Even though Buthus occitanus scorpions are found throughout the Mediterranean region, a lack of distinctive characteristics has hampered their classification into different subspecies. Yet, stings from this particular scorpion family are reported each year to result in pain followed by various toxic symptoms. In order to determine the toxicity origin of the rare French B. occitanus Amoreux scorpion, we collected several specimens and studied their venom composition using a nano ultra high performance liquid chromatography and matrix assisted laser desorption/ionisation time-of-flight mass spectrometry (nano UHPLC/MALDI-TOF-MS) automated workflow combined with an enzyme-linked immunosorbent assay (ELISA) approach. Moreover, we compared this dataset to that obtained from highly lethal Androctonus australis and Androctonus mauretanicus scorpions collected in North Africa. As a result, we found that the B. occitanus Amoreux venom is toxic to mice, an observation that is most likely caused by venom components that inhibit voltage-gated sodium channel inactivation. Moreover, we identified similarities in venom composition between B. occitanus scorpions living in the South of France and other Buthidae collected in Morocco and Algeria. As such, the results of this study should be taken into consideration when treating stings from the B. occitanus species living in the South of France. [Copyright &y& Elsevier]

Abstract: The availability of a large variety of specific blockers, which inhibit different K+ currents, would help to elucidate their differences in physiological function. Short peptide toxins isolated from scorpion venoms are able to block voltage-dependent or Ca2+-activated K+ channels. Here, we have studied the venom of the Moroccan scorpion Buthus occitanus Paris (BoP) in order to find new peptides, which could enlarge our structure–function relationship knowledge on the Kv1.3 blocker Kaliotoxin (KTX) that belongs to the α-KTx3.1 family. Indeed and since more a decade, KTX is widely used by international investigators because it exhibits a quite sharp specificity and a high-affinity for the Kv1.3 channel, which is not only a neuronal channel but also a therapeutic target for diverse autoimmune diseases such as multiple sclerosis, type 1 diabetes, and rheumatoid arthritis. The BoP venom was first investigated using HPLC and MALDI-TOF/MS. Further, the HPLC fractions were screened by ELISA with antibodies raised against KTX. These antibodies recognized at least three components toxic in mice by intracerebroventricular injection. They were further pharmacologically characterized by competition using 125I-KTX bound to its specific binding sites on rat brain synaptosomes. A single component (4161 Da) inhibited totally the 125I-KTX binding and with high-affinity (IC50 = 0.1 nM), while the two other components poorly competed with (IC50 > 100 nM). These toxins were sequenced in full by Edman's degradation. The high-affinity ligand (BoPKTX) shares 86% sequence identity with KTX and was classified as toxin α-KTx3.17. The two others peptides (BoP1 and BoP2, 4093 Da and 4121 Da, respectively) only differ by a Lys/Arg mutation. Their amino acid sequences were related to Martentoxin, which has been characterized from the Chinese scorpion Buthus martenzi Karch and described as both a BKCa and Kv1.3 blocker. Accordingly, they belong to the α-KTx16 family. [Copyright &y& Elsevier]

Abstract: During the past decade mass spectrometry (MS) has become one of the major suitable techniques for peptide and protein analysis from animal venoms, not only to identify new toxins within these complex mixtures, but also for toxin structural studies. The goal of the experiments presented here was to test the ultimate limit for producing scorpion venom mass fingerprinting (VMF; i.e. a comprehensive list of all masses in presence). Thanks to recent developments in nano ultra high performance liquid chromatography (UHPLC) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS), we sought the experimental conditions for setting up an automated data acquisition workflow. As the main result, we demonstrate the possibility to easily achieve VMF having in hand only tens of nanogram of venom. ELISA on-line was also beneficial to monitor in the picogram range for toxin families using specific antisera raised against major toxins. [Copyright &y& Elsevier]

Abstract: The present study analyzes the involvement of the endogenous opioid system in the antinociceptive effects produced in mammals after α- or β- scorpion toxin injections. The analgesic effects on mice of the α-anatoxin Amm VIII, a weak modulator of Nav1.2 channel, and the depressant insect-selective β-toxin LqqIT2 were evaluated by intraperitoneal route. The two toxins increased hot plate and tail flick latencies in a dose-dependent manner. We also compared the effects of the toxins with those obtained after acetic acid administration or cold-water tail immersion, which both induce pain relief through the activation of diffuse noxious inhibitory controls (DNIC) and the release of endogenous opioids. The increased latencies obtained with the toxins, acetic acid, or cold-water tail immersion were partly reversed by the co-administration of the opioid receptor antagonist naloxone. Finally, AmmVIII, LqqIT2, or acetic acid, induced increased c-fos mRNA expression in spinal cord. This increase disappeared when the toxins were co-injected with acetic acid. In conclusion, we show for the first time that an α-anatoxin exhibits a potent analgesic activity and confirm that depressant β-toxins are able to reduce nociception. We hypothesize that pain relief induced by these scorpion toxins may implicate the activation of an endogenous opioid system and may be partly the result of a counter irritation phenomenon, which could be due to the activation of DNIC. [ABSTRACT FROM AUTHOR]

Abstract: LqqIT2 is a depressant neurotoxin present in the venom of the Leiurus quinquestriatus quinquestriatus scorpion, one of the world''s most dangerous scorpions endemic to dry habitats in Africa and Asia. In order to determine its efficacy, potency and selectivity, LqqIT2 was subjected for the first time to an electrophysiological and pharmacological comparison between two different cloned sodium channels expressed in Xenopus laevis oocytes. Aside from typical β-toxin effects, LqqIT2 also affected the inactivation process and ion selectivity of the insect voltage-gated sodium channel. The most interesting feature of LqqIT2 is its total insect-selectivity. At a concentration of 1μM, the insect-voltage-gated sodium channel, para, was profoundly modulated while its mammalian counterpart, the rat brain Nav1.2 channel, was not affected. This trait offers excellent prospects for the development of novel insecticides. [Copyright &y& Elsevier]

The α-subunits of Kv1 channels display characteristic distributions and restricted co-assembly in mammalian brain. The heterogeneous composition of Kv1 channels has made it difficult to use specific toxins to label brain structures. We used autoradiography to analyse the competitive behaviour of three Kv1 channel toxins—α-dendrotoxin, kaliotoxin, and mast cell degranulating peptide—for binding to kaliotoxin binding sites in various brain structures. IC50 varied considerably between brain regions (by up to three orders of magnitude) for each ligand. α-dendrotoxin and kaliotoxin competed equally in some regions and to different extents in others, identifying two types of structure. Mast cell degranulating peptide competed with 125I-kaliotoxin less efficiently than α-dendrotoxin and kaliotoxin, in all regions. Thus, differences in the capacity of these three toxins to bind to kaliotoxin binding sites provide evidence of major differences in the composition of the Kv1 channels constituting the kaliotoxin binding sites. [Copyright &y& Elsevier]

BmTX3 is a toxin recently characterised from the venom of the Chinese scorpion Buthus martensi Karch, which specifically blocks a transient A-type K+ current in striatum neurons in culture and binds to rat brain synaptosomes with high affinity. With Aa1 and AmmTX3, it belongs to the new α-KTx15 subfamily from ‘short-chain’ scorpion toxins, which specifically block different types of K+ channels. Here, a highly specific polyclonal antiserum was raised in rabbit against a C-terminal deleted BmTX3 analogue (BmTX-del YP). Using liquid-phase radioimmunoassay, we have studied its selectivity for the toxins from the α-KTx15 subfamily. We have also demonstrated that no/or poor cross-reactivity was observed with a panel of ‘short-chain’ scorpion toxins representative of other structurally different subfamilies. These results suggest that a wide antigenic polymorphism, similar to that previously observed for ‘long-chain’ scorpion toxins acting as modulators of voltage-activated Na+ channels, is also the rule for the ‘short-chain’ scorpion toxins able to block K+ channels. [Copyright &y& Elsevier]

Androctonus australis is primarily involved in envenomations in North Africa, notably in Tunisia and Algeria, and constitutes a significant public health problem in this region. The toxicity of the venom is mainly due to various neurotoxins that belong to two distinct structural and immunological groups, group I (the AahI and AahIII toxins) and group II (AahII). Here, we report the use of a diabody mixture in which the molar ratio matches the characteristics of toxins and polymorphism of the venom. The mixture consists of the Db9C2 diabody (anti-group I) and the Db4C1op diabody (anti-AahII), the latter being modified to facilitate in vitro production and purification. The effectiveness of the antivenom was tested in vivo under conditions simulating scorpion envenomation. The intraperitoneal injection of 30 μg of the diabody mixture protected almost all the mice exposed to 3 LD50 s.c. of venom. We also show that the presence of both diabodies is necessary for the animals to survive. Our results are the first demonstration of the strong protective power of small quantities of antivenom used in the context of severe envenomation with crude venom. [ABSTRACT FROM AUTHOR]

Abstract: Immunotherapy is the only effective treatment for scorpion stings. However the efficiency of this treatment varies depending on the forms of the antibodies and route of administration used. The antibodies are mostly injected as F(ab´)2 fragments. In this study, we investigated damage to the heart and lung tissue and the inflammatory response caused by Androctonus australis hector venom, its toxic fraction after molecular filtration or the isolated main alpha toxin (Aah II) in the presence or absence of different antibody molecules. A mixture of antibody fragments, F(ab´)2 and Fab, significantly reduced local leukocytosis, hemorrhage and inflammatory oedema induced by the A. australis hector venom and its toxins. [Copyright &y& Elsevier]

Abstract: While α-KTx peptides are generally known for their modulation of the Shaker-type and the Ca2+-activated potassium channels, γ-KTxs are associated with hERG channels modulation. An exception to the rule is BmTx3 which belongs to subfamily α-KTx15 and can block hERG channels. To explain the peculiar behavior of BmTx3, a tentative “hot spot” formed of 2 basic residues (R18 and K19) was suggested but never further studied [Huys I, et al. BmTx3, a scorpion toxin with two putative functional faces separately active on A-type K+ and HERG currents. Biochem J 2004;378:745–52]. In this work, we investigated if the “hot spot” is a commonality in subfamily α-KTx15 by testing the effect of (AmmTx3, Aa1, discrepin). Furthermore, single mutations altering the “hot spot” in discrepin, have introduced for the very first time a hERG blocking activity to a previously non-active α-KTx. Additionally, we could extend our results to other α-KTx subfamily members belonging to α-KTx1, 4 and 6, therefore, the “hot spot” represents a common pharmacophore serving as a predictive tool for yet to be discovered α-KTxs. [Copyright &y& Elsevier]

Aa1 is a toxin purified from the venom of the North African scorpion Androctonus australis. It blocks fast K+ currents in cerebellar granular cells [Biochim. Biophys. Acta 1468 (2000) 203]. Two full-length cDNAs (about 250 bp) encoding the precursors of putative Aa1 isoforms (AaTX1 and AaTX2) were amplified by PCR from a venom gland cDNA library of A. australis. The deduced precursors were composed of 59 amino acid residues including a signal peptide of 22 residues and a mature toxin of 37 residues. The peptides display 94% sequence identity with Aa1. Intron–exon organisation of the gene corresponding to theAaTX1 cDNAs was also depicted. [Copyright &y& Elsevier]

A potent insecticidal toxin, β/δ - PrIT1, molecular mass of 5598.86 [M + H] + , was characterized from Phoneutria reidyi spider venom. Its partial amino acid sequence showed high similarity with insecticidal spider toxins from the genus Phoneutria . β/δ-PrIT1 was very toxic (LD 50 = 4 nmol/g) to flies ( Musca domestica ), but not to mice ( Mus musculus ). Kinetic studies showed that 125 I-β/δ-PrIT1 binds to two distinct sites in insect sodium channels, with close affinity (Kd 1 = 34.7 pM and Kd 2 = 35.1 pM). Its association is rather fast (t 1/2(1) = 1.4 min, t 1/2(2) = 8.5 min) and its dissociation is a slower process (t 1/2(1) = 5.4 min, t 1/2(2) = 32.8 min). On rat brain synaptosomes β/δ-PrIT1 partially competed (∼30%) with the beta-toxin 125 I-CssIV, but did not compete with the alpha-toxin of reference 125 I-AaII, nor with the beta-toxin 125 I-TsVII. On cockroach nerve cord synaptosomes, β/δ-PrIT1 did not compete with the anti-insect toxin 125 I-LqqIT1, but it competed (IC 50 = 80 pM) with the “alpha-like” toxin 125 I-BomIV. In cockroach neurons, β/δ-PrIT1 inhibited the inactivation of Nav-channels and it shifted the sodium channel activation to hyperpolarizing potentials. These results indicate two different binding sites for β/δ-PrIT1, leading to two different pharmacological responses. β/δ-PrIT1 is one of the most toxic spider toxins to insects without apparent toxicity to mammals, and provide new model for the development of insecticides. [ABSTRACT FROM AUTHOR]

The Old World scorpion Androctonus australis hector (Aah) produces one of the most lethal venoms for humans. Peptidic α-toxins AahI to AahIV are responsible for its potency, with AahII accounting for half of it. All four toxins are high affinity blockers of the fast inactivation phase of mammalian voltage activated Na+ channels. However, the high antigenic polymorphism of α-toxins prevents production of a polyvalent neutralizing antiserum, whereas the determinants dictating their trapping by neutralizing antibodies remain elusive. From an anti-AahII mAb, we generated an antigen binding fragment (Fab) with high affinity and selectivity for AahII and solved a 2.3 Å-resolution crystal structure of the complex. Sequestering of the C-terminal region of the bound toxin within a groove formed by the Fab combining loops is associated with a toxin orientation and main and side chain conformations that dictate the AahII antigenic specificity and efficient neutralization. From an anti-AahI mAb, we also preformed and crystallized a high affinity AahI-Fab complex. The 1.6 Å-resolution structure solved revealed a Fab molecule devoid of a bound AahI and with combining loops involved in packing interactions, denoting expulsion of the bound antigen upon crystal formation. Comparative analysis of the groove-like combining site of the toxin unbound anti-AahI Fab along with complementary data from a flexible docking approach suggests occurrence of distinctive trapping orientations for the two toxins relative to their respective Fab. This study provides complementary templates for designing new molecules aimed at capturing Aah α-toxins and suitable for immunotherapy. [ABSTRACT FROM AUTHOR]

Abstract: The venom of the North African scorpion Androctonus amoreuxi (Aam) was analyzed using a combination of gel filtration, C18 reverse phase HPLC together with mass spectrometry analysis and bioassays. Three novel Birtoxin-like (BTX-L) peptides of 58 amino acid residues comprising three disulfide bridges were isolated and chemically characterized. One peptide, AamBTX-L3, induced serious toxic symptoms in mice and was lethal at nanogram quantities using intracerebroventricular injection. The three BTX-L peptides were tested in competition experiments on rat brain synaptosomes against the 125I-labeled “classical” α- and β-toxins of reference, as well as with the 125I-KTX, a voltage-gated potassium channel blocker. Only AamBTX-L3 was able to prevent the equilibrium binding of the β-toxin 125I-Css IV to its receptor site 4 with a IC50 value of 189nM. Even if previous electrophysiological data allowed the classification of other BTX-L peptides among the β-type toxins, this report clearly shows that AamBTX-L3 is pharmacologically a β-toxin, which recognizes the voltage-gated Na+ (Nav) channels from central mammalian neurons. In order to uncover the residues functionally essential for interaction between the AamBTX-L3 with the putative receptor site of 125I-Css IV on Nav1.2, molecular models of the three novel Aam BTX-L molecules were made and their surfaces were compared to the already described Css IV biologically interactive surfaces. A hypothesis is given that in BTX-L3, three residues found in the α-helix play a key role during target binding. [Copyright &y& Elsevier]

Abstract: In this study, we have characterized the immunological and pharmacological properties of the three major α-type toxins from the scorpion Androctonus amoreuxi, AamH1, AamH2 and AamH3, which were previously described as putative toxins from cDNAs [Chen, T. et al., 2003. Regul. Pept. 115, 115–121]. The immunological tests (ELISA, RIA) have demonstrated that AamH1, AamH2 and AamH3 belong to the immunological groups 3 and 4 of α-type toxins. Analysis of the three toxin effects on currents through rat brain (rNav1.2), rat muscle (rNav1.4) and Drosophila (DmNav1) sodium channels expressed in Xenopus oocytes revealed that AamH1 and AamH2, but not AamH3, have anti-insect and anti-mammal activities and can be classified as α-like toxins. While AamH1 removes fast inactivation only in neuronal rNav1.2 channel and has no effect on muscular rNav1.4 channel, AamH2 affects both neuronal rNav1.2 and muscular rNav1.4 channels. AamH3 was lethal to mice by intracerebroventricular injection despite its lack of activity on the neuronal rNav1.2 channel. Finally, we have shown that the A. amoreuxi venom was better neutralized by the antiserum raised against the venom of Buthus occitanus tunetanus than by the antisera raised against scorpion venoms from the same genus Androctonus. [Copyright &y& Elsevier]

Abstract: Scorpion venoms are very complex mixtures of molecules, most of which are peptides displaying different kinds of biological activity. Indeed, these peptides specifically bind to a variety of pharmacological targets, in particular ionic channels located in prey tissues, resulting in neurotoxic effects. Toxins modulating Na+, K+, Ca2+ and Cl− currents have been described in scorpion venoms. In this work, we have used several specific antibodies raised against the most lethal scorpion toxins already described to screen the Moroccan scorpion Androctonus mauretanicus mauretanicus venom in order to characterize new compounds. This immunological screening was also implemented by toxicity tests in mice and with mass spectrometry study, providing new informations on the molecular composition of this venom. In fine, we were able to determine the molecular masses of 70–80 different compounds. According to the immunological data obtained, many toxins cross-react with three sera raised against the most lethal α-toxins found in North African scorpion venoms, but not at all with those raised against the main β-toxins from South and North American venoms. Some of the previously described toxins from Androctonus mauretanicus mauretanicus venom could thus be detected by combining immunological tests, toxicity in mice and molecular masses. Among these toxins, one of them, which showed a mild cross-reaction with the serum raised against AaH I (a highly potent toxin from the venom of Androctonus australis), was identified as Amm III and fully sequenced. [Copyright &y& Elsevier]

Abstract: The Buthidae is the most clinically important scorpion family, with over 500 species distributed worldwide. Taxonomical positions and phylogenetic relationships concerning the representative genera and species of this family have been mostly inferred based upon comparisons between morphological characters. Yet, some authors have performed such inferences by comparing some structural properties of a few selected molecules found in the venoms from these scorpions. Here, we propose a novel methodology pipeline designed to address these issues. We have analyzed the whole venoms from some species that exemplify peculiar cases in the Buthidae family (Tityus stigmurus, Tityus serrulatus, Tityus bahiensis, Leiurus quinquestriatus quinquestriatus and Leiurus quinquestriatus hebraeus), by means of a proteomic approach using a 2D-LC/MS technique. The molecules found in these venoms were clustered according to their physicochemical properties (molecular mass and hydrophobicity), by using the machine learning-based Weka software. The clusters assessment, along with the number of molecules found in a given cluster for each scorpion, which assigns for the venom and structural family complexities, respectively, was used to generate a phenetic correlation tree for positioning these species. Our results were in accordance with the classical taxonomy viewpoint, which places T. serrulatus and T. stigmurus as very close species, T. bahiensis as a less related species in the Tityus genus and L. q. quinquestriatus and L. q. hebraeus with small differences within the same species (L. quinquestriatus). Therefore, we believe that this is a well-suited method to determine venom complexities that reflect the scorpions'' evolutionary history, which can be crucial to reconstruct their phylogeny through the molecular evolution of their venoms. [Copyright &y& Elsevier]

Abstract: The genomic DNA sequence encoding the scorpion toxin Amm VIII was amplified from genomic DNA of the scorpion Androctonus mauretanicus mauretanicus from Morocco, subcloned and sequenced. An intron, with a high A+T content (73.5%), split a Gly codon at the end of the precursor signal peptide and the consensus GT/AG splice junction was identified in the Amm VIII gene. This intron of only 166bp is the smallest intron described so far for a long-chain scorpion toxin gene. In addition, this study led to the identification of three new toxin-related genes. From the deduced amino acid sequences of the encoded precursor proteins, we found that the mature putative toxins were highly similar to the scorpion toxins Leiurus quinquestriatus quinquestriatus IV and Odonthobuthus doriae 1. [Copyright &y& Elsevier]

Small conductance calcium activated potassium channels (SK) are crucial in the regulation of cell firing frequency in the nervous system and other tissues. In the present work, a novel SK channel blocker, designated BmSKTx1, was purified from the scorpion Buthus martensi Karsh venom. The sequence of the N-terminal 22 amino acid residues was determined by Edman degradation. Using this sequence information, the full-length cDNA and genomic gene of BmSKTx1 were cloned and sequenced. By these analyses, BmSKTx1 was found to be a peptide composed of 31 amino acid residues with three disulfide bonds. It shared little sequence homology with other known scorpion α-KTxs but showed close relationship with SK channel blockers in the phylogenetic tree. According to the previous nomenclature, BmSKTx1 was classified as α-KTx14.1. We examined the effects of BmSKTx1 on different ion channels of rat adrenal chromaffin cells (RACC) and locust dorsal unpaired median (DUM) neurons. BmSKTx1 selectively inhibited apamin-sensitive SK currents in RACC with Kd of 0.72 μM and Hill coefficient of 2.2. And it had no effect on Na+, Ca2+, Kv, and BK currents in DUM neuron, indicating that BmSKTx1 was a selective SK toxin. [Copyright &y& Elsevier]

Three novel scorpion toxins, Aa1 from Androctonus australis, BmTX3 from Buthus martensi and AmmTX3 from Androctonus mauretanicus were shown able to selectively block A-type K+ currents in cerebellum granular cells or cultured striatum neurons from rat brain. In electrophysiology experiments, the transient A-current completely disappeared when 1 μM of the toxins was applied to the external solution whereas the sustained K+ current was unaffected.The three toxins shared high sequence homologies (more than 94%) and constituted a new ‘short-chain’ scorpion toxin subfamily: α-KTx15. Monoiododerivative of 125I-sBmTX3 specifically bound to rat brain synaptosomes. Under equilibrium binding conditions, maximum binding was 14 fmol/mg of protein and the dissociation constant (Kd) was 0.21 nM. This Kd value was confirmed by kinetic experiments (kon=6.0×106 M-1 s-1 and koff=6.0×10-4 s-1). Competitions with AmmTX3 and Aa1 with 125I-sBmTX3 bound to its receptor on rat brain synaptosomes showed that they fully inhibited the 125I-sBmTX3 binding (Ki values of 20 and 44 pM, respectively), demonstrating unambiguously that the three molecules shared the same target in rat brain. A panel of toxins described as specific ligands for different K+, Na+ and Ca2+ channels were not able to displace 125I-sBmTX3 from its binding site. Thus, 125I-sBmTX3 is a new ligand for a still unidentified target in rat brain. In autoradiography, the distribution of 125I-sBmTX3 binding sites in the adult rat brain indicated a high density of 125I-sBmTX3 receptors in the striatum, hippocampus, superior colliculus, and cerebellum. [Copyright &y& Elsevier]