Small conductance calcium-activated K+ channels, SkCa, but not voltage-gated K+ (Kv) channels, are implicated in the antinociception induced by CGS21680, a A2A adenosine receptor agonist.

It has been shown that A2A adenosine receptors are implicated in pain modulation. The precise mechanism by which activation of A2A receptors produces analgesic effects, however, remains unclear. The aim of this study was to investigate the possible involvement of apamin-sensitive calcium-activated potassium channels (SKCa) and voltage-gated potassium (Kv) channels in A2A receptor activation-induced analgesic effects. Using mice, we evaluated the influence of apamin, a non specific blocker of SKCa channels, Lei-Dab7 (an analog of scorpion Leiurotoxin), a selective blocker of SKCa2 channels, and kaliotoxin (KTX) a Kv channel blocker, on the CGS 21680 (A2A adenosine receptor agonist)-induced increases in hot plate and tail pinch latencies. All drugs were injected in mice via the intracerebroventricular route. We found that apamin and Lei-Dab7, but not KTX, reduced antinociception produced by CGS21680 on the hot plate and tail pinch tests in a dose dependent manner. Lei-Dab 7 was more potent than apamin in this regard. We conclude that SKCa but not Kv channels are implicated in CGS 21680-induced antinociception.

Design and evaluation of a diabody to improve protection against a potent scorpion neurotoxin.

Diabodies are recombinant, dimeric, antibody-based molecules composed of two non-covalently associated single-chain antibody fragments that bind to an antigen in a divalent manner. In an attempt to develop more effective therapeutic molecules against scorpion venoms, we designed a diabody derived from monoclonal antibody 9C2, which neutralizes the toxicity of scorpion neurotoxin AahI in mammals. The recombinant diabody produced in the periplasm of Escherichia coli was purified to homogeneity in a single step by protein L-agarose affinity chromatography. It was functional, and possessed a high binding affinity to AahI (8 x 10(-11) M). The bivalence of the diabody was confirmed by size-exclusion chromatography, isoelectrofocussing and electron microscopic observations. Finally, the diabody showed high thermal stability in serum and demonstrated protective activity when injected intraperitoneally in mice experimentally envenomed with toxin AahI. In conclusion, the diabody format gives the 9C2 molecule advantageous properties that are particularly important for potential clinical applications in the treatment of envenomations.

[Theoretical and experimental bases for treatment of scorpion envenomations]. / Bases théoriques et expérimentales du traitement des envenimations scorpioniques.

Improving the efficacy of envenomation treatment depends on what is known about active molecules present in venoms. Regarding scorpions, studies carried out mainly on the most poisonous species have shown that the toxicity and mortality were due to small proteins-toxins--able to interfere with the normal process of the ionic channels. In certain cases, using the mouse model, it has been shown that over 90% of mortality was due to toxins operating on the sodium channels implicated in the action potential of the excitable cells. Pharmacokinetic studies have shown the diversity of their mode of action implying an adaptation of the means and tools intended to neutralise them. The toxins active on the sodium channels represent a family of proteins from 60 to 65 amino acids linked by 4 disulphide bridges with a very strong antigenic polymorphism; this has certain implications in terms of paraspecificity of antivenoms. The problem is even more complicated when one considers the variation of toxin quantity from one animal to another of the same species. Another approach is to identify the most active and represented toxins in venoms for each antigenic group and to develop a means of neutralizing them. It would also be possible to define toxoids for use either in the production of the antivenoms or as immunological protection for individuals at risk. Lastly, where symptomatic treatment is concerned, certain drugs such as aspirin, quinine or dandrolene have been shown definitely to increase the value of the LD50 in the mouse.

Chemical synthesis, molecular modeling, and antimicrobial activity of a novel bacteriocin, MMFII.

A new antimicrobial peptide, referred to as MMFII, was purified to homogeneity from lactic acid bacteria Lactococcus lactis, which were isolated from Tunisian dairy product. The complete amino acid sequence of the peptide has been established by amino acid analysis, Edman sequencing, and mass spectrometry and verified by solid-phase chemical synthesis. MMFII is a single-chain 37-residue polypeptide containing a single intramolecular disulfide bond, i.e., TSYGNGVHCNKSKCWIDVSELETYKAGTVSNPKDILW. It shares ca. 35% sequence identity with Leucocin A, a class IIa bacteriocin. Modeling based on the 3-D of Leucocin A shows three beta strands located in the N-terminal region (Thr1-Tyr3, Val7-Asn10, Lys13-Ile16) and an alpha helical domain from Asp17 to Asn31. When plotted as an alpha-helical wheel, the central alpha-helix of MMFII does not exhibit an amphipathic helical structure. The synthetic MMFII (sMMFII), obtained by the solid-phase method, was shown to be indistinguishable from the natural peptide. sMMFII is active against Lactococcus cremoris and Listeria ivanovii bacteria, whereas no activity was detected for any of the synthetic N-terminal truncated MMFII analogs Cys9-Trp37, Trp15-Trp37, and Val18-Trp37.

Parameters affecting in vitro oxidation/folding of maurotoxin, a four-disulphide-bridged scorpion toxin.

Maurotoxin (MTX) is a 34-mer scorpion toxin cross-linked by four disulphide bridges that acts on various K(+) channel subtypes. MTX adopts a disulphide bridge organization of the type C1-C5, C2-C6, C3-C4 and C7-C8, and folds according to the common alpha/beta scaffold reported for other known scorpion toxins. Here we have investigated the process and kinetics of the in vitro oxidation/folding of reduced synthetic L-MTX (L-sMTX, where L-MTX contains only L-amino acid residues). During the oxidation/folding of reduced L-sMTX, the oxidation intermediates were blocked by iodoacetamide alkylation of free cysteine residues, and analysed by MS. The L-sMTX intermediates appeared sequentially over time from the least (intermediates with one disulphide bridge) to the most oxidized species (native-like, four-disulphide-bridged L-sMTX). The mathematical formulation of the diffusion-collision model being inadequate to accurately describe the kinetics of oxidation/folding of L-sMTX, we have formulated a derived mathematical description that better fits the experimental data. Using this mathematical description, we have compared for the first time the oxidation/folding of L-sMTX with that of D-sMTX, its stereoisomer that contains only D-amino acid residues. Several experimental parameters, likely to affect the oxidation/folding process, were studied further; these included temperature, pH, ionic strength, redox potential and concentration of reduced toxin. We also assessed the effects of some cellular enzymes, peptidylprolyl cis-trans isomerase (PPIase) and protein disulphide isomerase (PDI), on the folding pathways of reduced L-sMTX and D-sMTX. All the parameters tested affect the oxidative folding of sMTX, and the kinetics of this process were indistinguishable for L-sMTX and D-sMTX, except when stereospecific enzymes were used. The most efficient conditions were found to be: 50 mM Tris/HCl/1.4 mM EDTA, pH 7.5, supplemented by 0.5 mM PPIase and 50 units/ml PDI for 0.1 mM reduced compound. These data represent the first report of potent stereoselective effects of cellular enzymes on the oxidation/folding of a scorpion toxin.

Neutralization of gating charges in domain II of the sodium channel alpha subunit enhances voltage-sensor trapping by a beta-scorpion toxin.

beta-Scorpion toxins shift the voltage dependence of activation of sodium channels to more negative membrane potentials, but only after a strong depolarizing prepulse to fully activate the channels. Their receptor site includes the S3-S4 loop at the extracellular end of the S4 voltage sensor in domain II of the alpha subunit. Here, we probe the role of gating charges in the IIS4 segment in beta-scorpion toxin action by mutagenesis and functional analysis of the resulting mutant sodium channels. Neutralization of the positively charged amino acid residues in the IIS4 segment by mutation to glutamine shifts the voltage dependence of channel activation to more positive membrane potentials and reduces the steepness of voltage-dependent gating, which is consistent with the presumed role of these residues as gating charges. Surprisingly, neutralization of the gating charges at the outer end of the IIS4 segment by the mutations R850Q, R850C, R853Q, and R853C markedly enhances beta-scorpion toxin action, whereas mutations R856Q, K859Q, and K862Q have no effect. In contrast to wild-type, the beta-scorpion toxin Css IV causes a negative shift of the voltage dependence of activation of mutants R853Q and R853C without a depolarizing prepulse at holding potentials from -80 to -140 mV. Reaction of mutant R853C with 2-aminoethyl methanethiosulfonate causes a positive shift of the voltage dependence of activation and restores the requirement for a depolarizing prepulse for Css IV action. Enhancement of sodium channel activation by Css IV causes large tail currents upon repolarization, indicating slowed deactivation of the IIS4 voltage sensor by the bound toxin. Our results are consistent with a voltage-sensor-trapping model in which the beta-scorpion toxin traps the IIS4 voltage sensor in its activated position as it moves outward in response to depolarization and holds it there, slowing its inward movement on deactivation and enhancing subsequent channel activation. Evidently, neutralization of R850 and R853 removes kinetic barriers to binding of the IIS4 segment by Css IV, and thereby enhances toxin-induced channel activation.

Disulfide bridge reorganization induced by proline mutations in maurotoxin.

Maurotoxin (MTX) is a 34-residue toxin that has been isolated from the venom of the chactidae scorpion Scorpio maurus palmatus, and characterized. Together with Pi1 and HsTx1, MTX belongs to a family of short-chain four-disulfide-bridged scorpion toxins acting on potassium channels. However, contrary to other members of this family, MTX exhibits an uncommon disulfide bridge organization of the type C1-C5, C2-C6, C3-C4 and C7-C8, versus C1-C5, C2-C6, C3-C7 and C4-C8 for both Pi1 and HsTx1. Here, we report that the substitution of MTX proline residues located at positions 12 and/or 20, adjacent to C3 (Cys(13)) and C4 (Cys(19)), results in conventional Pi1- and HsTx1-like arrangement of the half-cystine pairings. In this case, this novel disulfide bridge arrangement is without obvious incidence on the overall three-dimensional structure of the toxin. Pharmacological assays of this structural analog, [A(12),A(20)]MTX, reveal that the blocking activities on Shaker B and rat Kv1.2 channels remain potent whereas the peptide becomes inactive on rat Kv1.3. These data indicate, for the first time, that discrete point mutations in MTX can result in a marked reorganization of the half-cystine pairings, accompanied with a novel pharmacological profile for the analog.

Adenosine and hemodialysis in humans.

BACKGROUND: Infections and hypotension are serious complications that develop during hemodialysis (HD) treatment. Adenosine (ADO), a strong hypotensive and immunosuppressive agent, may participate in these two HD complications, because high concentrations of ADO metabolites are found in dialyzed human plasma. ADO, which is released by endothelial cells, is quickly transformed into inosine (INO) by plasmatic ADO deaminase (ADA) and mononuclear cell ADO deaminase (MCADA). In plasma, the degradation of ADO into INO and its uptake by red blood cells (RBC) are both very rapid, resulting in the short half-life of ADO in blood. METHODS: Using liquid chromatography, we evaluated ADO and INO plasma concentrations before and after HD session. RESULTS: Before the HD session, ADO and INO plasma concentrations were higher in hemodialyzed patients than in controls and in peritoneally dialyzed patients. At the end of the HD session, ADO plasma concentration was increased. ADO plasma concentration for the undialyzed patients was in the same range as that of the controls. Before HD, ADA activity was higher in hemodialyzed patients (559 +/- 349 IU) than in controls (219 +/- 48 IU), and the activity rose during the session (665 +/- 135 IU). ADA activity in the undialyzed patients (222 +/- 80 IU) was in the same range as that of the controls (219 +/- 48 IU). Before the HD session, the MCADA activity (247 +/- 144 IU) was lower than in controls (624 +/- 99 IU). HD did not modify ADO RBC uptake. ADO inhibited mononuclear cell proliferation and interferon-gamma production in humans. Finally, as much as 50 microM INO does not inhibit ADO uptake by RBC and does not modify ADA and MCADA activities. CONCLUSIONS: These data indicate that chronic HD inhibited MCADA activity and increased ADO plasma concentration. Both high ADO plasma concentration and low MCADA activity may be involved in dialysis-induced immune system failure and thereby favor infectious diseases.

In vivo neurotoxicity of Androctonus australis hector scorpion venom: evidence that the supra-thoracic nervous system is not implicated in the clinical manifestations.

The severity of scorpion stings is related to the highly active neurotoxins in the venom. In this study, rats whose supra-spinal central nervous system was deprived of its peripheral connections were experimentally poisoned by the venom of Androctonus australis hector scorpion. Clinical signs of severity were not modified when the rats had previously undergone high medullar section. These results suggest that the supra-thoracic nervous system is not implicated in the neurotoxicity manifestations of scorpion envenomation.

Purification, amino-acid sequence and partial characterization of two toxins with anti-insect activity from the venom of the South American scorpion Tityus bahiensis (Buthidae).

We report here the isolation by a two-step chromatographic procedure of two new toxins from the South American scorpion Tityus bahiensis. Their amino-acid sequences and some of their biological features were established. The two toxins have different biological properties. Toxin TbIT-I had almost no activity or pharmacological effects in vertebrate tissues whereas it was lethal to house flies (LD50 80.0 ng/house fly). In contrast, Tb2-II was active against both mammals (intracerebroventricular injection of 100 ng/mouse was lethal) and insects (LD50 40.0 ng/house fly). The amino-acid sequences of these toxins were established and found to be similar (60-95%) to previously described beta-toxins from the Tityus genus. Based on the available comparative information, this study attempts identify possible structure-function relationships that may be responsible for the differences in bioactivity displayed by these toxins.

Construction and functional evaluation of a single-chain antibody fragment that neutralizes toxin AahI from the venom of the scorpion Androctonus australis hector.

9C2 is a murine monoclonal IgG that participates in the neutralization of Androctonus australis hector scorpion venom. It recognizes AahI and AahIII, two of the three main neurotoxins responsible for almost all the toxicity of the venom when injected into mammals. Using PCR we cloned the antibody variable region coding genes from 9C2 hybridoma cells and constructed a gene encoding a single-chain antibody variable fragment molecule (scFv). This scFv was produced in the periplasm of Escherichia coli in a soluble and functional form and purified in a single step using protein L-agarose beads yielding 1-2 mg.L(-1) of bacterial culture. scFv9C2 was predominantly monomeric but also tended to form dimeric and oligomeric structures, all capable of binding toxin AahI. The affinity of scFv and the parental mAb for toxin AahI and homologous toxin AahIII was of the same magnitude, in the nanomolar range. Similarly, purified forms of scFv9C2 completely inhibited the binding of toxin AahI to rat brain synaptosomes. Finally, scFv9C2 was efficient in protecting mice against the toxic effects of AahI after injection of the toxin and scFv to mice by the intracerebroventricular route in a molar ratio as low as 0.36 : 1. Thus, we produced a recombinant scFv that reproduces the recognition properties of the parent antibody and neutralizes the scorpion neurotoxin AahI, thereby opening new prospects for the treatment of envenomation.

A recombinant scFv/streptavidin-binding peptide fusion protein for the quantitative determination of the scorpion venom neurotoxin AahI.

We created a construct encoding a peptide known to mimic the binding properties of biotin fused to the carboxy-terminus of a scFv fragment that binds a scorpion toxin (AahI). This fusion protein was produced in the periplasm of bacteria and purified to homogeneity by single-step affinity chromatography on streptavidin-agarose with a yield close to 1 mg/l. DNA sequencing, dot blot and mass spectrometric analyses demonstrated the integrity of the soluble immunoconjugate. Fusion to the streptavidin-binding peptide did not affect the ability of the scFv to recognize its antigen with a high affinity (Kd = 2.3 x 10(-10) M). Similarly, the streptavidin-binding property was not impaired in the fusion protein. Thus, the immunoconjugate was bifunctional and had a low molecular mass of 28 kDa. This enabled us to develop rapid and sensitive immunoassays for the specific detection of the toxin AahI accurately to 0.6 ng/ml, opening up new perspectives for the diagnosis of envenomations.

Lebetin peptides: potent platelet aggregation inhibitors.

Lebetins from Macrovipera lebetina snake venom constitute a new class of inhibitors of platelet aggregation. There are two groups of peptides: lebetin 1 (L1; 11- to 13-mer) and lebetin 2 (L2; 37- to 38-mer). The short lebetins are identical to the N-terminal segments of the longer ones. They inhibit platelet aggregation induced by various agonists (e.g. thrombin, PAF-acether or collagen). The shortest lebetin (11-mer) shows potent inhibition of rabbit (IC(50) = 7 nM) and human (IC(50) = 5 nM) platelets. They prevent collagen-induced thrombocytopenia in rats. N- and C-terminal-truncated synthetic L1gamma (sL1gamma; 11-mer) is less active in inhibiting platelet aggregation than the native peptide. Results from Ala scan studies of the sL1gamma peptide indicated that replacement of the residues (P3, G7, P8, P9 or N10) resulted in a remarkable drop in the activity, whereas replacement of residues K2, P4 or K6 by Ala resulted in enhancement of the antiplatelet activity by at least 10-fold. To examine the activity of multimeric L1gamma, several multimeric peptides were synthesized using the multiple-antigen peptide system assembled on a branched lysine core and their antiplatelet activity was evaluated in vitro. The largest multimeric peptides showed a 1,000-fold increase in antiplatelet activity.

Maurotoxin versus Pi1/HsTx1 scorpion toxins. Toward new insights in the understanding of their distinct disulfide bridge patterns.

Maurotoxin (MTX) is a scorpion toxin acting on several K(+) channel subtypes. It is a 34-residue peptide cross-linked by four disulfide bridges that are in an "uncommon" arrangement of the type C1-C5, C2-C6, C3-C4, and C7-C8 (versus C1-C5, C2-C6, C3-C7, and C4-C8 for Pi1 or HsTx1, two MTX-related scorpion toxins). We report here that a single mutation in MTX, in either position 15 or 33, resulted in a shift from the MTX toward the Pi1/HsTx1 disulfide bridge pattern. This shift is accompanied by structural and pharmacological changes of the peptide without altering the general alpha/beta scaffold of scorpion toxins.

Chemical synthesis and characterization of Pi1, a scorpion toxin from Pandinus imperator active on K+ channels.

Pi1 is a 35-residue toxin cross-linked by four disulfide bridges that has been isolated from the venom of the chactidae scorpion Pandinus imperator. Due to its very low abundance in the venom, we have chemically synthesized this toxin in order to study its biological activity. Enzyme-based proteolytic cleavage of the synthetic Pi1 (sPi1) demonstrates half-cystine pairings between Cys4-Cys25, Cys10-Cys30, Cys14-Cys32 and Cys20-Cys35, which is in agreement with the disulfide bridge organization initially reported on the natural toxin. In vivo, intracerebroventricular injection of sPi1 in mice produces lethal effects with an LD50 of 0.2 microgram per mouse. In vitro, the application of sPi1 induces drastic inhibition of Shaker B (IC50 of 23 nM) and rat Kv1.2 channels (IC50 of 0.44 nM) heterologously expressed in Xenopus laevis oocytes. No effect was observed on rat Kv1.1 and Kv1.3 currents upon synthetic peptide application. Also, sPi1 is able to compete with 125I-labeled apamin for binding onto rat brain synaptosomes with an IC50 of 55 pM. Overall, these results demonstrate that sPi1 displays a large spectrum of activities by blocking both SK- and Kv1-types of K+ channels; a selectivity reminiscent of that of maurotoxin, another structurally related four disulfide-bridged scorpion toxin that exhibits a different half-cystine pairing pattern.

Purification, characterization and molecular modelling of two toxin-like proteins from the Androctonus australis Hector venom.

Two toxin-like proteins (AahTL1 and AahTL3) were purified from the venom of the scorpion Androctonus australis Hector (Aah). AahTL1 and AahTL3 are the first non toxic proteins cross-reacting with AahI toxins group which indicates that these proteins can be used as a model of vaccins. In order to study structure-function relationships, their complete amino-acid sequences (66 residues) were determined, by automated Edman degradation. They show more than 50% of similarity with both AahI and AahIII antimammal toxins. Three-dimensional structural models of AahTL1 and AahTL3 constructed by homology suggest that the two proteins are structurally similar to antimammal scorpion alpha-toxins specific to voltage dependent Na+ channels. The models showed also that amino-acid changes between potent Aah toxins and both AahTL1 and AahTL3 disrupt the electrostatic potential gradient at their surface preventing their interaction with the receptor, which may explain their non toxicity.

Effect of maurotoxin, a four disulfide-bridged toxin from the chactoid scorpion Scorpio maurus, on Shaker K+ channels.

Maurotoxin is a 34-residue toxin isolated from the venom of the Tunisian chactoid scorpion Scorpio maurus palmatus and contains four disulfide bridges that are normally found in long-chain toxins of 60-70 amino acid residues, which affect voltage-gated sodium channels. However, despite the unconventional disulfide-bridge pattern of maurotoxin, the conformation of this toxin remains similar to that of other toxins acting on potassium channels. Here, we analyzed the effects of synthetic maurotoxin on voltage-gated Shaker potassium channels (ShB) expressed in Xenopus oocytes. Maurotoxin produces a strong, but reversible, inhibition of the ShB K+ current with an IC50 of 2 nM. Increasing concentrations of the toxin induce a progressively higher block at saturating concentrations. At nonsaturating concentrations of the toxin (5-20 nM), the channel block appears slightly more pronounced at threshold potentials suggesting that the toxin may have a higher affinity for the closed state of the channel. At the single channel level, the toxin does not modify the unitary current amplitude, but decreases ensemble currents by increasing the number of depolarizing epochs that failed to elicit any opening. A point mutation of Lys23 to alanine in maurotoxin produces a 1000-fold reduction in the IC50 of block by the toxin suggesting the importance of this charged residue for the interaction with the channel. Maurotoxin does not affect K+ currents carried by Kir2.3 channels in oocytes or Na+ currents carried by the alphaIIa channel expressed in CHO cells.

Modulation of adenosine concentration by opioid receptor agonists in rat striatum.

There is evidence that adenosine and morphine interact in the striatum. However, little is known about the precise role of the opioid receptor subtypes implicated in the modulation of adenosine tissue concentration and in adenosine receptor expression and function. We sought to evaluate, in the absence of withdrawal symptoms, the effects of the short-term administration of selective mu-, delta- or kappa-opioid receptor agonists on adenosine concentration and on adenosine A(2A) receptor function in rat striatum. Adenosine A(2A) receptor was chosen because the neuronal sub-population expressing this receptor coexpresses enkephalin, suggesting that adenosine A(2A) receptor may be regulated by opioid receptor agonists. Oxymorphone hydrochloride mu-opioid receptor agonist, 6 mg/kg/day), +[-(5 alpha,7 alpha, 8 beta)-(-)-N-methyl-N(7-(1-pyrrolidinyl)1-oxaspiro (4.5)dec-8-yl) benzenacetamide] (U69593) (kappa-opioid receptor agonist, 0.75 mg/kg/day), and (+)-4[(alpha R)-alpha-((2S,5R)-4-allyl-2, 5-dimethyl-1-piperazinyl)-3-methoxybenzyl]-N,N-diethylbenzamide) (SNC80) (delta-opioid receptor agonist, 9 mm/kg/day), or vehicle, were administered i.p 3 x daily during 5 days to groups of rats (n=6). We also investigated the effects of opioid receptor agonists on adenosine uptake by striatal cell extracts. We found that administration of mu- or delta-opioid receptor agonists significantly decreased adenosine uptake in striatal cell extracts and increased adenosine concentration (mean+24% and +45% for mu- and delta-opioid receptor agonist, respectively, relative to controls). None of the receptor agonists tested induced obvious modifications of adenosine A(2A) receptor function. However, the delta-opioid receptor agonist induced an increase in adenosine A(2A) mRNA expression (mean 44%). We conclude that mu and delta receptor agonists inhibit adenosine uptake by striatal cell extracts and increase adenosine concentrations in rat striatum.

Synthesis, 1H NMR structure, and activity of a three-disulfide-bridged maurotoxin analog designed to restore the consensus motif of scorpion toxins.

Maurotoxin (MTX) is a 34-residue toxin that has been isolated from the venom of the chactidae scorpion Scorpio maurus palmatus. The toxin displays an exceptionally wide range of pharmacological activity since it binds onto small conductance Ca(2+)-activated K(+) channels and also blocks Kv channels (Shaker, Kv1.2 and Kv1.3). MTX possesses 53-68% sequence identity with HsTx1 and Pi1, two other K(+) channel short chain scorpion toxins cross-linked by four disulfide bridges. These three toxins differ from other K(+)/Cl(-)/Na(+) channel scorpion toxins cross-linked by either three or four disulfide bridges by the presence of an extra half-cystine residue in the middle of a consensus sequence generally associated with the formation of an alpha/beta scaffold (an alpha-helix connected to an antiparallel beta-sheet by two disulfide bridges). Because MTX exhibits an uncommon disulfide bridge organization among known scorpion toxins (C1-C5, C2-C6, C3-C4, and C7-C8 instead of C1-C4, C2-C5, and C3-C6 for three-disulfide-bridged toxins or C1-C5, C2-C6, C3-C7, and C4-C8 for four-disulfide-bridged toxins), we designed and chemically synthesized an MTX analog with three instead of four disulfide bridges ([Abu(19),Abu(34)]MTX) and in which the entire consensus motif of scorpion toxins was restored by the substitution of the two half-cystines in positions 19 and 34 (corresponding to C4 and C8) by two isosteric alpha-aminobutyrate (Abu) derivatives. The three-dimensional structure of [Abu(19), Abu(34)]MTX in solution was solved by (1)H NMR. This analog adopts the alpha/beta scaffold with now conventional half-cystine pairings connecting C1-C5, C2-C6, and C3-C7 (with C4 and C8 replaced by Abu derivatives). This novel arrangement in half-cystine pairings that concerns the last disulfide bridge results mainly in a reorientation of the alpha-helix regarding the beta-sheet structure. In vivo, [Abu(19),Abu(34)]MTX remains lethal in mice as assessed by intracerebroventricular injection of the peptide (LD(50) value of 0. 25 microg/mouse). The structural variations are also accompanied by changes in the pharmacological selectivity of the peptide, suggesting that the organization pattern of disulfide bridges should affect the three-dimensional presentation of certain key residues critical to the blockage of K(+) channel subtypes.

Maurotoxin and the Kv1.1 channel: voltage-dependent binding upon enantiomerization of the scorpion toxin disulfide bridge Cys31-Cys34.

Maurotoxin (MTX) is a 34-amino acid polypeptide cross-linked by four disulfide bridges that has been isolated from the venom of the scorpion Scorpio maurus palmatus and characterized. Maurotoxin competed with radiolabeled apamin and kaliotoxin for binding to rat brain synaptosomes and blocked K+ currents from Kv1 channel subtypes expressed in Xenopus oocytes. Structural characterization of the synthetic toxin identified half-cystine pairings at Cys3-Cys24, Cys9-Cys29, Cys13-Cys19 and Cys31-Cys34 This disulfide bridge pattern is unique among known scorpion toxins, particularly the existence of a C-terminal '14-membered disulfide ring' (i.e. cyclic domain 31-34), We therefore studied structure-activity relationships by investigating the structure and pharmacological properties of synthetic MTX peptides either modified at the C-terminus ¿i.e. MTX(1-29), [Abu31,34]-MTX and [Cys31,34, Tyr32]D-MTX) or mimicking the cyclic C-terminal domain [i.e. MTX(31-34)]. Unexpectedly, the absence of a disulfide bridge Cys31-Cys34 in [Abu 31,34]-MTX and MTX(1-29) resulted in MTX-unrelated half-cystine pairings of the three remaining disulfide bridges for the two analogs, which is likely to be responsible for their inactivity against Kv1 channel subtypes. Cyclic MTX(31-34) was also biologically inactive. [Cys31,34, Tyr32]D-MTX, which had a 'native', MTX-related, disulfide bridge organization, but a D-residue-induced reorientation of the C-terminal disulfide bridge, was potent at blocking the Kv1.1 channel. This peptide-induced Kv1.1 blockage was voltage-dependent (a property not observed for MTX), maximal in the low depolarization range and associated with on-rate changes in ligand binding. Thus, the cyclic C-terminal domain of MTX seems to be crucial for recognition of Kv1.3, and to a lesser extent, Kv1.2 channels and it may contribute to the stabilization and strength of the interaction between the toxin and the Kv1.1 channel.