Hervé Rochat

Voltage sensor-trapping

Polypeptide neurotoxins alter ion channel gating by binding to extracellular receptor sites, even though the voltage sensors are in their S4 transmembrane segments. By analysis of sodium channel chimeras, a beta-scorpion toxin is shown here to negatively shift voltage dependence of activation and enhance closed state inactivation by binding to a receptor site that requires glycine 845 (Gly-845) in the S3-S4 loop at the extracellular end of the S4 segment in domain II of the alpha subunit. Toxin action requires prior depolarization to drive the S4 voltage sensors outward, but these effects are lost in the mutant G845N. The results reveal a voltage sensor-trapping model of toxin action in which the IIS4 voltage sensor is trapped in its outward, activated position by toxin binding.

Two types of scorpion toxin receptor sites, one related to the activation, the other to the inactivation of the action potential sodium channel

The action of the neurotoxin in Buthinae scorpion venoms (Androctonus, Buthus or Leiurus genera) has been extensively studied. These proteins induce a prolongation of the action potential of nerves and muscles by slowing down inactivation of the sodium channel. Their affinity for their receptor site depends on membrane potential. In the present report we describe a toxin from a Centrurinae scorpion, Centruroides suffusus, which binds rat brain synaptosomes at a receptor site distinct from the Buthinae scorpion site independently of voltage. We name Androctonus-like toxins, alpha-scorpion toxins (alpha-ScTX), and Centruroides-like toxins, beta-scorpion toxins (beta-ScTX). We further report that beta-ScTX induces repetitive firing in frog myelinated nerve fibres by producing an abnormal sodium permeability. The beta-toxin binds specifically to rat brain synaptosomes (Kd = 3 nM) and induces an inhibition of the uptake and a stimulation of the release of GABA at concentrations which are in good agreement with the Kd value. These effects are blocked by tetrodotoxin. The binding site of beta -ScTX is distinct from those of other neurotoxins acting on the sodium channel like tetrodotoxin, alpha-ScTX and veratridine. The alpha-ScTX/beta-ScTX binding site capacities decreases as development of rat brain synaptosomes progresses ; at day 7 after birth, it is 1.1. and at day 39, 0.3.

Two types of scorpion neurotoxins characterized by their binding to two separate receptor sites on rat brain synaptosomes

Summary The neurotoxin II from the venom of the American scorpion Centruroides suffusus suffusus was iodinated using the lactoperoxydase method. The labeled toxin binds specifically to a single class of non interacting binding sites on rat brain synaptosomes. The dissociation constant is 3–4 nM and the capacity 800–1400 fmoles per mg of protein. No competition between this toxin and neurotoxin II from the African scorpion Androctonus australis Hector has been found. There are 6–10 times more sites for Centruroides than for Androctonus neurotoxin. Moreover, unlike the binding of the Androctonus neurotoxin, the binding of Centruroides neurotoxin II is not potential dependent. Therefore a distinction between scorpion neurotoxins is proposed according to their binding properties to rat brain synaptosomes.

An excitatory and a depressant insect toxin from scorpion venom both affect sodium conductance and possess a common binding site

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.

Purification and properties of the insect toxin from the venom of the scorpion Androctonus australis Hector

Summary The purification of a protein toxic to insects from the venom of the scorpion Androctonus australis Hector has been performed by recycling Sephadex G-50 gel filtration and equilibrium chromatography on DEAE-Sephadex A-50 and Amberlite CG-50. The final product was purified 267-fold as compared to the crude venom. The toxicity yield was 95 percent. Purity of the insect toxin was assessed by amino acid analysis, N-terminal sequential degradation, C-terminal amino acid determination and zone electrophoresis. Its molecular weight is 7498. The insect toxin differs from the proteins toxic to mammals contained in the same venom. The sequence of the first 15 amino acid residues from the N-terminal of both types of toxins is compared. The physiological significance of these neurotoxic proteins is discussed.

Two neurotoxins (BmK I and BmK II) from the venom of the scorpion Buthus martensi Karsch: purification, amino acid sequences and assessment of specific activity

Two neurotoxins, BmK I and BmK II, were purified from the venom of the Chinese scorpion Buthus martensi Karsch. The complete amino acid sequences of both toxins, each containing 64 amino acid residues, were determined by the automatic sequencing of reduced and S-carboxymethylated toxins and their peptides, obtained after cleavage with TPCK-treated trypsin and Staphylococcus aureus V8 protease, respectively. Toxicity as minimum lethal dose tested by i.c.v. injection in mice showed that BmK I was six times more potent than BmK II. Only two amino acid replacements were found: at position 59 Val in BmK I was replaced by Ile in BmK II, and at position 62 a basic Lys residue in BmK I was substituted by a neutral Asn residue in BmK II. These features suggest that the positively charged residue (Lys or Arg) in the C-terminal position 62 (or 61 or 63) may also play an important role in facilitating the interaction between scorpion neurotoxins and the receptor on sodium channels. The effects of BmK I on nerve excitability were examined with the crayfish axon using intracellular recording and voltage-clamp conditions. The results indicate that BmK I preferentially blocks the sodium channel inactivation process. Thus, functional and structural similarities suggest that BmK I and BmK II belong to group 3 of scorpion alpha-type toxins.

Tityus serrulatus toxin VII bears pharmacological properties of both β-toxin and insect toxin from scorpion venoms

Some beta-toxins from the South American scorpion Tityus serrulatus (e.g. Ts VII) are highly toxic both for mouse and fly larva. Radioiodinated Ts VII and the insect toxin from the North African scorpion Androctonus australis Hector (AaH IT) bind to the same site on a house fly head synaptosomal fraction. These results reinforce the hypothesis about the existence of a correlated series of scorpion toxins as previously defined by amino acid compositions and sequences, and immunological and circular dichroism studies, in suggesting that Ts VII constitutes a link which may fill the pharmacological gap existing between beta-toxins and insect toxins such as AaH IT.

Chemical synthesis and characterization of maurocalcine, a scorpion toxin that activates Ca2+ release channel/ryanodine receptors.

Maurocalcine is a novel toxin isolated from the venom of the chactid scorpion Scorpio maurus palmatus. It is a 33‐mer basic peptide cross‐linked by three disulfide bridges, which shares 82% sequence identity with imperatoxin A, a scorpion toxin from the venom of Pandinus imperator. Maurocalcine is peculiar in terms of structural properties since it does not possess any consensus motif reported so far in other scorpion toxins. Due to its low concentration in venom (0.5% of the proteins), maurocalcine was chemically synthesized by means of an optimized solid‐phase method, and purified after folding/oxidation by using both C18 reversed‐phase and ion exchange high‐pressure liquid chromatographies. The synthetic product (sMCa) was characterized. The half‐cystine pairing pattern of sMCa was identified by enzyme‐based cleavage and Edman sequencing. The pairings were Cys3‐Cys17, Cys10‐Cys21, and Cys16‐Cys32. In vivo, the sMCa was lethal to mice following intracerebroventricular inoculation (LD50, 20 μg/mouse). In vitro, electrophysiological experiments based on recordings of single channels incorporated into planar lipid bilayers showed that sMCa potently and reversibly modifies channel gating behavior of the type 1 ryanodine receptor by inducing prominent subconductance behavior.

Scorpion neurotoxins: A family of homologous proteins.

Eleven neurotoxins have been purified from the venoms of three scorpion sub-species, Androctonus australis Hector, Buthus occitanus tunetanus and Leiurus quinquestriatus quinquestriatus [ 1 ] . These proteins all consist of a single polypeptide chain of 57 to 66 amino acid residues cross-linked by four disulfide bridges. The complete amino acid sequence of toxins I and I’ of A. australis Hector has been recently determined [2,3]. In this communication we report the sequence of the first 22 to 26 amino acid residues from the N-terminal end of six additional neurotoxins. It is shown that scorpion neurotoxins form a new set of homologous proteins. Furthermore, this set can be divided in three subgroups when additional amino acid sequence homologies and specific toxities are taken into account.

Maurotoxin, a four disulfide bridge toxin from Scorpio maurus venom: purification, structure and action on potassium channels.

A new toxin acting on K+ channels, maurotoxin (MTX), has been purified to homogeneity from the venom of the chactoid scorpion Scorpio maurus. MTX is a basic single chain 34 amino acid residue polypeptide, amidated at its C terminal, and crosslinked by four disulfide bridges. It shows 29–68% sequence identity with other K+ channel toxins, and presents an original disulfide pattern, the last two half‐cystine residues (31–34) being connected. Although the first three disulfide bonds have not been defined experimentally, modelling based on the structure of charybdotoxin favored two combinations out of six, one of which has two bridges (3–24 and 9–29) in common with the general motif of scorpion toxins. The last bridge would connect residues 13 and 19. MTX inhibits the binding to rat brain synaptosomal membranes of both [125I]apamin, a SKCa channel blocker (IC50 5 nM), and [125I]kaliotoxin, a Kv channel blocker (IC50 30 pM). MTX blocks the Kv1.1, Kv1.2 and Kv1.3 currents expressed in Xenopus oocytes with IC50 of 45, 0.8 and 180 nM, respectively. MTX represents a member of a new class of short toxins with 4 disulfide bridges, active on voltage‐dependent K+ channel and also competing with apamin for binding to its receptor.