NMR structures and activity of a novel alpha-like toxin from the scorpion Leiurus quinquestriatus hebraeus.

NMR structures of a new toxin from the scorpion Leiurus quinquestriatus hebraeus (Lqh III) have been investigated in conjunction with its pharmacological properties. This toxin is proposed to belong to a new group of scorpion toxins, the alpha-like toxins that target voltage-gated sodium channels with specific properties compared with the classical alpha-scorpion toxins. Electrophysiological analysis showed that Lqh III inhibits a sodium current inactivation in the cockroach axon, but induces in addition a resting depolarization due to a slowly decaying tail current atypical to other alpha-toxin action. Binding studies indicated that radiolabeled Lqh III binds with a high degree of affinity (Ki=2.2 nM) on cockroach sodium channels and that the alpha-toxin from L quinquestriatus hebraeus highly active on insects (LqhalphaIT) and alpha-like toxins compete at low concentration for its receptor binding site, suggesting that the alpha-like toxin receptor site is partially overlapping with the receptor site 3. Conversely, in rat brain, Lqh III competes for binding of the most potent anti-mammal alpha-toxin from Androctonus australis Hector venom (AaH II) only at very high concentration. The NMR structures were used for the scrutiny of the similarities and differences with representative scorpion alpha-toxins targeting the voltage-gated sodium channels of either mammals or insects. Three turn regions involved in the functional binding site of the anti-insect LqhalphaIT toxin reveal significant differences in the Lqh III structure. The electrostatic charge distribution in the Lqh III toxin is also surprisingly different when compared with the anti-mammal alpha-toxin AaH II. Similarities in the electrostatic charge distribution are, however, recognized between alpha-toxins highly active on insects and the alpha-like toxin Lqh III. This affords additional important elements to the definition of the new alpha-like group of scorpion toxins and the mammal versus insect scorpion toxin selectivities.

Sequence and electrophysiological characterization of two insect-selective excitatory toxins from the venom of the Chinese scorpion Buthus martensi.

The two insecticidal peptides Bm32-VI and Bm33-I, isolated from the venom of the Chinese scorpion Buthus martensi induce paralytical symptoms typical of insect contractive toxins. They show, respectively, 74% and 77% homology with AaIT from Androctonus australis, comparable insecticidal activity and no vertebrate toxicity. Under voltage-clamp conditions, both toxins induced (1) an increased fast Na(+) current, (2) a shift in voltage dependence of Na(+) current activation, (3) the occurrence of a delayed current, and (4) a slow development of a holding current. Increased Na(+) conductance at negative potential values is responsible for axonal hyperexcitability and the contractive paralysis of insect prey.

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 microns) 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 microns) 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.

Octopaminergic dorsal unpaired median (DUM) neurones innervating the colleterial glands of the female cockroach Periplaneta americana

The musculature of the colleterial glands receives innervation from branch 4B4a of the nerves designated 4B, which arise from the posterior part of the terminal abdominal ganglion in the female cockroach Periplaneta americana (L). Using Methylene Blue staining, the gross anatomy of the colleterial gland innervation has been described. Cobalt backfilling via branch 4B4 of nerve 4B revealed about 21 dorsal unpaired median (DUM) neurones located on both median and posterior parts of the terminal abdominal ganglion. Octopamine immunohistochemistry has shown that at least 15 octopamine-immunoreactive DUM neurones from median and posterior groups projected via branch 4B4a to the left and right colleterial glands. These data, together with results reporting the presence of octopamine-immunoreactive branches supplying these colleterial glands, make octopaminergic DUM neurones suitable candidates to modulate the muscle activity of the colleterial glands in female Periplaneta americana.

Postsynaptic effects of 4-aminopyridine in the sixth abdominal ganglion of the cockroach.

The synaptic action of 4-aminopyridine (4-AP) was studied in the sixth abdominal ganglion of the cockroach Periplaneta americana L. The drug 4-AP did not modify the affinity of cholinergic receptors. At concentrations above 10(-4)M, 4-AP depolarized the postsynaptic membranes even after blockage of muscarinic and nicotinic receptors by antagonistic substances.

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.

Action of babycurus-toxin 1 from the east African scorpion Babycurus centrurimorphus on the isolated cockroach giant axon.

A toxin named babycurus-toxin 1 (mol. wt 8191), from telson extracts of the scorpion Babycurus centrurimorphus, was found to depolarize the cockroach giant axon. It progressively blocked the evoked action potentials after a short period of limited repetitive activity and after 30 min of toxin action it became impossible to evoke responses to current stimulations. Voltage-clamp experiments on the sodium current indicated that the toxin in micromolar concentrations progressively decreased the transient inward peak sodium current, but also slowed the activation phase of this sodium current and maintained an inward current during the voltage pulses, which deactivated slowly. The toxin also induced in the insect axon a slowly activating-deactivating component of the sodium current. This suggests that the toxin modifies both activation and inactivation mechanisms of sodium channels. Thus there is some similarity in the electrophysiological effects between BcTx1 and the beta-toxins active on mammals.

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.

Primary structure and electrophysiological characterization of two almost identical isoforms of toxin from Isometrus vittatus (family: Buthidae) scorpion venom.

Two almost identical proteins with 70 amino acid residues each, closely packed by four disufide bridges, and molecular masses of 7899.5 and 7884.7 were isolated and sequenced from the venom of the scorpion Isometrus vittatus from Pakistan. They differ by an acidic amino acid residue (glutamic or aspartic) at the same position 55 of the peptide chain, however, they exhibit the same length, the same charge and are undistinguishable when separated by C(18) reverse phase HPLC. The mixture of the two proteins called IsomTx1 depolarizes the cockroach isolated axon; artificial repolarization is followed by sustained repetitive activity, artificial hyperpolarization facilitates bursting activity observed as an answer to rapid depolarization to -60 mV. The depolarization is antagonized by TTX. In voltage-clamp experiments IsomTx1 increases axonal sodium permeability which has a particular importance between resting and threshold potentials and moderately slows down the fast inactivation. These characteristics closely resemble those of other anti-insect scorpion toxins classified as contractive toxins from Androctonus and Buthotus venoms.

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.

Pre- and postsynaptic effects of taurine and GABA in the cockroach central nervous system.

Taurine resembles GABA in its synaptic effects in the cockroach cercal nerve giant fiber synapse where it exerts a depressant action upon synaptic transmission. Both taurine and GABA produce an increased conductance of pre- and postsynaptic membranes through changes in the permeability of chloride ions.

Effect of K+ accumulation removal and high extracellular osmolarity on K+ current in insect axonal membrane.

The effect of a removal of K+ accumulation on K+ current in insect axonal membrane was observed. Experiments were performed on isolated giant axon of a cockroach using double oil gap technique. K+ accumulation was reduced by: (1) an outward water flow induced by non-electrolytes (urea, glucose) added to extracellular saline and (2) by an increase of non-specific permeability of axonal glial layer obtained after the application of DMSO. The conclusions are: (1) osmolar effect depends on the type of molecule used for osmotic shock, (2) increase of outward K+ current in conditions of high extraaxonal osmotic pressure is attributed to the decrease of K+ accumulation and outward water flow, (3) removal of K+ accumulation doesn't affect the kinetics and the time course of K+ current, (4) experiments confirmed the presence of an inactivating component in the axonal outward K+ current, (5) DMSO must be used cautiously as a solvent in electrophysiological experiments.

Peptides of arachnid venoms with insecticidal activity targeting sodium channels.

Arachnids have a venom apparatus and secrete a complex chemical mixture of low molecular mass organic molecules, enzymes and polypeptide neurotoxins designed to paralyze or kill their prey. Most of these toxins are specific for membrane voltage-gated sodium channels, although some may also target calcium or potassium channels and other membrane receptors. Scorpions and spiders have provided the greatest number of the neurotoxins studied so far, for which, a good number of primary and 3D structures have been obtained. Structural features, comprising a folding that determines a similar spatial distribution of charged and hydrophobic side chains of specific amino acids, are strikingly common among the toxins from spider and scorpion venoms. Such similarities are, in turn, the key feature to target and bind these proteins to ionic channels. The search for new insecticidal compounds, as well as the study of their modes of action, constitutes a current approach to rationally design novel insecticides. This goal tends to be more relevant if the resistance to the conventional chemical products is considered. A promising alternative seems to be the biotechnological approach using toxin-expressing recombinant baculovirus. Spider and scorpion toxins having insecticidal activity are reviewed here considering their structures, toxicities and action mechanisms in sodium channels of excitable membranes.

Effect of toxin 1 from Androctonus australis Hector on sodium currents in giant axons of Loligo forbesi.

The effects of external application of micromolar concentrations of toxin 1 of the scorpion, Androctonus australis Hector, on the sodium conductance of squid giant axons have been studied quantitatively using the voltage clamp technique. Toxin concentrations which induce long plateau action potentials under current clamp conditions were found to simultaneously decrease the peak conductance and increase the delayed sodium conductance. Return to holding potential level after step depolarizations was accompanied by large exponential tails of current. The toxin-induced maintained sodium conductance increased with membrane depolarization independently of the peak conductance. Depolarizing conditioning prepulses to - 30 mV were found to almost totally inactivate the peak sodium current but to leave the delayed conductance unaffected. This property was taken as an indication that the total current is made of the added contributions of two distinct populations on sodium channels : fast activating and inactivating channels and slow activating channels. These two channel populations were separated from each other and analysed. It was found that the fast channels were almost identical to normal channels whereas the slow channels had a much slower (nearly exponential) kinetics and activated for more positive values of membrane potential. These observations strongly support the second hypothesis of Gillespie and Meves (1980) that the peak conductance and maintained conductance reflect the existence of two separate populations of channels. They further indicate that slow channels probably originate from the modification by the toxin of normal voltage-sensitive channels.

Actions of insect toxin and other toxins derived from the venom of the scorpion Androctonus australis on isolated giant axons of the cockroach (Periplaneta americana).

1. Insect toxin, mammal toxins I and II and crustacean toxin were obtained from the venom of the scorpion Androctonus australis. Their effects on the isolated giant axon of the cockroach Periplaneta americana were investigated by current-clamp and voltage-clamp techniques. 2. In current-clamp conditions, mammal toxins and crustacean toxin (1.3-13 microM) induced a large prolongation of the falling phase of the evoked action potentials. Insect toxin (0.13-3.3 microM) induced a progressive slow depolarization of the membrane potential and repetitive firing of action potentials. No changes in the time-course of the action potential were induced by insect toxin. 3. In voltage-clamp conditions, mammal and crustacean toxins induced a slowing of the turn-off of the transient inward sodium current, with either no change or a small increase in the peak sodium current. Insect toxin by contrast induced an increase in the peak sodium current and a slowing of the sodium current turn-off, this effect being greatest at lower values of the clamped membrane voltage. 4. It is concluded that the repetitive activity induced by insect toxin results from a voltage-dependent modulation of sodium inactivation coupled with an increase in both the resting and active sodium permeabilities of the cockroach axonal membrane.

Outward chloride/potassium co-transport in insect neurosecretory cells (DUM neurones).

The mechanism underlying outward chloride transport in the cell body and in the neuritic field of cockroach Dorsal Unpaired Median (DUM) neurones was assessed using the intracellular microelectrode technique. The chloride equilibrium potential was indirectly estimated from the reversal potentials of responses to gamma-aminobutyric acid (GABA) pressure ejections and of inhibitory postsynaptic potential (IPSP) evoked by electrical stimulation of the anterior connectives. Changes in intracellular chloride concentration [Cl-]i following various treatments were estimated from the amplitude changes of soma GABA responses and IPSP. Decreasing external Cl- concentration reduced the amplitude of GABA-mediated inhibitory events without affecting the membrane potential. Cl-/K+ co-transport was assessed by increasing external K+ concentration. The rate of outward Cl- movement was reduced furosemide but not by SITS or DIDS. All these results suggest that Cl- is not passively distributed in DUM neurones and that an active outwardly directed Cl-/K+ co-transport is implicated in the regulation of [Cl-]i.