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A diversity of marine invertebrates, such as cnidarians are rich sources of large bio-active molecules. This chemo-diversity of bio-active compounds has a promising potential in several biotechnological and therapeutic applications. On the basis of a comparative bibliographic approach, we intend in this review to present and discuss an overview of: i) the diversity of marine invertebrates as a candidate for bio-active molecules production; ii) the diversity of bio-active compounds and venom derived from these organisms; iii) the relationship between, the diversity of these marine organisms and the structure of the toxins they secrete. In this bibliographic study, a focus is going to be made on protein neurotoxins targeting ion channels. We also discuss the potential link between the bioecological characteristics of cnidarians and the diversity of toxins.

Cakile maritima is a halophytic plant model that is well known by its ability to tolerate high salt concentrations. Salinity was reported to improve the tolerance of halophytes to several abiotic stresses; however, the involvement of salt in the tolerance to biotic stress is still scant. In the present work, the effect of salt on C. maritima responses towards the pathogenic Alternaria alternata was investigated. For that, C. maritima seeds were germinated for four weeks. Plants were then divided into four groups: i) Plants irrigated with salt (200mM NaCl); ii) Plants infested by fungus; iii) Plants irrigated with salt and infested by fungus and finally control plants (0mM NaCl, without inoculation). Our results showed that upon salt stress or fungal attack, plants reduced biomass production, hydration status and photosynthetic performance which were associated with a decrease in the gas exchange and chlorophyll fluorescence parameters, with a more pronounced effect upon fungal attack. However, under combined stress, a significant increase of these parameters was noticed, with a level close to that of control. Concerning nutrient contents, K, Zn, Fe, Cu and Mg decreased in the C. maritima leaves exposed to both stresses applied individually. In contrast, all these nutrients were increased in plants grown under combined stress. Taken together, we can conclude that plants grown under combined stresses had better growth rate and physiological performance compared to all other treated plants, and that salt may be the key in improving the C. maritima ability to tolerate fungal attack.

The aim of this study is to optimize the pretreatment of Alfa fibers Stipa tenacissima for the production of bioethanol second generation. The Alfa is a complex lignocellulosic substrate. It is mainly composed by cellulose (49.09% ± 0.015), hemicellulose (18.20% ± 0.3) and lignins (16.25% ± 0.475). In the current study, process of production of the biofuel was carried out through three successive steps: a pretreatment performed with dilute acid (3%) at 120 °C during 30 min, an enzymatic saccharification by a commercial enzyme (AP2) and fermentation steps. Dilute acid pretreatment of the biomass resulted in an efficient degradation of cellulose and hemicellulose by increasing the total sugars (39.951 g/L) and reducing sugars (22.36 g/L) yields. These yields were increased after enzymatic saccharification to reach 22.7 g/L of reducing sugars. The fermentation of the aforementioned hydrolysate was efficient with ethanol production being 36.36% of theoretical maximum.

Contamination by micro and nano plastics is actually considered as a global environmental preoccupation. The quantification of microplastics in natural habitats and the characterization of their potential effects in marine wild organisms is currently of high importance. The main objective of this work was to investigate the fate and the effects of a microplastic mixture (ratio of 1: polyethylene (PE), 1: polypropylene (PP)) in the wedge clam Donax trunculus. The assimilation kinetics of microplastics particles was assessed in different organs (gills, digestive gland and flesh) using three different protocols (direct observation, H2O2, and HNO3/HCl digestion) in order to compare method's efficacity. The main biological endpoints studied were Aceylcholinesterase (AChE) inhibition as a neurotoxicity biomarker, the Catalase (CAT) enzymatic activity and the Gluthation-S-Transfereases (GSTs) activities as oxidative stress and phase II detoxification phase markers, respectively. Results showed that the H2O2 digestion method was more efficient to assess particles assimilation than the direct observation and acid digestion. In all cases no particles were detected in clam's flesh and gills were the first target organ for micro-plastics accumulation. The exposure of Donax truculus to PP/PE mixture (0.06 g/Kg of sand) induce a significant inhibition of AChE activity in both gills and digestive gland and oxidative stress in all organs studied. This study brings new results on the potential accumulation of PP and PE associated to neurotoxicity and oxidative stress of the wedge clam Donax trunculus.

Multiresistant Gram-negative bacteria are the prime mover of nosocomial infections. Some are naturally resistant to antibiotics, their genetic makes them insensitive to certain families of antibiotics and they transmit these resistors to their offspring. Moreover, when bacteria are subjected to antibiotics, they eventually develop resistance against drugs to which they were previously sensitive. In recent years, many bacteriocins active against gram-negative bacteria have been identified proving their efficacy in treating infections. While further investigation remains necessary before the possibilities for bacteriocins in clinical practice can be described more fully, this review provides an overview of bacteriocins acting on the most common infectious gram negative bacteria (Klebsiella, Acinetobacter, Pseudomonas aeruginosa and E. coli).

The striatum, a major input structure of basal ganglia, integrates glutamatergic cortical and thalamic inputs to control psychomotor behaviors. Nigrostriatal dopamine (DA) neurodegeneration in Parkinson's disease causes a loss of spinal and glutamatergic synapses in the striatal medium spiny neurons (MSNs). Adaptive responses, a form of homeostatic plasticity, to these changes are caused by a decrease in a potassium Kv4 channel-dependent inactivating A-type potassium (KIA) current that increases the intrinsic excitability of MSNs. Nevertheless, the functional outcome of these compensatory mechanisms does not allow adequate behavioral recovery in vivo. We thus addressed the question of whether further blockade of Kv4 activity could enhance the striatal responsiveness of MSNs to DA depletion and restore normal function in vivo. To test this hypothesis, we examined the effects of a selective blocker of Kv4 channels, AmmTX3, on the motor, cognitive, and emotional symptoms produced by 6-hydroxydopamine lesions of the nigrostriatal DA pathway in rats. Striatal infusion of AmmTX3 (0.2-0.4 μg) reduced motor deficits, decreased anxiety, and restored short-term social and spatial memories. These results underlie the importance of Kv4 channels as players in the homeostatic responses, and, more importantly, provide a potential target for adjunctive therapies for Parkinson's disease.

The present study investigates the biochemical and morphological responses induced in the liver and gills of Nile tilapia Oreochromis niloticus by exposure to various Red 195 dye concentrations (0.05, 0.1 and 0.2 mg L−1) for various durations (7, 14 and 21 days). The histology and antioxidant activities of catalase (CAT), glutathione reductase (GR) and glutathione S-transferase (GST) were monitored and evaluated during exposure and after recovery in clean water. The results revealed that CAT activity decreased after 7 and 21 days of exposure to 0.2 mg L−1 concentrations and increased only after 7 days of exposure to high concentrations compared to the liver and gill control samples, respectively. Both organs were, however, noted to undergo a decrease in GST activity after 7 days of exposure to low Red 195 dye concentrations. Compared to the control, the gills of the tilapia exposed to 0.05 and 0.1 mg L−1 concentrations for longer periods underwent an increase in GST activity. Similarly, GR activity was higher in the liver of tilapia exposed to this dye at the sampling days, except for the highest concentration (0.2 mg L−1) after 21 days of exposure. The GR activity in the gills decreased significantly after 7 and 21 days of exposure to 0.05 and 0.1 mg L−1 concentrations, respectively. The results of the recovery group revealed that the liver and gills displayed insignificant differences in antioxidant enzyme activities. The liver and gill tissues of the fish exposed to Red 195 showed several histopathological changes. Liver damages included an increase in cytoplasmic vacuolization, disruption of endothelial lining, metabolic zonation, necrotic cells, rupture of hepatocyte membrane, and decline of higher eosinophilia. The gills also exhibited some necrotic cells, edema, lifting of filaments and lamellar epithelium, and vascular disorders, such as extreme vasodilatation and proliferation of filament epithelium. A correlation between the biochemical and histological changes of the liver and gill tissues was established, attributing the tissue and cell damages to the accumulation of hydrogen peroxide or production of other radicals via a Fenton reaction.

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.

Small-conductance calcium-activated potassium channels (K Ca 2) generating the medium afterhyperpolarization seen after an action potential modulate the neuronal integration signal. The effects of two K Ca 2 channel blockers, apamin, specific to K Ca 2.2 and K Ca 2.3 channels, and lei-Dab7, which binds to K Ca 2.2 channels only, were compared to evaluate the involvement of K Ca 2 channel subunits in behavior, spatial learning and memory in rats. Intracerebroventricular (9–5 ng) injections of lei-dab7 decreased locomotor activity, food intake and body weight in rats deprived of food. A dose of 3 ng lei-Dab7 had no effect on these types of behavior. We therefore used this dose for attention and memory tasks. No modification to attention or memory was observed in a spatial radial-arm maze task with rats given 3 ng lei-Dab7, whereas apamin (0.3 ng) improved reference memory and accelerated changes of strategy from egocentric to allocentric. These findings suggest that K Ca 2.3 blockade improves memory in rats. Lei-Dab7 entirely outcompeted the binding of iodinated apamin to 64 brain structures (mean IC 50 : 34.5 nM), although IC 50 values were highly variable. By contrast, overall IC 50 values for apamin were close to mean values (11.3 pM). The very low affinity of the hippocampus and neocortex for lei-Dab7 may account for the absence of a behavioral effect of this compound. The variability of IC 50 values suggests that K Ca 2 channel composition varies considerably as a function of the brain structure considered.

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.

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.

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.

Adenosine is a nucleoside displaying various biological effects via stimulation of four G-protein–coupled receptors, A1, A2A, A2B, and A3. Adenosine also modulates voltage-gated (Kv) and small conductance calcium-activated (SKCa) potassium channels. The effect of these potassium channels on the expression of adenosine receptors is poorly understood. We evaluated the action of BgK (a natural Kv channel blocker) and Lei-Dab7 (a synthetic SKCa channel blocker) on the expression of adenosine A2A receptors (A2AR) in Jurkat human T cells. We found that Lei-Dab7, but not BgK, increased the maximal binding value of the tritiated ligand ZM241385 to A2AR in a dose-dependent manner (+45% at 5 nM; +70% at 50 nM as compared to control). These results were further confirmed by Western blotting using a specific monoclonal antibody to human A2AR. The ligand affinity-related dissociation constant and A2AR mRNA amount were not significantly modified by either drug. We suggest that modulation of SKCa channels can influence membrane expression of A2AR and thus has a therapeutic potential.

Maurotoxin (MTX) is a 34-mer scorpion toxin cross-linked by four disulphide bridges that acts on various K+ channel types. It folds according to an alpha/beta scaffold, i.e., a helix connected to a two stranded beta-sheet by two disulphide bridges. In a former study, various parameters that affect the oxidation and folding of the reduced form of synthetic MTX were investigated in vitro. It was found that MTX achieves its final 3-D structure by evolving over time through a series of oxidation intermediates, from the least to the most oxidized species. MTX oxidative intermediates can be studied by iodoacetamide alkylation of free cysteine residues followed by mass spectrometry analysis. Here, we have analysed the effect of Cu2+ (0.1 to 50 mM) on the kinetics of MTX oxidative folding and found that it dramatically speeds up the formation of the four-disulphide bridged, native-like, MTX (maximal production within 30 minutes instead of > 60 hours). This catalysing effect of Cu2+ was found to be concentration-dependent, reaching a plateau at 10 mM copper ions. Cu2+ was also found to prevent the slow transition of a three disulphide-bridged MTX intermediate towards the final four disulphide-bridged product (12% of total MTX). The data are discussed in light of the potential effects of Cu2+ on MTX secondary structure formation, disulphide bridging and peptidyl prolyl cis-trans isomerization.

We asked whether blockade of voltage-gated K+ channel Kv1.1, whose altered axonal localization during myelin insult and remyelination may disturb nerve conduction, treats experimental autoimmune encephalomyelitis (EAE).Electrophysiological, cell proliferation, cytokine secretion, immunohistochemical, clinical, brain magnetic resonance imaging, and spectroscopy studies assessed the effects of a selective blocker of Kv1.1, BgK-F6A, on neurons and immune cells in vitro and on EAE-induced neurological deficits and brain lesions in Lewis rats.BgK-F6A increased the frequency of miniature excitatory postsynaptic currents in neurons and did not affect T-cell activation. EAE was characterized by ventriculomegaly, decreased apparent diffusion coefficient, and decreased (phosphocreatine + beta-adenosine triphosphate)/inorganic phosphate ratio. Reduced apparent diffusion coefficient and impaired energy metabolism indicate astrocytic edema. Intracerebroventricularly BgK-F6A-treated rats showed attenuated clinical EAE with unexpectedly reduced ventriculomegaly and preserved apparent diffusion coefficient values and (phosphocreatine + beta-adenosine triphosphate)/inorganic phosphate ratio. Thus, under BgK-F6A treatment, brain damage was dramatically reduced and energy metabolism maintained.Kv1.1 blockade may target neurons and astrocytes, and modulate neuronal activity and neural cell volume, which may partly account for the attenuation of the neurological deficits. We propose that Kv1.1 blockade has a broad therapeutic potential in neuroinflammatory diseases (multiple sclerosis, stroke, and trauma).

International audience; SK channels are responsible for long-lasting hyperpolarization following action potential and contribute to the neuronal integration signal. This study evaluates the involvement of SK channels on learning and memory in rats, by comparing the effects of two SK channel blockers, i.e., apamin which recognizes SK2 and SK3 channels, and lei-Dab7 which binds SK2 channels only. lei-Dab7 totally competes and contests apamin binding on whole brain sections (IC(50): 11.4 nM). Using an olfactory associative task, intracerebroventricular blocker injections were tested on reference memory. Once the task was mastered with one odor pair, it was then tested with a new odor pair. Apamin (0.3 ng), injected before or after the acquisition session, improved new odor pair learning in a retention session 24 hours later, whereas lei-Dab7 (3 ng) did not significantly affect the mnesic processes. These results indicated that the blockage of SK channels by apamin facilitates consolidation on new odor associations; lei-Dab7, containing only SK2 subunits, remains without effect suggesting an involvement of SK3 channels in the modulation of the mnesic processes.

Maurocalcine (MCa) is a 33 amino acid residue peptide toxin isolated from the scorpion Scorpio maurus palmatus. MCa and mutated analogues were chemically synthesized, and their interaction with the skeletal muscle ryanodine receptor (RyR1) was studied on purified RyR1, sarcoplasmic reticulum (SR) vesicles, and cultured myotubes. MCa strongly potentiates [3H]ryanodine binding on SR vesicles (7-fold at pCa 5) with an apparent EC50 of 12 nm. MCa decreases the sensitivity of [3H]ryanodine binding to inhibitory high Ca2+ concentrations and increases it to the stimulatory low Ca2+ concentrations. In the presence of MCa, purified RyR1 channels show long-lasting openings characterized by a conductance equivalent to 60% of the full conductance. This effect correlates with a global increase in Ca2+ efflux as demonstrated by MCa effects on Ca2+ release from SR vesicles. In addition, we show for the first time that external application of MCa to cultured myotubes produces a cytosolic Ca2+ increase due to Ca2+ release from 4-chloro-m-cresol-sensitive intracellular stores. Using various MCa mutants, we identified a critical role of Arg24 for MCa binding onto RyR1. All of the other MCa mutants are still able to modify [3H]ryanodine binding although with a decreased EC50 and a lower stimulation efficacy. All of the active mutants produce both the appearance of a subconductance state and Ca2+ release from SR vesicles. Overall, these data identify some amino acid residues of MCa that support the effect of this toxin on ryanodine binding, RyR1 biophysical properties, and Ca2+ release from SR.

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 (IC50 = 7 nM) and human (IC50 = 5 nM) platelets. They prevent collagen-induced thrombocytopenia in rats. N- and C-terminal-truncated synthetic L1γ (sL1γ; 11-mer) is less active in inhibiting platelet aggregation than the native peptide. Results from Ala scan studies of the sL1γ 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 L1γ, 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 (MTX) is a 34-mer scorpion toxin cross-linked by four disulfide bridges that acts on both Ca(2+)-activated (SK) and voltage-gated (Kv) K(+) channels. A 38-mer chimera of MTX, Tsk-MTX, has been synthesized by the solid-phase method. It encompasses residues from 1 to 6 of Tsk at N-terminal, and residues from 3 to 34 of MTX at C-terminal. As established by enzyme cleavage, Tsk-MTX displays half-cystine pairings of the type C1-C5, C2-C6, C3-C7 and C4-C8 which, contrary to MTX, correspond to a disulfide bridge pattern common to known scorpion toxins. The 3-D structure of Tsk-MTX, solved by (1)H NMR, demonstrates that it adopts the alpha/beta scaffold of scorpion toxins. In vivo, Tsk-MTX is lethal by intracerebroventricular injection in mice (LD(50) value of 0.2 microg/mouse). In vitro, Tsk-MTX is as potent as MTX, or Tsk, to interact with apamin-sensitive SK channels of rat brain synaptosomes (IC(50) value of 2.5 nM). It also blocks voltage-gated K(+) channels expressed in Xenopus oocytes, but is inactive on rat Kv1.3 contrary to MTX.

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 α/β 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: 50mM Tris/HCl/1.4mM EDTA, pH7.5, supplemented by 0.5mM PPIase and 50units/ml PDI for 0.1mM reduced compound. These data represent the first report of potent stereoselective effects of cellular enzymes on the oxidation/folding of a scorpion toxin.

Apamin-sensitive small conductance calcium-activated potassium channels (SKCa1-3) mediate the slow afterhyperpolarization in neurons, but the molecular identity of the channel has not been defined because of the lack of specific inhibitors. Here we describe the structure-based design of a selective inhibitor of SKCa2. Leiurotoxin I (Lei) and PO5, peptide toxins that share the RXCQ motif, potently blocked human SKCa2 and SKCa3 but not SKCa1, whereas maurotoxin, Pi1, Tskappa, and PO1 were ineffective. Lei blocked these channels more potently than PO5 because of the presence of Ala(1), Phe(2), and Met(7). By replacing Met(7) in the RXCQ motif of Lei with the shorter, unnatural, positively charged diaminobutanoic acid (Dab), we generated Lei-Dab(7), a selective SKCa2 inhibitor (K(d) = 3.8 nm) that interacts with residues in the external vestibule of the channel. SKCa3 was rendered sensitive to Lei-Dab(7) by replacing His(521) with the corresponding SKCa2 residue (Asn(367)). Intracerebroventricular injection of Lei-Dab(7) into mice resulted in no gross central nervous system toxicity at concentrations that specifically blocked SKCa2 homotetramers. Lei-Dab(7) will be a useful tool to investigate the functional role of SKCa2 in mammalian tissues.