Imed Regaya

Design and Characterization of a Highly Selective Peptide Inhibitor of the Small Conductance Calcium-activated K+Channel, SkCa2

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, Tsκ, and PO1 were ineffective. Lei blocked these channels more potently than PO5 because of the presence of Ala1, Phe2, and Met7. By replacing Met7 in the RXCQ motif of Lei with the shorter, unnatural, positively charged diaminobutanoic acid (Dab), we generated Lei-Dab7, 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-Dab7 by replacing His521 with the corresponding SKCa2 residue (Asn367). Intracerebroventricular injection of Lei-Dab7 into mice resulted in no gross central nervous system toxicity at concentrations that specifically blocked SKCa2 homotetramers. Lei-Dab7 will be a useful tool to investigate the functional role of SKCa2 in mammalian tissues.

Critical Amino Acid Residues Determine the Binding Affinity and the Ca2+ Release Efficacy of Maurocalcine in Skeletal Muscle Cells

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.

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

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

Differential effects of two blockers of small conductance Ca2+-activated K+ channels, apamin and lei-Dab7, on learning and memory in rats.

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.

Small-conductance Ca2+-activated K+ channels : Heterogeneous affinity in rat brain structures and cognitive modulation by specific blockers

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.

Kv4 channel blockade reduces motor and neuropsychiatric symptoms in rodent models of Parkinson's disease.

The striatum, a major input structure of basal ganglia, integrates glutamatergic cortical and thalamic inputs to control psychomotor behaviors. Nigrostriatal dopamine (DA) neurodegeneration in Parkinsons 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 Parkinsons disease.

Bacteriocins Active Against Multi-Resistant Gram Negative Bacteria Implicated in Nosocomial Infections

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).

Evolution of maurotoxin conformation and blocking efficacy towards Shaker B channels during the course of folding and oxidation in vitro

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 80 h: (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 alpha-helix within 5 h, followed by the formation of the beta-sheet within 22 h. 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 alpha-helix and two- or three-disulphide-bridged sMTX for the beta-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.

Genetic Characterization of Lactic Acid Bacteria Isolated from Tunisian Milk Waste and their Antimicrobial Activity Against some Bacteria Implicated in Nosocomial Infections.

BACKGROUND A total of 94 lactic acid bacteria (LAB) were isolated from Tunisian artisanal (Ricotta cheeses 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.

Effect of Cu2+ on the Oxidative Folding of Synthetic Maurotoxin In Vitro

Abstract 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 α/β scaffold, i.e., a helix connected to a two stranded β-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.