Marcel Pelhate

The Putative Bioactive Surface of Insect-selective Scorpion Excitatory Neurotoxins

Scorpion neurotoxins of the excitatory group show total specificity for insects and serve as invaluable probes for insect sodium channels. However, despite their significance and potential for application in insect-pest control, the structural basis for their bioactivity is still unknown. We isolated, characterized, and expressed an atypically long excitatory toxin, Bj-xtrIT, whose bioactive features resembled those of classical excitatory toxins, despite only 49% sequence identity. With the objective of clarifying the toxic site of this unique pharmacological group, Bj-xtrIT was employed in a genetic approach using point mutagenesis and biological and structural assays of the mutant products. A primary target for modification was the structurally unique C-terminal region. Sequential deletions of C-terminal residues suggested an inevitable significance of Ile73 and Ile74 for toxicity. Based on the bioactive role of the C-terminal region and a comparison of Bj-xtrIT with a Bj-xtrIT-based model of a classical excitatory toxin, AaHIT, a conserved surface comprising the C terminus is suggested to form the site of recognition with the sodium channel receptor.

Pharmacological properties of insect axons: A review

NUMEROUS synthetic chemicals currently in use as agrochemical and pharmaceutical agents, together with a wide range of toxic molecules of both plant and animal origins. exert specific actions on excitable tissues. Amongst these chemically diverse molecules are many which act by modifying the properties of the voltage-dependent sodium and potassium channels involved in action potential generation in neurones and in heart and skeletal muscle. Considerable progress has been achieved in the physiology and pharmacology of the nerve axon membrane in the past forty years, as a result of experiments on the giant axons of invertebrate animals, in particular those of molluscs and crustaceans (NARAHASHI, 1974, 1975). In recent years, advances in methods for recording the electrical properties of insect giant axons h?ve revealed their value in determining the molecular mechanisms of action of novel neuroactive chemicals including insecticides. The majority of investigations on insect axons have been performed using the giant axons of interganglionit interneurones, located in the connectives linking the abdominal ganglia in the central nervous system of the cockroach Periplaneta americana. In the initial studies, axons were impaled by means of microelec-

Poneratoxin, a novel peptide neurotoxin from the venom of the ant, Paraponera clavata.

1. At concentrations varying from 10(-8) to 10(-6) M synthetic poneratoxin (PoTX) is a strong, but very slowly acting agonist for smooth muscles and its blocks synaptic transmission in the insect CNS in a concentration-dependent manner and depolarizes giant interneurons. 2. However, in isolated dorsal unpaired median cells 10(-6) M PoTX causes only a reversible hyperpolarization of about 5 mV. 3. At concentrations from 10(-8) to 10(-6) M PoTX affects the electrical activity of isolated cockroach axons, as well as isolated frog and rat skeletal muscle fibres. 4. PoTX prolongs action potentials and induces slow automatic activity, due to a slow Na(+)-current activation at very negative values of potential and due to slow deactivation.

Pyrethroid insecticides: Actions of deltamethrin and related compounds on insect axonal sodium channels

Abstract The actions of deltamethrin and eight other pyrethroids were tested on isolated giant axons of the cockroach Periplaneta americana, using microelectrode and oil-gap, single-fibre electrophysiological recording techniques. Deltamethrin at micromolar concentrations induced a slow progressive depolarization of the axon membrane accompanied by a gradual reduction in action potential amplitude. The deltamethrin-induced depolarization was enhanced by an increase in stimulation frequency and was reduced in the presence of the sodium channel blocking agent saxitoxin (1 × 10−7 M). Other synthetic pyrethroids (biopermethrin and its 1S enantiomer, biotetramethrin, s-bioallethrin, bioresmethrin and its 1S enantiomer, cismethrin and kadethrin) were also studied. In contrast to the findings with deltamethrin all other compounds, apart from the 1S isomers which were inactive, induced prolonged negative (depolarizing) after-potentials. Deltamethrin appears to affect a small fraction of sodium channels which are held in a modified open-state, whereas the pyrethroids which generate large negative after-potentials appear to induce a brief alteration of the open-state sodium channels with a larger number of channels affected. Differences between the actions of pyrethroids on insect axonal sodium channels and whole insects are discussed.

Purification, structure and activity of three insect toxins from Buthus occitanus tunetanus venom

One contractive and two depressant toxins active on insect were purified by high-performance liquid chromatography from the venom of Buthus occitanus tunetanus (Bot). The two depressant toxins, BotIT4 and BotIT5, differ only at position 6 (Arg for Lys) and are equally toxic to insects (LD50 to Blatella germanica = 110 ng/100 mg body weight). They show a strong antigenic cross-reaction with a depressive toxin from Leiurus quinquestriatus quinquestriatus (LqqIT2). The two toxins are able to inhibit with high affinity (K0.5 between 2 and 3 nM) the specific binding of the radioiodinated excitatory insect toxin (125I-AaHIT) on its receptor site on Periplaneta americana synaptosomal membranes. These toxins depolarize the cockroach axon, irreversibly block the action potential, and slow down and very progressively block the transmembrane transient Na+ current. The contracturant toxin BotIT1 is highly toxic to B. germanica (LD50 = 60 ng/ 100 mg body weight) and barely toxic to mice (LD50 = 1 microgram/20 g body weight) when injected intracerebroventricularly. It does not compete with 125I-AaHIT for its receptor site on P. americana synaptosomal membranes. On cockroach axon, BotIT1 develops plateau potentials and slows down the inactivation mechanism of the Na+ channels. Thus, BotIT1 belongs to the group of alpha insect-selective toxins and shows a strong sequence identity (> 90%) with Lqh alpha IT and LqqIII, two insect alpha-toxins previously purified from the venom of L. q. hebraeus and L. q. quinquestriatus. respectively.

Anatomy and targets of dorsal unpaired median neurones in the terminal abdominal ganglion of the male cockroach Periplaneta americana L.

The morphology of the Dorsal Unpaired Median (DUM) neurones in the Terminal Abdominal Ganglion (TAG) of the adult male cockroach Periplaneta americana were described based on wholemount preparations and paraffin sections and by using anterograde and retrograde cobalt mapping, octopamine‐like immunohistochemistry, and double immunofluorescence technique with both conjugated gamma‐aminobutyric acid (GABA) and octopamine antisera. Among 60 ± 6 neurones with large somata (diameter 40 to 60 μm) on the dorsal midline surface of the TAG that were stained with toluidine blue, about 36 efferent DUM neurones exhibited octopamine‐like immunoreactivity. The DUM neurones were arranged in three clusters (anterior, median and posterior) corresponding to the 7th–11th abdominal ganglia of the fused TAG. Anterior efferent DUM neurones with one, two, and four pairs of lateral neurites entered segmental nerves VIIB; VIIB and phallic nerves; IXB and phallic nerves; VIIIA, IXA, X, and IX, respectively. Three octopamine‐like immunoreactive DUM neurones innervating heart chambers via segmental nerves (VIIA, VIIIA, and IXA) in the last abdominal segments occurred within abdominal ganglia 7, 8, and 9. Together with octopamine‐like immunoreactive efferent DUM neurones, GABA‐like immunoreactive dorsal midline neurones with small somata (10 to 20 μm) also occurred within the median group. The spatial distribution of DUM neurones in the TAG suggested that they had their origins in the median neuroblast, as for DUM neurones in the grasshopper.

Block of synaptic transmission in insect CNS by toxins from the venom of the WASP Megascolia Flavifrons (FAB.)

1. The effects of the venom and its fractions of Megascolia flavifrons have been studied on synaptic transmission and axonal excitability of the giant interneuron of the cockroach. 2. The venom does not affect axonal excitability, but blocks synaptic transmission, and induces postsynaptic depolarization with a delay. 3. Five different active fractions have been recognized. 4. Three fractions of them contain substances already identified as histamine, Thr6 bradykinin and Thr6 bradykinin-Lys-Ala (megascoliakinin). 5. Three fractions contain activities, which have not yet been chemically identified. 6. All of them, and also bradykinin block synaptic transmission; histamine was not active.

Refined electrophysiological analysis suggests that a depressant toxin is a sodium channel opener rather than a blocker.

The effects of a recombinant depressant insect toxin from Leiurus quinquestriatus hebraeus, Lqh IT2-r, have been studied in current and voltage-clamp conditions on the isolated axonal and DUM neuron preparations of the cockroach Periplaneta americana. Lqh IT2-r depolarizes the axon, blocks the evoked action potentials, and modifies the amplitude and the kinetics of the sodium current. The inward transient peak current is greatly decreased and is followed by a maintained slow activating-deactivating sodium current. The slow component develops at membrane potentials more negative than the control, and has a time constant of activation of several tens of milliseconds. The flaccid properties of Lqh IT2-r do not correspond to a blockage of the Na+ channels, but may be attributed to modified Na+ channels which open at more negative potential, activate slowly and do not inactivate normally.

Poneratoxin, a new toxin from an ant venom, reveals an interconversion between two gating modes of the Na channels in frog skeletal muscle fibres

The effects of synthetic poneratoxin (PoTX), a new toxin isolated from the venom of the ant Paraponera clavata, were studied under current- and voltage-clamp conditions in frog skeletal muscle fibres. PoTX induces a concentration-dependent (10−9 M–5×10−6 M) prolongation of the action potentials and, at saturating concentration, a slow repetitive activity developing at negative potentials. PoTX specifically acts on voltage-dependent Na channels by decreasing the peak Na current (INa) and by simultaneously inducing a slow INa which starts to activate at −85 mV and inactivates very slowly. Both the fast and the slow components of INa are suppressed by tetrodotoxin and reverse at the same potential corresponding to the equilibrium potential for Na ions. The fast component of INa has voltage dependence, activation and steady-state inactivation almost similar to those of the control INa. The voltage dependence of the slow Na conductance is 40 mV more negative than that of the fast one. The results suggest that PoTX affects all the Na channels and that the fast and the slow INa components originate from a possible PoTX-induced interconversion between a fast and a slow operating mode of the Na channels.

Effects of a centipede venom fraction on insect nervous system, a native Xenopus oocyte receptor and on an expressed Drosophila muscarinic receptor.

Centipede venoms are complex protein mixtures; very few is known about their pharmacological actions. Application of a Scolopendra sp. venom fraction (SC1) on the cockroach giant axon induced an increase in the leak current correlated with a decrease in the membrane resistance, suggesting the presence in SC1 of components opening non-specific pores in the axonal membrane. On a cockroach central cholinergic synapse, microinjection of SC1 induced a small transient depolarization of the postsynaptic membrane, followed by a slow stable depolarization and a drastic decrease in the evoked subthreshold excitatory postsynaptic potential amplitude. A pretreatment of the ganglion with atropine or scopolamine reduced the amplitude of the SC1-induced depolarizing wave, suggesting a possible cholinergic muscarinic target. On control Xenopus oocytes, SC1 induced an inward oscillatory Ca2(+)-dependent Cl- current mediated through the activation of native lysophosphatidic acid receptors (LPAr). Indeed, pretreatment of oocytes with 1 microM N-palmitoyl-tyrosine phosphoric acid, a selective competitive antagonist of LPAr, decreased responses to SC1 by 70%. Application of SC1 to oocytes expressing a cloned Drosophila muscarinic receptor (Dml) induced a biphasic response comprising: (1) a large fast Cl- current that was abolished by pretreatment with atropine and scopolamine and (2) a slow and small oscillating Cl- current corresponding to the response observed in control oocytes. These observations confirm the presence of muscarinic agonists in SCI and reveal their direct action on an insect muscarinic receptor subtype homologous to mammalian M1-M3 receptors.