Changing the structural context of a functional beta-hairpin. Synthesis and characterization of a chimera containing the curaremimetic loop of a snake toxin in the scorpion alpha/beta scaffold

An approach to obtain new active proteins is the incorporation of all or a part of a well defined active site onto a natural structure acting as a structural scaffold. According to this strategy we tentatively engineered a new curaremimetic molecule by transferring the functional central loop of a snake toxin, sequence 26-37, sandwiched between two hairpins, onto the structurally similar beta-hairpin of the scorpion toxin charybdotoxin, stabilized by a short helix. The resulting chimeric molecule, only 31 amino acids long, was produced by solid phase synthesis, refolded, and purified to homogeneity. As shown by structural analysis performed by CD and NMR spectroscopy, the chimera maintained the expected alpha/beta fold characteristic of scorpion toxins and presented a remarkable structural stability. The chimera competitively displaces the snake curaremimetic toxin alpha from the acetylcholine receptor at 10(-5) M concentrations. Antibodies, elicited in rabbits against the chimera, recognize the parent snake toxin and prevent its binding to the acetylcholine receptor, thus neutralizing its toxic function. All these data demonstrate that the strategy of active site transfer to the charybdotoxin scaffold has general applications in the engineering of novel ligands for membrane receptors and in vaccine design.     

Molecular determinants of high affinity binding of alpha-scorpion toxin and sea anemone toxin in the S3-S4 extracellular loop in domain IV of the Na+ channel alpha subunit

alpha-Scorpion toxins and sea anemone toxins bind to a common extracellular site on the Na+ channel and inhibit fast inactivation. Basic amino acids of the toxins and domains I and IV of the Na+ channel alpha subunit have been previously implicated in toxin binding. To identify acidic residues required for toxin binding, extracellular acidic amino acids in domains I and IV of the type IIa Na+ channel alpha subunit were converted to neutral or basic amino acids using site-directed mutagenesis, and altered channels were transiently expressed in tsA-201 cells and tested for 125I-alpha-scorpion toxin binding. Conversion of Glu1613 at the extracellular end of transmembrane segment IVS3 to Arg or His blocked measurable alpha-scorpion toxin binding, but did not affect the level of expression or saxitoxin binding affinity. Conversion of individual residues in the IVS3-S4 extracellular loop to differently charged residues or to Ala identified seven additional residues whose mutation caused significant effects on binding of alpha-scorpion toxin or sea anemone toxin. Moreover, chimeric Na+ channels in which amino acid residues at the extracellular end of segment IVS3 of the alpha subunit of cardiac Na+ channels were substituted into the type IIa channel sequence had reduced affinity for alpha-scorpion toxin characteristic of cardiac Na+ channels. Electrophysiological analysis showed that E1613R has 62- and 82-fold lower affinities for alpha-scorpion and sea anemone toxins, respectively. Dissociation of alpha-scorpion toxin is substantially accelerated at all potentials compared to wild-type channels. alpha-Scorpion toxin binding to wild type and E1613R had similar voltage dependence, which was slightly more positive and steeper than the voltage dependence of steady-state inactivation. These results indicate that nonidentical amino acids of the IVS3-S4 loop participate in alpha-scorpion toxin and sea anemone toxin binding to overlapping sites and that neighboring amino acid residues in the IVS3 segment contribute to the difference in alpha-scorpion toxin binding affinity between cardiac and neuronal Na+ channels. The results also support the hypothesis that this region of the Na+ channel is important for coupling channel activation to fast inactivation.     

The prolyl aminopeptidase from Lactobacillus delbrueckii subsp. bulgaricus belongs to the alpha/beta hydrolase fold family

Prolyl aminopeptidase (PepIP) of Lactobacillus delbrueckii subsp. bulgaricus displays the Gly-x-Ser-x-Gly-Gly consensus motif surrounding the catalytic serine of the prolyl oligopeptidases family. Sequence comparison revealed that this motif and two other domains appear well conserved among bacterial PepIPs and members of the alpha/beta hydrolase fold family. Secondary structural predictions of PepIP were performed from amino acid sequence and corroborated by circular dichroism analysis. These predictions well matched the core structure of alpha/beta hydrolases organised in eight beta-sheets connected by alpha-helices. We obtained 26 mutants of PepIP by chemical or site-directed mutagenesis. Most substitutions associated with stable and inactive mutant proteins were mainly located in the three conserved boxes (including the catalytic serine motif). Taken together, our results strongly suggest that PepIP belongs to the alpha/beta hydrolase fold family and that Ser107, Asp246 and His273 constitute the catalytic triad of the enzyme.     

Cytokine synthesis of human monocytes stimulated by triple or single helical conformer of an antitumour (1-->3)-beta-D-glucan preparation, sonifilan

We have cloned and sequenced the fission yeast (Schizosaccharomyces pombe) fas1+ gene, which encodes the fatty acid synthetase (FAS) beta subunit, by applying a PCR technique to conserved regions in the beta subunit of the alpha6beta6 types of FAS among different organisms. The deduced amino acid sequence of the Fas1 polypeptide, consisting of 2073 amino acids (Mr = 230,616), exhibits the 48.1% identity with the beta subunit from the budding yeast (Saccharomyces cerevisiae). This subunit, with five different catalytic activities, bears four distinct domains, while the alpha subunit, the sequence of which was previously reported by Saitoh et al. (S. Saitoh et al., 1996, J. Cell Biol. 134, 949-961), carries three domains. We have developed a co-expression system of the FAS alpha and beta subunits by cotransformation of two expression vectors, containing the lsd1+/fas2+ gene and the fas1+ gene, into fission yeast cells. The isolated FAS complex showed quite high specific activity, of more than 4000 mU/mg, suggesting complete purification. Its molecular weight was determined by dynamic light scattering and ultracentrifugation analysis to be 2.1-2.4 x 10(6), and one molecule of the FAS complex was found to contain approximately six FMN molecules. These results indicate that the FAS complex from S. pombe forms a heterododecameric alpha6beta6 structure. Electron micrographs of the negatively stained molecule suggest that the complex adopts a unique barrel-shaped cage architecture.     

Macromolecular derivatives of N,N-di-(2-chloroethyl)-4-phenylene diamine mustard. 2. In vitro cytotoxicity and in vivo anticancer efficacy

Scorpions have survived successfully over millions of years without detectable changes in their morphology. Instead, they have developed an efficient alomonal machinery and a stinging device supporting their needs for prey and defense. They produce a large variety of polypeptidic toxins that bind and modulate ion channel conductance in excitable tissues. The binding site, mode of action, and chemical properties of many toxins have been studied extensively, but little is known about their genomic organization and diversity. Genes representing each of the major classes of Buthidae scorpion toxins, namely, "long" toxins, affecting sodium channels (alpha, depressant, and excitatory), and "short" toxins, affecting potassium and chloride channels, were isolated from a single scorpion segment and analyzed. Each toxin type was found to be encoded by a gene family. Regardless of toxin length, 3-D structure, and site of action, all genes contain A+T-rich introns that split, at a conserved location, an amino acid codon of the signal sequence. The introns vary in length and sequence but display identical boundaries, agree with the GT/AG splice junctions, and contain T-runs downstream of a putative branch point, 5'-TAAT-3'. Despite little sequence similarity among all toxin classes, the conserved gene organization, intron features, and common cysteine-stabilized alpha-helical (CSH) core connecting an alpha-helix to a three-stranded beta-sheet suggest, that they all evolved from an ancestral common progenitor. Furthermore, the vast diversity found among genomic copies, cDNAs, and their protein products for each toxin suggests an extensive evolutionary process of the scorpion "pharmaceutical factory," whose success is due, most likely, to the inherent permissiveness of the toxin exterior to structural alterations.     

Depressant insect selective neurotoxins from scorpion venom: chemistry, action, and gene cloning

The present study examines the similarity in the symptoms and binding properties between the depressant and excitatory insect-selective neurotoxins, derived from scorpion venom. A comparison of their primary structures and neuromuscular effects is presented. A new depressant toxin (LqhIT2) was purified from the venom of the scorpion Leiurus quinquestriatus hebraeus. The effects of this toxin on a prepupal housefly neuromuscular preparation mimic its effects on the intact insect, i.e, a brief period of repetitive bursts of regular junction potentials (JPs) is followed by reduced amplitude JPs ending with a block of the neuromuscular transmission. "Loose" patch clamp recordings indicate that the repetitive activity has a presynaptic origin (the motor nerve) and resembles the effect of the excitatory toxin AaIT. The final synaptic block is supposed to be the end result of neuronal membrane depolarization. Such an effect is not caused by an excitatory toxin, which induces long "trains" of repetitive firing. The amino acid sequences of three depressant toxins were determined by automatic Edman degradation indicating a high degree of sequence homology. This conservation differs from those of other groups of scorpion toxins. The opposing pharmacological effects of depressant toxins are discussed in light of the above neuromuscular effects and sequence analysis. A genetic approach in the study of the structure-function relationships of the depressant toxins was initiated by isolating cDNA clones encoding the LqhIT2 and BjIT2 toxins. Their sequence analysis revealed the precursor form of these toxins: A 21 amino acid residue signal peptide followed by a 61 amino acid region of the mature toxin, and three additional amino acids at the carboxy terminus.     

Analysis of the requirements for class II-restricted T cell recognition of a single determinant reveals considerable diversity in the T cell response and degeneracy of peptide binding to I-Ed

The amino acid sequences recognized by five I-E(d)-restricted and one E alpha A beta d-restricted murine T cell clones were determined. The clones had been raised to a synthetic peptide representing amino acids 305-328 of influenza virus hemagglutinin. It was found that although all of the T cell clones recognized a single 10-residue region of the peptide, 307KYVKQNTLKL316, different clones could recognize minimal ("core") determinants spanning 8, 9, or 10 of these amino acids. To see whether particular amino acids within the sequence 307-316 were universally important for T cell recognition, the six clones were assayed for their ability to tolerate single amino acid substitutions of the 10 residue peptide. In all, 190 analogues of the peptide in which each amino acid in the sequence was replaced, in turn, by each of the other 19 naturally occurring amino acids were tested. It was shown that 1) the six T cell clones had very different requirements for recognition of the peptide, 2) substitutions at every single position within the peptide could be shown to affect recognition in a T cell-specific manner, and 3) every single position within the peptide could be replaced by a large number of amino acids and still be recognized by at least one T cell clone. These results demonstrate the great diversity exhibited by the T cell repertoire in recognizing a 10-amino acid determinant, as well as the degeneracy of peptide binding to I-E(d).     

Globin synthesis in normal human bone marrow

Globin synthesis has been studied by in vitro labelling with radioactive amino acids in 60 normal human bone-marrow samples. Under the conditions routinely used to fractionate alpha and beta chains by chromatography alpha/beta production ratios ranging from 0.5 to 1.0 were obtained, depending on the method of sample treatment. This variation was due entirely to the presence of non-haem proteins derived from white cells which chromagraphy with globin on CM-cellulose. Purification of globin on Sephadex G100 and fractionation of alpha and beta globin chains by a modified chromatographic system resulted in alpha/beta ratios of unity. The relevance of these findings to the study of marrows in which there is unbalanced globin chain production is discussed.     

The CDNA and genomic DNA sequences of a mammalian neurotoxin from the scorpion Buthus martensii Karsch

The cDNA library of venomous glands of the scorpion Buthus martensii Karsch (BmK) was constructed. A cDNA encoding a mammalian neurotoxin corresponding to the known alpha-type toxin, BmK M1, was amplified by polymerase chain reaction (PCR) and cloned, and its full-length sequence was determined. The open reading frame encoded the precursor of BmK M1 with 84 amino acid residues, including a signal peptide of 19 residues, a mature toxin of 64 residues and an additional C-terminal residue Arg which might be cleaved off by proteinase postprocessing immediately after protein synthesis. Based on the determined cDNA sequence and using the total DNA of the scorpion as a template, the gene of BmK M1 was also amplified by PCR and sequenced. The genomic DNA sequence revealed an intron of 408 base pairs present within the signal peptide region. Both the intron and exon of BmK M1 share about 75% similarity with those of AaH I' another alpha-type mammalian neurotoxin in the scorpion Androctonus australis Hector.     

New toxins acting on sodium channels from the scorpion Leiurus quinquestriatus hebraeus suggest a clue to mammalian vs insect selectivity

Two new toxins were purified from Leiurus quinquestriatus hebraeus (Lqh) scorpion venom, Lqh II and Lqh III. Lqh II sequence reveals only two substitutions, as compared to AaH II, the most active scorpion alpha-toxin on mammals from Androctounus australis Hector. Lqh III shares 80% sequence identity with the alpha-like toxin Bom III from Buthus occitanus mardochei. Using bioassays on mice and cockroach coupled with competitive binding studies with 125I-labeled scorpion alpha-toxins on rat brain and cockroach synaptosomes, the animal selectivity was examined. Lqh II has comparable activity to mammals as AaH II, but reveals significantly higher activity to insects attributed to its C-terminal substitution, and competes at low concentration for binding on both mammalian and cockroach sodium channels. Lqh II thus binds to receptor site 3 on sodium channels. Lqh III is active on both insects and mammals but competes for binding only on cockroach. The latter indicates that Lqh III binds to a distinct receptor site. Thus, Lqh II and Lqh III represent two different scorpion toxin groups, the alpha- and alpha-like toxins, respectively, according to the structural and pharmacological criteria. These new toxins may serve as a lead for clarification of the structural basis for insect vs mammal selectivity of scorpion toxins.     

NMR solution structure of a two-disulfide derivative of charybdotoxin: structural evidence for conservation of scorpion toxin alpha/beta motif and its hydrophobic side chain packing

The alpha/beta scorpion fold consisting of a short alpha-helix and beta-sheet is a structural motif common to scorpion toxins, insect defensins, and plant gamma-thionins that invariably contains three disulfides. CHABII is a two-disulfide derivative of the scorpion toxin charybdotoxin (ChTX), chemically synthesized by inserting two L-alpha-aminobutyric acids in place of the two half-cystine residues involved in the disulfide 13-33. This disulfide is one of the two disulfides which connect the alpha-helix to the beta-sheet. The solution structure of CHABII was determined at pH 6.3 and 5 degrees C using 2D NMR and simulated annealing from 513 distance and 46 dihedral angle constraints. The NMR structure of CHABII is well-defined as judged from the low value of the averaged backbone rms deviation between the 30 lowest energy structures and the energy-minimized mean structure ((rmsd) = 0.65 A for the entire sequence and 0.48 A for the segment 3-36). Analysis and comparison of the solution structures of CHABII and ChTX lead to the following conclusions: (i) the fold of CHABII is similar to that of ChTX as indicated by the low value of the averaged backbone atomic rms deviation between the 10 lowest energy solution structures of the two proteins (1.44 A); (ii) the packing of the hydrophobic core is well-preserved, underlying the critical structural role of the hydrophobic interactions even for such a small and cysteine-rich protein as ChTX.     

An excitatory scorpion toxin with a distinctive feature: an additional alpha helix at the C terminus and its implications for interaction with insect sodium channels

BACKGROUND: Scorpion neurotoxins, which bind and modulate sodium channels, have been divided into two groups, the alpha and beta toxins, according to their activities. The beta-toxin class includes the groups of excitatory and depressant toxins, which differ in their mode of action and are highly specific against insects. The three-dimensional structures of several alpha and beta toxins have been determined at high resolution, but no detailed 3D structure of an excitatory toxin has been presented so far. RESULTS: The crystal structure of an anti-insect excitatory toxin from the scorpion Buthotus judaicus, Bj-xtrIT, has been determined at 2.1 A resolution and refined to an R factor of 0.209. The first 59 residues form a closely packed module, structurally similar to the conserved alpha and beta toxins ('long toxins') affecting sodium channels. The last 17 residues form a C-terminal extension not previously seen in scorpion toxins. It comprises a short alpha helix anchored to the N-terminal module by a disulfide bridge and is followed by a highly mobile stretch of seven residues, of which only four are seen in the electron-density map. This mobile peptide covers part of a conserved hydrophobic surface that is thought to be essential for interaction with the channel in several long toxins. CONCLUSIONS: Replacement of the last seven residues by a single glycine abolishes the activity of Bj-xtrIT, strongly suggesting that these residues are intimately involved in the interaction with the channel. Taken together with the partial shielding of the conserved hydrophobic surface and the proximity of the C terminus to an adjacent surface rich in charged residues, it seems likely that the bioactive surface of Bj-xtrIT is formed by residues surrounding the C terminus. The 3D structure and a recently developed expression system for Bj-xtrIT pave the way for identifying the structural determinants involved in the bioactivity and anti-insect specificity of excitatory toxins.     

Recombinant protein sequences can trigger methylation of N-terminal amino acids in Escherichia coli

Recombinant human hemoglobin rHb1.1 has been genetically engineered with the replacement of the wild-type valine residues at all N-termini with methionine, an Asn 108 Lys substitution on the beta globins, and a fusion of the two alpha globins with a glycine linker. When rHb1.1 was expressed in Escherichia coli, methylation of the N-terminal methionine of the alpha globin was discovered. Another mutant has been engineered with the alpha globin gene coding for N-terminal methionine followed by an insertion of alanine. Characterization of expressed hemoglobin from this variant revealed a methylated N-terminal alanine that occurred through two posttranslational events: initial excision of the N-terminal methionine, followed by methylation of alanine as the newly generated N-terminus. No methylation was observed for variants expressed with wild-type valine at the N-terminus of the alpha globin. The methylation of N-terminal amino acids was attributed to a specific protein sequence that can trigger methylation of proteins expressed in E. coli. Here we demonstrate that proline at position 4 in the protein sequence of alpha globin seems an essential part of that signaling. Although N-terminal methylation has been observed previously for native E. coli proteins with similar N-terminal sequences, methylation of the recombinant globins has allowed further delineation of the recognition sequence, and indicates that methylation of heterologous proteins can occur in E. coli.     

Two similar peptides from the venom of the scorpion Pandinus imperator, one highly effective blocker and the other inactive on K+ channels

Two novel peptides, named Pi4 and Pi7, were purified from the venom of the scorpion Pandinus imperator, and their primary structures were determined. These peptides have 38 amino acids residues, compacted by four disulfide bridges, instead of the normal three found in most K+-channel specific toxins. Both peptides contain 25 identical amino acid residues in equivalent positions (about 66% identity), including all eight half-cystines. Despite the fact that their C-terminal sequence comprising amino acid residues 27 to 37 are highly conserved (10 out of 11 amino acids are identical), Pi4 blocks completely and reversibly Shaker B K+ -channels (a Kv1.1 sub-family type of channel) at 100nM concentration, whereas Pi7 is absolutely inactive at this concentration. Similar effects were observed in binding and displacement experiments to rat brain synaptosomal membranes using 125I-Noxiustoxin, a well known K+-channel specific toxin. In this preparation Pi4 displaces the binding of radiolabeled Noxiustoxin with Ic50 in the order of 10 nM, whereas Pi7 is ineffective at same concentration. Comparative analysis of Pi4 and Pi7 sequences with those obtained by site directed mutagenesis of Charybdotoxin, another very well studied K -channel blocking toxin, shows that the substitution of lysine (in Pi4) for arginine (in Pi7) at position 26, might be one of the important 'point mutations' responsible for such impressive variation in blocking properties of both toxins, here described.     

Homology of the N-terminal domain of the petH gene product from Anabaena sp. PCC 7119 to the CpcD phycobilisome linker polypeptide

The complete nucleotide sequence of the petH gene encoding ferredoxin-NADP+ reductase from the nitrogen-fixing cyanobacterium Anabaena sp. PCC 7119 has been determined. The encoded polypeptide is 136 amino acids longer than the enzyme obtained after purification to homogeneity. The extended N-terminal domain consists of 80 amino acids which shows homology to the CpcD phycobilisome linker polypeptide, through which FNR might be anchored to the thylakoid-bound phycobilisomes. A 56 amino acid interdomain fragment is found which could be a target for proteolysis.     

Extensive random mutagenesis analysis of the Na+/K+-ATPase alpha subunit identifies known and previously unidentified amino acid residues that alter ouabain sensitivity--implications for ouabain binding

Random mutagenesis with ouabain selection has been used to comprehensively scan the extracellular and transmembrane domains of the alpha1 subunit of the sheep Na+/K+-ATPase for amino acid residues that alter ouabain sensitivity. The four random mutant libraries used in this study include all of the transmembrane and extracellular regions of the molecule as well as 75% of the cytoplasmic domains. Through an extensive number of HeLa cell transfections of these libraries and subsequent ouabain selection, 24 ouabain-resistant clones have been identified. All previously described amino acids that confer ouabain resistance were identified, confirming the completeness of this random mutagenesis screen. The amino acid substitutions that confer the greatest ouabain resistance, such as Gln111-->Arg, Asp121-->Gly, Asp121-->Glu, Asn122-->Asp, and Thr797-->Ala were identified more than once in this study. This extensive survey of the extracellular and transmembrane regions of the Na+/K+-ATPase molecule has identified two new regions of the molecule that affect ouabain sensitivity: the H4 and the H10 transmembrane regions. The new substitutions identified in this study are Leu330-->Gln, Ala331-->Gly, Thr338-->Ala, and Thr338-->Asn in the H4 transmembrane domain and Phe982-->Ser in the H10 transmembrane domain. These substitutions confer modest increases in the concentration of cardiac glycoside needed to produce 50% inhibition of activity (IC50 values), 3.1-7.9-fold difference. The results of this extensive screening of the Na+/K+-ATPase alpha1 subunit to identify amino acids residues that are important in ouabain sensitivity further supports our hypothesis that the H1-H2 and H4-H8 regions represent the major binding sites for the cardiac glycoside class of drugs.     

Three-dimensional structure of human protein kinase C interacting protein 1, a member of the HIT family of proteins

The three-dimensional structure of protein kinase C interacting protein 1 (PKCI-1) has been solved to high resolution by x-ray crystallography using single isomorphous replacement with anomalous scattering. The gene encoding human PKCI-1 was cloned from a cDNA library by using a partial sequence obtained from interactions identified in the yeast two-hybrid system between PKCI-1 and the regulatory domain of protein kinase C-beta. The PKCI-1 protein was expressed in Pichia pastoris as a dimer of two 13.7-kDa polypeptides. PKCI-1 is a member of the HIT family of proteins, shown by sequence identity to be conserved in a broad range of organisms including mycoplasma, plants, and humans. Despite the ubiquity of this protein sequence in nature, no distinct function has been shown for the protein product in vitro or in vivo. The PKCI-1 protomer has an alpha+beta meander fold containing a five-stranded antiparallel sheet and two helices. Two protomers come together to form a 10-stranded antiparallel sheet with extensive contacts between a helix and carboxy terminal amino acids of a protomer with the corresponding amino acids in the other protomer. PKCI-1 has been shown to interact specifically with zinc. The three-dimensional structure has been solved in the presence and absence of zinc and in two crystal forms. The structure of human PKCI-1 provides a model of this family of proteins which suggests a stable fold conserved throughout nature.     

Identification and characterization of a novel beta subunit of soluble guanylyl cyclase that is active in the absence of a second subunit and is relatively insensitive to nitric oxide

Previously characterized soluble guanylyl cyclases form alpha-beta heterodimers that can be activated by the gaseous messenger, nitric oxide. In mammals, four subunits have been cloned, named alpha1, alpha2, beta1, and beta2. We have identified a novel soluble guanylyl cyclase isoform from the nervous system of the insect Manduca sexta that we have named M. sexta guanylyl cyclase beta3 (MsGC-beta3). It is most closely related to the mammalian beta subunits but has several features that distinguish it from previously identified soluble cyclases. Most importantly, MsGC-beta3 does not need to form heterodimers to form an active enzyme because guanylyl cyclase activity can be measured when it is expressed alone in COS-7 cells. Moreover, this activity is only weakly enhanced in the presence of the nitric oxide donor, sodium nitroprusside. Several of the amino acids in rat beta1 subunits, previously identified as being important in heme binding or necessary for nitric oxide activation, are substituted with nonsimilar amino acids in MsGC-beta3. There are also an additional 315 amino acids C-terminal to the catalytic domain of MsGC-beta3 that have no sequence similarity to any known protein. Northern blot analysis shows that MsGC-beta3 is primarily expressed in the nervous system of Manduca.     

Anti-insect scorpion toxins: historical account, activities and prospects

Some toxins from scorpion venoms, much more toxic to insects than to other animal classes, possess high affinity to Na+ channels. These anti-insect scorpion toxins have been divided into: 1) alpha toxins which lack strict selectivity for insects, do not compete with following groups of anti-insect toxins, resemble other alpha scorpion toxins by their structure and their ability, as alpha anemone toxins, to prolong insect axonal action potential durations through a drastic slowing down of the Na+ current inactivation, 2) excitatory insect selective scorpion toxins which induce in blowfly larvae an immediate fast paralysis; in isolated cockroach axons, they depolarize and induce a sustained repetitive activity of short (normal) action potentials through a shift of Na+ activation mechanism towards more negative potentials and some decrease of inactivation at these potential values, 3) depressant insect selective neurotoxins which cause a slow progressive flaccid paralysis of larvae, depolarize insect axons and reduce or even suppress evoked action potentials; resting depolarizations which are antagonized by a post-application of TTX, are due to the opening of sodium channels at very negative potential values and to the suppression of their inactivation mechanism. The decrease of the maximal Na+ conductance following flaccid toxin action may be understood if toxin-modified channels opened at very negative potentials values remain open (or re-open) for much longer times than in control conditions and pass by substate less conductant states. Anti-insect scorpion toxins become of major interest into insect neurophysiology and also into insect pest control, due to their specific target sites and to the recent constructions of insecticidal baculovirus expressions of several of these toxins.