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Hemoscorpius lepturus is the most medically important scorpion in Iran. The clinical signs of H. lepturus envenomation are remarkably similar to those reported for brown spiders, including dermonecrosis, hematuria, renal failure and even death. The lethality and toxicity of brown spiders’ venom have been attributed to its phospholipase D activity. This study aims to identify a phospholipase D with possible lethality and dermonecrotic activity in H. lepturus venom. In this study, a cDNA library of the venom glands was generated by Illumina RNA sequencing. Phospholipase D (PLD) from H. lepturus was characterized according to its significant similarity with PLDs from brown spiders. The main chain designated as Hl‐RecPLD1 (the first recombinant isoform of H. lepturus PLD) was cloned, expressed and purified. Sphingomyelinase, dermonecrotic and lethal activities were examined. Hl‐PLD1 showed remarkable sequence similarity and structural homology with PLDs of brown spiders. The conformation of Hl‐PLD1 was predicted as a “TIM beta/alpha‐barrel”. The lethal dose 50 (LD50) and dermonecrotic activities of Hl‐RecPLD1 were determined as 3.1 μg/mouse and 0.7 cm2 at 1 μg respectively. It is the first report indicating that a similar molecular evolutionary mechanism has occurred in both American brown spiders and this Iranian scorpion. In conclusion, Hl‐RecPLD1 is a highly active phospholipase D, which would be considered as the lethal dermonecrotic toxin in H. lepturus venom.

Androctonus mauretanicus (A. mauretanicus) and Buthus occitanus (B. occitanus) scorpions, which belong to the Buthidae family, are the most venomous scorpions in Morocco. For the first time, we investigated the effects of such scorpion venoms on serum electrolytes in subcutaneously injected rabbits. For this purpose, 3 groups of 6 albinos adult male rabbits (New Zealand) were used in this experiment. Two of the groups were given a single subcutaneous injection of either crude Am venom (5 μg/kg) or Bo venom (8 μg/kg) whereas the third group (control group) only received physiological saline solution (NaCl 0.9%). The blood samples were collected from injected rabbits via the marginal vein at time intervals of 30 min, 1 h, 2 h, 4 h, 6 h and 24 h after venom injection. The concentrations of electrolytes in the serum samples were measured. Our study indicates that scorpion envenomation in vivo, rabbit animal model, caused severe and persistent hypomagnesaemia and hypochloremia, which are accompanied of hypernatremia, hyperkalemia and hypercalcaemia. The intensity of electrolytes imbalance was clearly superior in the case of A. mauretanicus scorpion venom (although a lower quantity of venom was injected). This is coherent with the experimental data which indicate that A. mauretanicus venom is more toxic than B. occitanus venom.

Besides classical scorpion toxin-potassium channel binding modes, novel modes remain unknown. Here, we report a novel binding mode of native toxin BmKTX towards Kv1.3 channel. The combined experimental and computational data indicated that BmKTX-D33H analog used the classical anti-parallel β-sheet domain as the channel-interacting interface together with the conserved channel pore-blocking Lys(26). However, the wild-type BmKTX was found to use Arg(23) rather than Lys(26) as the new pore-blocking residue, and mainly adopt the turn motif between the α-helix and antiparallel β-sheet domains to recognize Kv1.3 channel. Together, these findings not only reveal that scorpion toxin-potassium channel interaction modes are more diverse than thought, but also highlight the functional role of toxin acidic residues in mediating diverse toxin-potassium channel binding modes.

The NADPH oxidase proteins catalyse the formation of superoxide anion which act as signalling molecules in physiological and pathological processes. Nox1-dependent NADPH oxidase is expressed in heart, lung, colon, blood vessels and brain. Different strategies involving Nox1 inhibition based on diphenylene iodonium derivatives are currently tested for colorectal cancer therapy. Here, after peptides screening on Nox1-dependent NADPH oxidase assay in HT-29 cells, we identify a peptide (referred to as NF02), cell-active, that potently block Nox1-dependent reactive oxygen species generation. Study of DEPMPO adduct formation by electron paramagnetic resonance showed that NF02 has no superoxide scavenging activity and no impact on cellular reactive oxygen species-producing enzymes such xanthine oxidase. NF02 was not cytotoxic, inhibited reactive oxygen species production of reconstituted Nox1/Noxo1/Noxa1 complex in HEK293 and did not decrease Nox2 dependent cellular NADPH oxidase reactive oxygen species production. Finally, NF02 inhibited cell migration and invasion of colorectal cancer cells which is consistent with the described impact of Nox1 inhibitors on cell migration. NF02 peptide is a new NADPH oxidase inhibitor specific for Nox1 over Nox2 and xanthine oxidase which might represent a useful Nox1 tool with potential therapeutic insights.

Background: A total of 94 lactic acid bacteria (LAB) were isolated from Tunisian artisanal (Ricotta cheese’s whey) and industrial (bactofugate) milk waste, identified and then screened for their antimicrobial activity against some bacteria implicated on nosocomial infections. Objective: Bacterial genera and species identification was performed using molecular tools. The antimicrobial activity was tested against 7 strains of Gram-negative bacteria and 4 strains of Gram-positive bacteria as well as 6 yeasts. Method: The Crude extract was found to have a narrow antimicrobial spectrum on Gram-positive bacteria mainly Listeria monocytogenes. Among the strains which showed antibacterial activity, four were determined to be bacteriocins-producers. They were identified as Lactococcus lactis. Results: Brain Heart Infusion (BHI) Agar was found more adapted than Man, Rogosa and Sharpe (MRS) to investigate the antimicrobial activity of L. actococcus lactis against L. isteria monocytogenes. The genetic determinants encoding the antimicrobial peptides were targeted by specific PCR. Conclusion: All L. lactis bacteriocin producing strains possessed the Nisine Z gene (nisZ) except for one, which contained both Nisine A and Nisine Z genes (nisA and nisZ). They have been identified as antilisterial agentS.

A new bacteriocin, lacticin LC14, produced by Lactococcus lactis BMG6.14, was isolated and characterized. It was purified to homogeneity from overnight broth culture by ammonium sulfate precipitation, Sep-Pak chromatography, and two steps of reversed-phase HPLC. Lacticin LC14 showed bactericidal-type antimicrobial activity against several lactic acid bacteria and pathogenic strains including Listeria monocytogenes. It was inactivated by proteinase K and pronase E, but was resistant to papain, lysozyme, lipase and catalase. Lacticin LC14 was heat resistant, stable over a wide range of pH (2-10) and after treatment by solvents and detergents. Its N-terminal end was found unreactive towards Edman sequencing. Based on MALDI-TOF mass spectrometry, its molecular mass was 3333.7 Da. LC14 amino acid composition revealed a high proportion of hydrophobic residues, but no modified ones. LC14 may be able to challenge other well known other bacteriocins in probiotic and therapeutic applications.

Maurotoxin (MTX) is a 34-residue toxin that was isolated initially from the venom of the scorpion Scorpio maurus palmatus. Unlike the other toxins of the α-KTx6 family (Pi1, Pi4, Pi7, and HsTx1), MTX exhibits a unique disulfide bridge organization of the type C(1) C(5) , C(2) C(6) , C(3) C(4) , and C(7) C(8) (instead of the conventional C(1) C(5) , C(2) C(6) , C(3) C(7) , and C(4) C(8) , herein referred to as Pi1-like) that does not prevent its folding along the classic α/β scaffold of scorpion toxins. MTX(Pi1) is an MTX variant with a conventional pattern of disulfide bridging without any primary structure alteration of the toxin. Here, using MTX and/or MTX(Pi1) as models, we investigated how the type of folding influences toxin recognition of the Shaker B potassium channel. Amino acid residues of MTX that were studied for Shaker B recognition were selected on the basis of their homologous position in charybdotoxin, a three disulfide-bridged scorpion toxin also active on this channel type. These residues favored either an MTX- or MTX(Pi1) -like folding. Our data indicate clearly that Lys(23) and Tyr(32) (two out of ten amino acid residues studied) are the most important residues for Shaker B channel blockage by MTX. For activity on SKCa channels, the same amino acid residues also affect, directly or indirectly, the recognition of SK channels. The molecular modeling technique and computed docking indicate the existence of a correlation between the half cystine pairings of the mutated analogs and their activity on the Shaker B K(+) channel. Overall, mutations in MTX could, or could not, change the reorganization of disulfide bridges of this molecule without affecting its α/β scaffold. However, changing of the peptide backbone (cross linking disulfide bridges from MTX-like type vs MTX(Pi1) -like type) appears to have less impact on the molecule activity than mutation of certain key amino acids such as Lys(23) and Tyr(32) in this toxin.

Protein-protein control recognition remains a huge challenge, and its development depends on understanding the chemical and biological mechanisms by which these interactions occur. Here we describe a protein-protein control recognition technique based on the dominant electrostatic interactions occurring between the proteins. We designed a potassium channel inhibitor, BmP05-T, that was 90.32% identical to wild-type BmP05. Negatively charged residues were translocated from the nonbinding interface to the binding interface of BmP05 inhibitor, such that BmP05-T now used BmP05 nonbinding interface as the binding interface. This switch demonstrated that nonbinding interfaces were able to control the orientation of protein binding interfaces in the process of protein-protein recognition. The novel function findings of BmP05-T peptide suggested that the control recognition technique described here had the potential for use in designing and utilizing functional proteins in many biological scenarios.

Overdistension of lung tissue during mechanical ventilation causes cytokine release, which may be facilitated by the autonomic nervous system. We used mechanical ventilation to cause lung injury in rats, and studied how cervical section of the vagus nerve, or substance P (SP) antagonism, affected the injury. The effects of 40 or 25 cmH2O high airway pressure injurious ventilation (HV40 and HV25) were studied and compared with low airway pressure ventilation (LV) and spontaneous breathing (controls). Lung mechanics, lung weight, gas exchange, lung myeloperoxidase activity, lung concentrations of interleukin (IL)-1β and IL-6, and amounts of lung SP were measured. Control rats were intact, others were bivagotomized, and in some animals we administered the neurokinin-1 (NK-1) receptor blocking agent SR140333. We first determined the durations of HV40 and HV25 that induced the same levels of lung injury and increased lung contents of IL-1β and IL-6. They were 90 min and 120 min, respectively. Both HV40 and HV25 increased lung SP, IL-1β and IL-6 levels, these effects being markedly reduced by NK-1 receptor blockade. Bivagotomy reduced to a lesser extent the HV40- and HV25-induced increases in SP but significantly reduced cytokine production. Neither vagotomy nor NK-1 receptor blockade prevented HV40-induced lung injury but, in the HV25 group, they made it possible to maintain lung injury indices close to those measured in the LV group. This study suggests that both neuronal and extra-neuronal SP might be involved in ventilator-induced lung inflammation and injury. NK-1 receptor blockade could be a pharmacological tool to minimize some adverse effects of mechanical ventilation.

Leiurotoxin I is a neurotoxin, blocker of Ca2+-activated apamin-sensitive K+ channel, purified from the venom of the scorpion Leiurus quinquestriatus hebraeus. It is a 31-residue polypeptide reticulated by three disulfide bridges, i.e. Cys3-Cys21, Cys8-Cys26 and Cys12-Cys28. To investigate the role of these disulfide bridges in the folding of this toxin, analogs lacking one disulfide bridge were synthesized. The structures of two analogs in which two half-cystines were replaced by α-aminobutyrate residues to suppress one disulfide bridge, were analyzed by 1H NMR. The NMR studies reveal a three-dimensional structure identical with the native toxin for the analog lacking disulfide bridge Cys3-Cys21 and a loss of organized structure for another analog lacking disulfide bridge Cys12-Cys28. These analogs are, respectively, fully active and only weakly active (2% of the residual activity) when tested in vitro for their ability to interact with their receptor channel and in vivo for their neurotoxic activity in mice. This suggests that disulfide bridge Cys12-Cys28 is essential for the folding process. In contrast, the lack of disulfide bridge Cys3-Cys21 does not affect the folding and the maintenance of bioactive conformation of Leiurotoxin I.

A multibranched peptide construct (SPC3) derived from the conserved sequence of the third variable domain (V3) of the human immunodeficiency virus (HIV) envelope (Env) inhibits HIV infectivity. It is being tested in phase II clinical trials (FDA protocol 257A). Because some Env-derived peptides inhibit HIV infectivity through alteration of Env biosynthetic pathway, we studied whether SPC3 displays its activity through interference with Env biosynthesis or with its functions at the membrane. Syncytium formation was impaired when human CD4+ cells expressed recombinant HIV Env in the presence of SPC3. This inhibition was not due to an effect of SPC3 on the amount of Env expressed at the cell membrane. As assessed using antibodies, the conformation of the receptor binding site and of V3 presented on membrane Env was not affected by the presence of SPC3 during biosynthesis. Finally, despite the ability of SPC3 to bind to CD4+ cell membrane, SPC3 did not interfere with Env binding to CD4. These data suggest that SPC3 interferes with the infection process at a post-CD4 binding step, and not with the folding of Env.

In the present study, a 'novel' toxin, called Am IT from the venom of scorpion Androctonus mauretanicus is isolated and characterized. A detailed analysis of the action of Am IT on insect axonal sodium currents is reported. Am IT was purified through gel filtration followed by C18 reversed-phase HPLC. Toxicity of Am IT in vivo was assessed on male German cockroach (Blattella germanica) larvae and C57/BL6 mice. Cross-reactivity of Am IT with two β-toxins was evidenced using (125)I-iodinated toxin-based radioimmunoassays with synaptosomal preparations from rat brain. The complete amino acid sequence of Am IT was finally determined by Edman sequencing. Am IT was observed to compete with AaH IT4 purified from the venom of scorpion Androctonus australis in binding assays. It was recognized by an antibody raised against a β-type toxin, which indicated some structural similarity with β-toxins (or related toxin family). The 'novel' toxin exhibited dual activity since it competed with anti-mammal toxins in binding assays as well as showed contracting activity to insect. The toxin competed with radio-labeled β-toxin Css IV by binding to Na(+) channels of rat brain synaptosomes. Analysis of toxin amino acid sequences showed that Am IT shares high structural identity (92%) with AaH IT4. In conclusion, Am IT not only reveals an anti-insect compound properties secreted by 'Old World' scorpions, paralyzing insect larvae by binding to Na(+) channels on larvae's nerve-cell membranes, but also exerts toxic activity in mice, which is similar to anti-mammal toxins from 'New World' scorpions (North and South Americas). Therefore, Am IT appears to be structurally and functionally similar to AaH IT4.

Small-conductance, Ca2+ activated K+ channels (SK channels) are expressed at high levels in brain regions responsible for learning and memory. In the current study we characterized the contribution of SK2 channels to synaptic plasticity and to different phases of hippocampal memory formation. Selective SK2 antisense-treatment facilitated basal synaptic transmission and theta-burst induced LTP in hippocampal brain slices. Using the selective SK2 antagonist Lei-Dab7 or SK2 antisense probes, we found that hippocampal SK2 channels are critical during two different time windows: 1) blockade of SK2 channels before the training impaired fear memory, whereas, 2) blockade of SK2 channels immediately after the training enhanced contextual fear memory. We provided the evidence that the post-training cleavage of the SK2 channels was responsible for the observed bidirectional effect of SK2 channel blockade on memory consolidation. Thus, Lei-Dab7-injection before training impaired the C-terminal cleavage of SK2 channels, while Lei-Dab7 given immediately after training facilitated the C-terminal cleavage. Application of the synthetic peptide comprising a leucine-zipper domain of the C-terminal fragment to Jurkat cells impaired SK2 channel-mediated currents, indicating that the endogenously cleaved fragment might exert its effects on memory formation by blocking SK2 channel-mediated currents. Our present findings suggest that SK2 channel proteins contribute to synaptic plasticity and memory not only as ion channels but also by additionally generating a SK2 C-terminal fragment, involved in both processes. The modulation of fear memory by down-regulating SK2 C-terminal cleavage might have applicability in the treatment of anxiety disorders in which fear conditioning is enhanced.

International audience; In skeletal muscle, sarcoplasmic reticulum (SR) calcium release is controlled by the plasma membrane voltage through interactions between the voltage-sensing dihydropyridine receptor (DHPr) and the ryanodine receptor (RYr) calcium release channel. Maurocalcine (MCa), a scorpion toxin peptide presenting some homology with a segment of a cytoplasmic loop of the DHPr, has been previously shown to strongly affect the activity of the isolated RYr. We injected MCa into mouse skeletal muscle fibers and measured intracellular calcium under voltage-clamp conditions. Voltage-activated calcium transients exhibited similar properties in control and in MCa-injected fibers during the depolarizing pulses, and the voltage dependence of calcium release was similar under the two conditions. However, MCa was responsible for a pronounced sustained phase of Ca(2+) elevation that proceeded for seconds following membrane repolarization, with no concurrent alteration of the membrane current. The magnitude of the underlying uncontrolled extra phase of Ca(2+) release correlated well with the peak calcium release during the pulse. Results suggest that MCa binds to RYr that open on membrane depolarization and that this interaction specifically alters the process of repolarization-induced closure of the channels.

International audience; OSK1, a toxin from the venom of the Asian scorpion Orthochirus scrobiculosus, is a 38-residue peptide cross-linked by three disulfide bridges. A structural analog of OSK1, [Lys(16),Asp(20)]-OSK1, was found previously to be one of the most potent blockers of the voltage-gated K(+) channel Kv1.3 hitherto characterized. Here, we demonstrate that progressive trimming of the N-terminal domain of [Lys(16),Asp(20)]-OSK1 results in marked changes in its pharmacological profile, in terms of both K(+) channel affinity and selectivity. Whereas the affinity to Kv1.1 and Kv1.3 did not change significantly, the affinity to Kv1.2 and K(Ca)3.1 was drastically reduced with the truncations. It is surprising that a striking gain in potency was observed for Kv3.2. In contrast, a truncation of the C-terminal domain, expected to partially disrupt the toxin beta-sheet structure, resulted in a significant decrease or a complete loss of activity on all channel types tested. These data highlight the value of structure-function studies on the extended N-terminal domain of [Lys(16),Asp(20)]-OSK1 to identify new analogs with unique pharmacological properties.

Background/aims One of the main issues in the development of antiviral therapy is the emergence of drug-resistant viruses. In the case of hepatitis C virus (HCV), selection of drug-resistant mutants was evidenced by in vitro studies on protease inhibitors (PIs); for example, BILN-2061, VX-950 and SCH-6. Four mutations in the HCV protease (R155Q, A156T, D168A and D168V) have been identified in vitro in the HCV replicon system that confer resistance to BILN-2061 (a reference inhibitor). However, the molecular mechanism of drug resistance is still unknown. The aim of this study is to unravel, using an molecular modelling strategy, the structural basis of such molecular mechanism of HCV resistance to PIs. We focused on protease mutations conferring HCV resistance to BILN-2061 and described for the first time such mechanism at a molecular level. Methods The structures of drug-resistant NS3 proteases were obtained by mutation of selected residues (R155Q, A156T, D168A and D168V) and the ternary complexes formed between NS3–4A and BILN-2061 were optimized using GenMol software ( www.3dgenoscience.com ; Genoscience, Marseille, France). Results Two mechanisms were evidenced for viral resistance to BILN-2061. A ‘direct’ resistance mechanism is based on contacts between the mutated R155Q and A156T protease residues and its inhibitor. In the ‘indirect’ resistance mechanism, the mutated D168A/V residue is not in close contact with the drug itself but interacts with other residues connected to the drug. Conclusions These data provide new insights in the understanding of the mechanisms of HCV drug escape, and may allow predicting potential cross-resistance phenomenon with other PIs. This approach can be used as a basis for future rational PI drug design candidates.

Maurocalcine (MCa), a 33 amino acid toxin obtained from scorpion venom, has been shown to interact with the isolated skeletal-type ryanodine receptor (RyR1) and to strongly modify its calcium channel gating. In this study, we explored the effects of MCa on RyR1 in situ to establish whether the functional interaction of RyR1 with the voltage-sensing dihydropyridine receptor (DHPR) would modify the ability of MCa to interact with RyR1. In developing skeletal muscle cells the addition of MCa into the external medium induced a calcium transient resulting from RyR1 activation and strongly inhibited the effect of the RyR1 agonist chloro-m-cresol. In contrast, MCa failed to affect the depolarization-induced Ca2+ release. In intact adult fibres MCa did not induce any change in the cytosolic Ca2+ concentration. However, when the surface membrane was permeabilized and calcium release events were readily observable, MCa had a time-dependent dual effect: it first increased event frequency, from 0.060 ± 0.002 to 0.150 ± 0.007 sarcomere−1 s−1, and reduced the amplitude of individual events without modifying their spatial distribution. Later on it induced the appearance of long-lasting events resembling the embers observed in control conditions but having a substantially longer duration. We propose that the functional coupling of DHPRs and RyR1s within a Ca2+ release unit prevents MCa from either reaching its binding site or from being able to modify the gating not only of the RyR1s physically coupled to DHPRs but all RyR1s within the Ca2+ release unit.

ancestor (a species of fossil scorpion from Silurian to lower Devonian) [2]. From the fossils of the late Silurian period, scorpions were found to have developed and adapted their venomous systems [1]. Evolving over the past 400 million years, scorpion venoms contain a va riety of compounds, including the more or less potent and selective peptide toxins acting on the diverse and ubiquitous ion channel proteins. The latter are widely distributed in many systems, such as nervous, skeletal and cardiovascular in the scorpion’s prey (or predator). Basically, the severity of scorpion envenomation ranges from local pain and paresthesia to lethal cardiotoxicity and encephalopathy [3]. At present, it is a serious cause of mortality in several Latin countries, in South America, southern and western Asia, and Africa. The number of scorpion stings is estimated to be

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.

Maurotoxin (MTX) and HsTx1 are two scorpion toxins belonging to the alpha-KTx6 structural family. These 34-residue toxins, cross-linked by four disulfide bridges, share 59% sequence identity and fold along the classical alpha/beta scaffold. Despite these structural similarities, they fully differ in their pharmacological profiles. MTX is highly active on small (SK) and intermediate (IK) conductance Ca(2+)-activated (K(+)) channels and on voltage-gated Kv1.2 channel, whereas HsTx1 potently blocks voltage-gated Kv1.1 and Kv1.3 channels only. Here, we designed and chemically produced MTX-HsTx1, a chimera of both toxins that contains the N-terminal helical region of MTX (sequence 1-16) and the C-terminal beta-sheet region of HsTx1 (sequence 17-34). The three-dimensional structure of the peptide in solution was solved by (1)H NMR. MTX-HsTx1 displays the activity of MTX on SK channel, whereas it exhibits the pharmacological profile of HsTx1 on Kv1.1, Kv1.2, Kv1.3, and IK channels. These data demonstrate that the helical region of MTX exerts a key role in SK channel recognition, whereas the beta-sheet region of HsTx1 is crucial for activity on all other channel types tested.

Aah I is a 63-residue alpha-toxin isolated from the venom of the Buthidae scorpion Androctonus australis hector, which is considered to be the most dangerous species. We report here the first chemical synthesis of Aah I by the solid-phase method, using a Fmoc strategy. The synthetic toxin I (sAah I) was renatured in DMSO-Tris buffer, purified and subjected to thorough analysis and comparison with the natural toxin. The sAah I showed physico-chemical (CD spectrum, molecular mass, HPLC elution), biochemical (amino-acid composition, sequence), immunochemical and pharmacological properties similar to those of the natural toxin. The synthetic toxin was recognized by a conformation-dependent monoclonal anti-Aah I antibody, with an IC50 value close to that for the natural toxin. Following intracerebroventricular injection, the synthetic and the natural toxins were similarly lethal to mice. In voltage-clamp experiments, Na(v) 1.2 sodium channel inactivation was inhibited by the application of sAah I or of the natural toxin in a similar way. This work describes a simple protocol for the chemical synthesis of a scorpion alpha-toxin, making it possible to produce structural analogues in time.

Animal toxins acting on ion channels of excitable cells are principally highly potent short peptides that are present in limited amounts in the venoms of various unrelated species, such as scorpions, snakes, sea anemones, spiders, insects, marine cone snails and worms. These toxins have been used extensively as invaluable biochemical and pharmacological tools to characterize and discriminate between the various ion channel types that differ in ionic selectivity, structure and/or cell function. Alongside the huge molecular and functional diversity of ion channels, a no less impressive structural diversity of animal toxins has been indicated by the discovery of an increasing number of polypeptide folds that are able to target these ion channels. Indeed, it appears that these peptide toxins have evolved over time on the basis of clearly distinct architectural motifs, in order to adapt to different ion channel modulating strategies (pore blockers compared with gating modifiers). Herein, we provide an up-to-date overview of the various types of fold from animal toxins that act on ion channels selective for K+, Na+, Ca2+ or Cl− ions, with special emphasis on disulphide bridge frameworks and structural motifs associated with these peptide folds.

Maurotoxin (MTX) is a 34-residue toxin that has been isolated initially from the venom of the scorpion Scorpio maurus palmatus. It presents a large number of pharmacological targets, including small conductance Ca2+-activated and voltage-gated K+ channels. Contrary to other toxins of the alpha-KTx6 family (Pi1, Pi4, Pi7, and HsTx1), MTX exhibits a unique disulfide bridge organization of the type C1-C5, C2-C6, C3-C4, and C7-C8 (instead of the conventional C1-C5, C2-C6, C3-C7, and C4-C8, herein referred to as Pi1-like) that does not prevent its folding along the classic alpha/beta scaffold of scorpion toxins. Here, we developed an innovative strategy of chemical peptide synthesis to produce an MTX variant (MTXPi1) with a conventional pattern of disulfide bridging without any alteration of the toxin chemical structure. This strategy was used solely to address the impact of half-cystine pairings on MTX structural properties and pharmacology. The data indicate that MTXPi1 displays some marked changes in affinities toward the target K+ channels. Computed docking analyses using molecular models of both MTXPi1 and the various voltage-gated K+ channel subtypes (Shaker B, Kv1.2, and Kv1.3) were found to correlate with MTXPi1 pharmacology. A functional map detailing the interaction between MTXPi1 and Shaker B channel was generated in line with docking experiments.

Pi4 is a 38-residue toxin cross-linked by four disulfide bridges that has been isolated from the venom of the Chactidae scorpion Pandinus imperator. Together with maurotoxin, Pi1, Pi7 and HsTx1, Pi4 belongs to the alpha KTX6 subfamily of short four-disulfide-bridged scorpion toxins acting on K+ channels. Due to its very low abundance in venom, Pi4 was chemically synthesized in order to better characterize its pharmacology and structural properties. An enzyme-based cleavage of synthetic Pi4 (sPi4) indicated half-cystine pairings between Cys6-Cys27, Cys12-32, Cys16-34 and Cys22-37, which denotes a conventional pattern of scorpion toxin reticulation (Pi1/HsTx1 type). In vivo, sPi4 was lethal after intracerebroventricular injection to mice (LD50 of 0.2 microg per mouse). In vitro, addition of sPi4 onto Xenopus laevis oocytes heterologously expressing various voltage-gated K+ channel subtypes showed potent inhibition of currents from rat Kv1.2 (IC50 of 8 pm) and Shaker B (IC50 of 3 nm) channels, whereas no effect was observed on rat Kv1.1 and Kv1.3 channels. The sPi4 was also found to compete with 125I-labeled apamin for binding to small-conductance Ca(2+)-activated K+ (SK) channels from rat brain synaptosomes (IC50 value of 0.5 microm). sPi4 is a high affinity blocker of the Kv1.2 channel. The toxin was docked (BIGGER program) on the Kv channel using the solution structure of sPi4 and a molecular model of the Kv1.2 channel pore region. The model suggests a key role for residues Arg10, Arg19, Lys26 (dyad), Ile28, Lys30, Lys33 and Tyr35 (dyad) in the interaction and the associated blockage of the Kv1.2 channel.

Maurotoxin, a 34-amino acid toxin from Scorpio maurus scorpion venom, was examined for its ability to inhibit cloned human SK (SK1, SK2, and SK3), IK1, and Slo1 calcium-activated potassium (K(Ca)) channels. Maurotoxin was found to produce a potent inhibition of Ca(2+)-activated (86)Rb efflux (IC(50), 1.4 nM) and inwardly rectifying potassium currents (IC(50), 1 nM) in CHO cells stably expressing IK1. In contrast, maurotoxin produced no inhibition of SK1, SK2, and SK3 small-conductance or Slo1 large-conductance K(Ca) channels at up to 1 microM in physiologically relevant ionic strength buffers. Maurotoxin did inhibit (86)Rb efflux (IC(50), 45 nM) through, and (125)I-apamin binding (K(i), 10 nM) to SK channels in low ionic strength buffers (i.e., 18 mM sodium, 250 mM sucrose), which is consistent with previous reports of inhibition of apamin binding to brain synaptosomes. Under similar low ionic strength conditions, the potency for maurotoxin inhibition of IK1 increased by approximately 100-fold (IC(50), 14 pM). In agreement with its ability to inhibit recombinant IK1 potassium channels, maurotoxin was found to potently inhibit the Gardos channel in human red blood cells and to inhibit the K(Ca) in activated human T lymphocytes without affecting the voltage-gated potassium current encoded by Kv1.3. Maurotoxin also did not inhibit Kv1.1 potassium channels but potently blocked Kv1.2 (IC(50), 0.1 nM). Mutation analysis indicates that similar amino acid residues contribute to the blocking activity of both IK1 and Kv1.2. The results from this study show that maurotoxin is a potent inhibitor of the IK1 subclass of K(Ca) potassium channels and may serve as a useful tool for further defining the physiological role of this channel subtype.

We have modelled the conformation of the III-IV loop of the Ca(v)2.1 subunit of P/Q calcium channels, a loop that is implicated in fast voltage-dependent inactivation. Change in channel inactivation requires its direct interaction with the I-II loop. This interaction occurs with an affinity in the order of 70 nm. Intracellular injection of a 40-mer III-IV loop-derived peptide produces an increase in the rate of fast inactivation. This alteration in channel kinetic is also accompanied by a hyperpolarizing shift in the steady-state voltage-dependence of inactivation. None of these effects are observed in the presence of a beta subunit, suggesting the existence of a competitive mechanism of action between the beta subunit and the III-IV loop. Amino acid sequence comparison using BLAST reveals that the III-IV loop shares 53% identity with the gamma subunit of G proteins. Because of the pivotal contribution of the III-IV loop to inactivation, an atomic model of the III-IV loop was generated by both homology modelling and molecular mechanics calculations. Using the X-ray structures of the betagamma dimer of the heterotrimeric G-proteins as templates, the III-IV loop is predicted to contain a well-structured alpha-helix at the amino-terminus with both the N- and C-termini having the same orientation in the plane of the inner lipid bilayer. We provide a hypothetical working model in which we propose that the III-IV loop interacts with the I-II loop via its Gbetagamma binding domain.

International audience; We have investigated the molecular mechanisms whereby the I-II loop controls voltage-dependent inactivation in P/Q calcium channels. We demonstrate that the I-II loop is localized in a central position to control calcium channel activity through the interaction with several cytoplasmic sequences; including the III-IV loop. Several experiments reveal the crucial role of the interaction between the I-II loop and the III-IV loop in channel inactivation. First, point mutations of two amino acid residues of the I-II loop of Ca(v)2.1 (Arg-387 or Glu-388) facilitate voltage-dependent inactivation. Second, overexpression of the III-IV loop, or injection of a peptide derived from this loop, produces a similar inactivation behavior than the mutated channels. Third, the III-IV peptide has no effect on channels mutated in the I-II loop. Thus, both point mutations and overexpression of the III-IV loop appear to act similarly on inactivation, by competing off the native interaction between the I-II and the III-IV loops of Ca(v)2.1. As they are known to affect inactivation, we also analyzed the effects of beta subunits on these interactions. In experiments in which the beta(4) subunit is co-expressed, the III-IV peptide is no longer able to regulate channel inactivation. We conclude that (i) the contribution of the I-II loop to inactivation is partly mediated by an interaction with the III-IV loop and (ii) the beta subunits partially control inactivation by modifying this interaction. These data provide novel insights into the mechanisms whereby the beta subunit, the I-II loop, and the III-IV loop altogether can contribute to regulate inactivation in high voltage-activated calcium channels.

Maurotoxin (MTX) is a 34-mer scorpion toxin cross-linked by four disulphide bridges that acts on various K+ channels, including the voltage-gated Shaker B subtype. In the present study, we have investigated over 80h: (1) the time-course of folding of synthetic MTX (sMTX) by CD analysis; (2) the kinetics of disulphide bridge formation by MS; and (3) the potency of MTX in blocking Shaker B currents during the combined process of its in vitro folding and oxidation. From the CD data, we show that stable secondary structures of sMTX evolve sequentially over time, with the appearance of the α-helix within 5h, followed by the formation of the β-sheet within 22h. Using MS analysis, the sMTX intermediates were also found to appear sequentially from the least (one-disulphide-bridged sMTX) to the most oxidized species (native-like, four-disulphide-bridged sMTX). The time course of formation of secondary structures coincides mainly with the occurrence of one-disulphide-bridged sMTX for the α-helix and two- or three-disulphide-bridged sMTX for the β-sheet. On-line electrophysiological recordings, which measure sMTX blocking efficacy on K+ currents during its folding and oxidation, were performed on Shaker B channels expressed in Xenopus oocytes. Unexpectedly, the results demonstrate that sMTX is highly potent at the initial stage of oxidation, whereas its blocking activity can be transiently and dramatically reduced at later stages during the course of folding/oxidation before it reaches full bioactivity. These data suggest that formation of disulphide bridges can both physically stabilize and alter the bioactive three-dimensional structure of sMTX.

This Special Issue of Molecules deals with the structure–activity relationship of natural compounds which possess some pharmacological/chemical properties of potential interest (from basic research to the clinical applications) in a wide range of areas, such as bacteriology, parasitology, cancerology, inflammation, etc.[...]

Aims Scorpion venoms contain complex mixtures of molecules, including peptides. These peptides specifically bind to various targets, in particular ion channels. Toxins modulating Na + , K + , Ca 2 + and Cl − currents were described from venoms. The Androctonus and Buthus geni of scorpions are widely distributed in Morocco. Their stings can cause pain, inflammation, necrosis, muscle paralysis and death. The myotoxicity is predominantly associated with neurotoxic effects and is a cause of mortality and morbidity. In this study, pharmacological effects of venoms were investigated in vitro on neuromuscular transmission. Main methods Effects of Androctonus mauretanicus (Am) and Buthus occitanus (Bo) venoms were investigated using the chick biventer cervicis nerve-muscle preparations. The protective activity of antivenom was also investigated. The antivenom was made from serum of horse that was hyperimmunized with Bo and Androctonus australis hector (Aah) venoms and one venom from Middle East species (Lq). The protective activity of the antivenom was assessed on the neuromuscular system by using stimulated chick nerve-muscle. The results were compared with lethal activity neutralization in mice. Key findings Am and Bo venoms contain myotoxins and postsynaptic neurotoxins. In agreement with lethal potencies of these venoms in mice, Am venom displays greater neurotoxicity and myotoxicity. The antivenom prevented lethality caused by Am, Bo and Aah venoms. The antivenom did not prevent toxic effects caused by Am venom whereas it neutralized Bo venom. Significance Am and Bo venoms contain distinct toxins that are responsible for myotoxicity and neurotoxicity. It would be appropriate to add Am venom to produce more efficient antivenom.

International audience; Small-conductance calcium-activated potassium channels (SK1-SK3 channels) are responsible for long-lasting hyperpolarization following action potential and contribute to the neuronal firing and integration signal. Two peptide toxins: apamin and Leiurotoxin 1, block this SK channels with high affinities. We generated a modified Leiurotoxin 1 (Lei-Dab7) that inhibits SK2 channels with a high selectivity. Competitive binding of radio-iodinated apamin to different rat brain structures, in the presence of native apamin and Lei-Dab7, has shown that dissociation constants differ by a factor of 1000 and thus demonstrated that ligand affinity is as important as ligand selectivity for a specific receptor. However, the lack of ligands discriminating between SK channel subunits is impeding the understanding of the role of each heteromeric SK channel type in different tissues. Our study aims to better understand the molecular combinations of SK channels and their association with specific functional implications. On this purpose, a clustering technique allows us to identify five groups of brain structures reflecting singular profiles of affinity and selectivity of Lei-Dab7 in comparison with apamin. The analysis of correspondences between Lei-Dab7 binding and distribution of SK subunits in these groups of brain structures suggests that functional heteromeric SK channels are involved in specific information processes.

International audience; Scorpion stings and snake bites are major health hazards that lead to suffering of victims and high mortality. Thousands of injuries associated with such stings and bites of venomous animals occur every year worldwide. In North Africa, more than 100,000 scorpion stings and snake bites are reported annually. An appropriate determination of the 50% lethal doses (LD50) of scorpion and snake venoms appears to be an important step to assess (and compare) venom toxic activity. Such LD50 values are also commonly used to evaluate the neutralizing capacity of specific anti-venom batches. In the present work, we determined experimentally the LD50 values of reference scorpion and snake venoms in Swiss mice, and evaluated the influence of two main venom injection routes (i.e., intraperitoneal (IP) versus intravenous (IV)). The analysis of experimental LD50 values obtained with three collected scorpion venoms indicates that Androctonus mauretanicus (Am) is intrinsically more toxic than Androctonus australis hector (Aah) species, whereas the latter is more toxic than Buthus occitanus (Bo). Similar analysis of three representative snake venoms of the Viperidae family shows that Cerastes cerastes (Cc) is more toxic than either Bitis arietans (Ba) or Macrovipera lebetina (Ml) species. Interestingly, the venom of Elapidae cobra snake Naja haje (Nh) is far more toxic than viper venoms Cc, Ml and Ba, in agreement with the known severity of cobra-related envenomation. Also, our data showed that viper venoms are about three-times less toxic when injected IP as compared to IV, distinct from cobra venom Nh which exhibited a similar toxicity when injected IP or IV. Overall, this study clearly highlights the usefulness of procedure standardization, especially regarding the administration route, for evaluating the relative toxicity of individual animal venoms. It also evidenced a marked difference in lethal activity between venoms of cobra and vipers, which, apart from the nature of toxins, might be attributed to the rich composition of high molecular weight enzymes in the case of viper venoms.

Human potassium channels are widely inhibited by peptide toxins from venomous animals. However, no human endogenous peptide inhibitor has been discovered so far. In this study, we demonstrate for the first time using electrophysiological techniques, that endogenous human β-defensin 2 (hBD2) is able to selectively and dose-dependently inhibit the human voltage-gated Kv1.3 channel at picomolar peptide concentration. The co-immunoprecipitation assays further supported the selective binding of hBD2 to Kv1.3 channel. Using mutagenesis experiments, we found that the outer pore domain of Kv1.3 channel was the binding site of hBD2, which is similar to the interacting site of Kv1.3 channel recognized by animal toxin inhibitors. The hBD2 was able to suppress IL-2 production through inhibition of Kv1.3 channel currents in human Jurkat cells, which was further confirmed by the lack of hBD2 activity on IL-2 production after Kv1.3 knockdown in these cells. More interestingly, hBD2 was also found to efficiently inhibit Kv1.3 channel currents and suppress IL-2 production in both human primary CD3(+) T cells and peripheral mononuclear cells from either healthy donors or psoriasis patients. Our findings not only evidenced hBD2 as the first characterized endogenous peptide inhibitor of human potassium channels, but also paved a promising avenue to investigate newly discovered function of hBD2 as Kv1.3 channel inhibitor in the immune system and other fields.

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.

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 (Cys13) and C4 (Cys19), 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, (A12,A20)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. fl 2001 Federation of European Biochemical Societies. Published by Elsevier Science B.V. All rights reserved.

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 α/β scaffold of scorpion toxins.

The auxiliary beta subunit importantly regulates voltage-dependent Ca(2+) channel activity through an interaction with the AID domain, a binding site located in the cytoplasmic I-II linker of the ion-conducting alpha(1) subunit. In the present study, we used two synthetic peptides corresponding to partial sequences of the I-II linker of alpha(1A) (AID(A)-peptides) as tools to disrupt the alpha(1)-beta interaction. In vitro binding experiments confirmed that these peptides exhibit a reasonable affinity to the neuronal beta(3) subunit to serve this purpose, although they failed to prevent immunoprecipitation of native N- and P/Q-type channels by anti-beta(3) antibodies. Together, our results (i) provide evidence for the reversibility of channel subunit association suggesting that the disruption of the alpha(1)-beta interaction may be a possible mechanism for Ca(2+) channel regulation in vivo, and (ii) support a model whereby the alpha(1)-beta association is based on multiple interaction sites.

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.

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.

Tityus kappa (Ts kappa), a novel toxin from the venom of the scorpion Tityus serrulatus, is a 35-residue polypeptide cross-linked by three disulphide bridges and acts on small-conductance calcium-activated potassium channels (SK channels). Ts K was chemically synthesized using the solid-phase method and characterized. The synthetic product, sTs kappa, was indistinguishable from the natural toxin when tested in vitro in competition assay with radiolabelled apamin for binding to rat brain synaptosomes (IC50 = 3 nM). The sTs kappa was further tested in vivo for lethal activity to mice following intracerebroventricular inoculation (LD50 = 70 ng per mouse). The half-cystine pairings were formerly established by enzyme-based cleavage of sTs kappa; they were between Cys7-Cys28, Cys13-CyS33 and Cys17-Cys35, which is a disulphide bridge pattern similar to that of other short scorpion toxins. According to previous studies on SK channel-acting toxins, the putative influence of certain basic residues of Ts kappa (i.e. Arg6, Arg9, Lys18, Lys19) in its pharmacological activity was investigated using synthetic point-mutated analogues of the toxin with an Ala substitution at these positions. Data from binding assay, together with conformational analysis of the synthetic analogues by 1H-NMR, suggest that Arg6, and to a lesser extent Arg9, are important residues for an high-affinity interaction of this toxin with SK channels; interestingly these residues are located outside the alpha-helical structure, whereas the pharmacologically important basic residues from other SK channel-specific toxins had been located inside the alpha-helix.

SPC3 is a peptide construct (eight branches of the GPGRAF motif) derived from the consensus sequence present at the apex of the third variable domain of the human immunodeficiency virus (HIV) envelope (Env). It presents a potent anti-HIV activity and is currently tested in phase II clinical trials (FDA protocol 257A). Its mode of action remains unclear. It was thought that SPC3 exerts its effect both during HIV interaction with CD4+ cells but also through interference either with a post-binding event or with Env processing. Accordingly, SPC3 was supposed to be able to bind and to enter CD4+ cells. In this work, we addressed these points. SPC3 was found to interact with CD4+ cell membrane with a K0.5 value in the range of 500 nM. The binding of SPC3 to CD4+ cells involves its interaction with a cell membrane associated protein which is pronase sensitive and different from CD4. This interaction was similar from 2 to 37 degrees C. The maximum binding occurred at acidic pH whereas the interaction was inhibited in alkaline conditions. We observed also that SPC3 was internalized rapidly into the cells - the maximal intracell amount was reached within 30 min - where it remained stable for at least 24 h. Altogether, these data suggest that SPC3 can exert its antiviral activity via interference with events occurring at the cell surface but also into the target cell.

Scorpion venom contains various toxins which block ion-channels, responsible for either sodium, potassium, calcium or chloride membrane permeation. This review focuses on the three-dimensional structure of scorpion toxins specific for potassium channels, and on their structure–activity relationships. The overall fold of all these toxins is similar, despite their various specificities towards different types of potassium channels. Fine studies of the influence of punctual mutations of both toxins and channels have converged on a precise description of the scorpion toxins functional maps. From this knowledge, it now becomes possible to predict the specificity of a newly described scorpion toxin. The way is thus now open that leads to the design of new potent synthetic potassium channel blockers which in turn could be used as therapeutic drugs.

TsKapa (TsK), purified from the Buthidae Tityus serrulatus is a very high potent ligand for small-conductance apamin-sensitive calcium-activated potassium channels (SK). It is able to efficiently compete with apamin for binding on this channel (K0.5 = 0.3 nM) [Legros, C. et al., FEBS Lett. 390:81-84, 1996]. The solution structure of TsK has been determined by 2D-NMR techniques, which led to the full description of its 3D conformation: a short alpha helix from residues 14 to 20 and a three-stranded antiparallel beta sheet (residues 2-3, 27-29, and 32-34). The interaction of TsK with the SK potassium channel has been modeled according to the charge anisotropy of the ligand. The resulting dipole moment orientates TsK so that it presents toward the receptor, a surface, mainly basic, encompassing residues K18 and K19 on one side and R9 and Y8 on the other. Despite its three-dimensional structure that is related with scorpion toxins active on voltage-gated potassium channels such as charybdotoxin, the pharmacological activity and specificity of TsK is related with shorter scorpion toxins (i.e., possessing an only two-stranded beta sheet) such as scyllatoxin (also named leiurotoxin I) or P05.

International audience; A synthetic peptide mimicking the North African scorpion Androctonus australis hector toxin II was designed and produced by chemical solid-phase synthesis. It contains the entire sequence of toxin II (64 amino acid residues), with each half-cystine being replaced by the isosteric residue a-aminobutyric acid, and was thus devoid of disulfide bridges. This construct was totally nontoxic in mice even if large amounts, equivalent to 1000 times the LD50 of the original toxin, were injected by the intracerebroventricular route. The synthetic peptide, either as a monomer or polymerized by means of glutaraldehyde, induced the production of antitoxin neutralizing antibodies in immunized mice and rabbits. After three injections with either the monomeric or polymerized synthetic peptide, the immunized mice were protected against several lethal doses of the corresponding native toxin or scorpion venom. Six months after immunization, the mice were completely protected against challenge with eight LD50 of the original toxin. The protection was better when the polymerized synthetic peptide was used. One month after the start of the immunization program, it showed a good correlation between antibody titer and protection. However, antibody titer decreased with time but protection remained high. This suggests that additional factors other than circulating antibodies play a role in protective activity.

International audience; Potassium channel functions are often deciphered by using selective and potent scorpion toxins. Among these toxins, only a limited subset is capable of selectively blocking small conductance Ca(2+)-activated K(+) (SK) channels. The structural bases of this selective SK channel recognition remain unclear. In this work, we demonstrate the key role of the electric charges of two conserved arginine residues (Arg-485 and Arg-489) from the SK3 channel outer vestibule in the selective recognition by the SK3-blocking BmP05 toxin. Indeed, individually substituting these residues with histidyl or lysyl (maintaining the positive electric charge partially or fully), although decreasing BmP05 affinity, still preserved the toxin sensitivity profile of the SK3 channel (as evidenced by the lack of recognition by many other types of potassium channel-sensitive charybdotoxin). In contrast, when Arg-485 or Arg-489 of the SK3 channel was mutated to an acidic (Glu) or alcoholic (Ser) amino acid residue, the channel lost its sensitivity to BmP05 and became susceptible to the "new" blocking activity by charybdotoxin. In addition to these SK3 channel basic residues important for sensitivity, two acidic residues, Asp-492 and Asp-518, also located in the SK3 channel outer vestibule, were identified as being critical for toxin affinity. Furthermore, molecular modeling data indicate the existence of a compact SK3 channel turret conformation (like a peptide screener), where the basic rings of Arg-485 and Arg-489 are stabilized by strong ionic interactions with Asp-492 and Asp-518. In conclusion, the unique properties of Arg-485 and Arg-489 (spatial orientations and molecular interactions) in the SK3 channel account for its toxin sensitivity profile.