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1.
J Physiol Pharmacol ; 66(2): 249-57, 2015 Apr.
Article in English | MEDLINE | ID: mdl-25903955

ABSTRACT

Many factors, including hyperglycemia, hypertension, obesity, dyslipidemia, and a sedentary lifestyle, contribute to a high prevalence of cardiovascular disease. Specific vascular impairment treatments in the context of diabetes and vascular risk need to be improved. Salidroside is the primary active component of Rhodiola rosea and has documented antioxidative, cardioprotective, and vasculoprotective properties. The aim of this study was to test the hypothesis that salidroside has protective effects against hyperglycemia, hypertension, and vasodilation impairment in the Goto-Kakizaki (GK) rat model of diabetes. We evaluated cardiovascular parameters (e.g., daytime/nighttime systolic and diastolic blood pressure, heart rate, and activity), metabolic parameters (e.g., body weight, food and water consumption, serum fructosamine level, glucose tolerance), eNOS / phospho-eNOS expression level and in vitro vascular reactivity of aorta and second-order mesenteric arteries in Wistar-Kyoto (control) and GK (diabetic) rats treated with salidroside (40 mg/kg) or placebo (water) for 5 weeks. GK rats showed hypertension, marked glucose intolerance, and impaired endothelium-dependent and endothelium-independent vasodilation capacity. Salidroside showed beneficial effects on endothelial and non-endothelial vasodilation and likely acts on the endothelium and smooth muscle cells through the soluble guanylyl cyclase pathway. Despite its vascular effects, salidroside had no effect on blood pressure and heart rate in GK and control rats, it did not improve glucose metabolism or limit hypertension in the GK model of type 2 diabetes.


Subject(s)
Cardiovascular Diseases/drug therapy , Cardiovascular System/drug effects , Diabetes Mellitus, Type 2/metabolism , Glucosides/pharmacology , Phenols/pharmacology , Animals , Aorta/drug effects , Aorta/metabolism , Blood Pressure/drug effects , Cardiotonic Agents/pharmacology , Cardiovascular Diseases/metabolism , Cardiovascular System/metabolism , Endothelium, Vascular/drug effects , Endothelium, Vascular/metabolism , Glucose Intolerance/metabolism , Guanylate Cyclase/metabolism , Heart Rate/drug effects , Hyperglycemia/drug therapy , Hyperglycemia/metabolism , Hypertension/metabolism , Male , Myocytes, Smooth Muscle/drug effects , Myocytes, Smooth Muscle/metabolism , Nitric Oxide Synthase Type III/metabolism , Rats , Rats, Inbred WKY , Receptors, Cytoplasmic and Nuclear/metabolism , Soluble Guanylyl Cyclase , Vasodilation/drug effects
2.
Arch Inst Pasteur Tunis ; 85(1-4): 9-19, 2008.
Article in French | MEDLINE | ID: mdl-19469412

ABSTRACT

The lactococcin B (LnB) is a hydrophobic, positively charged bacteriocin, produced by Lactococcus lactis ssp. cremoris 9B4. It consists of a peptidic chain made up of 47 amino acid residues, and inhibits Lactococcus exclusively. In order to study its biological activity a synthetic lactococcin B (LnBs) was obtained by solid-phase chemical synthesis using a Fmoc strategy. LnBs was shown to be indistinguishable from the natural peptide. In addition, a synthetic (7-47) LnBst analogue was obtained by withdrawal of peptidyl-resin after the 41 cycle of LnBs peptide chain assembly. The synthetic N-terminal truncated (7-47) LnBst analogue was found to be inactive on indicator strains. Our results strongly suggest that the first six N-terminal amino acid residues are involved in the bactericidal activity of LnB.


Subject(s)
Bacteriocins/chemical synthesis , Bacteriocins/genetics , Amino Acid Sequence/genetics , Amino Acids/genetics , Bacteriocins/chemistry , Bacteriocins/isolation & purification , Chromatography, High Pressure Liquid , Fluorenes , Hydrophobic and Hydrophilic Interactions , Lactococcus lactis/chemistry , Lactococcus lactis/genetics , Lactococcus lactis/physiology , Molecular Sequence Data , Peptides/chemical synthesis , Peptides/chemistry , Peptides/genetics , Protein Structure, Secondary/genetics , Sequence Alignment , Sequence Analysis, Protein , Sequence Homology, Amino Acid , Solid Phase Extraction/methods
3.
Mol Pharmacol ; 63(2): 409-18, 2003 Feb.
Article in English | MEDLINE | ID: mdl-12527813

ABSTRACT

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.


Subject(s)
Potassium Channel Blockers/pharmacology , Potassium Channels, Calcium-Activated , Potassium Channels/metabolism , Scorpion Venoms/pharmacology , Amino Acid Sequence , Animals , Apamin/metabolism , CHO Cells , Cricetinae , Humans , Intermediate-Conductance Calcium-Activated Potassium Channels , Iodine Radioisotopes , Molecular Sequence Data , Sequence Homology, Amino Acid , T-Lymphocytes/drug effects , T-Lymphocytes/metabolism , Transfection
4.
Biochem J ; 358(Pt 3): 681-92, 2001 Sep 15.
Article in English | MEDLINE | ID: mdl-11535129

ABSTRACT

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.


Subject(s)
Protein Folding , Scorpion Venoms/chemistry , Scorpion Venoms/metabolism , Alkylation , Disulfides , Humans , Indicators and Reagents , Iodoacetamide , Kinetics , Models, Theoretical , Neurotoxins/chemistry , Oxidation-Reduction , Peptidylprolyl Isomerase/metabolism , Protein Disulfide-Isomerases/metabolism , Recombinant Proteins/metabolism , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
5.
J Biol Chem ; 276(46): 43145-51, 2001 Nov 16.
Article in English | MEDLINE | ID: mdl-11527975

ABSTRACT

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.


Subject(s)
Peptides/chemistry , Potassium Channel Blockers , Potassium Channels, Calcium-Activated/antagonists & inhibitors , Potassium Channels/chemistry , Scorpion Venoms/pharmacology , Alanine/pharmacology , Amino Acid Sequence , Animals , Arginine/chemistry , COS Cells , Cell Line , Cloning, Molecular , Dose-Response Relationship, Drug , Electrophysiology , Humans , Kinetics , Methionine/pharmacology , Models, Molecular , Molecular Sequence Data , Mutagenesis, Site-Directed , Mutation , Neurotoxins/pharmacology , PC12 Cells , Peptides/pharmacology , Phenylalanine/pharmacology , Protein Binding , Rats , Scorpion Venoms/chemistry , Sequence Homology, Amino Acid , Small-Conductance Calcium-Activated Potassium Channels , Threonine/chemistry , Transfection , Valine/chemistry , gamma-Aminobutyric Acid/pharmacology
6.
FEBS Lett ; 489(2-3): 202-7, 2001 Feb 02.
Article in English | MEDLINE | ID: mdl-11165250

ABSTRACT

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


Subject(s)
Disulfides/chemistry , Potassium Channels, Voltage-Gated , Proline/chemistry , Scorpion Venoms/chemistry , Amino Acid Sequence , Animals , Apamin/metabolism , Binding, Competitive , Dose-Response Relationship, Drug , Female , Iodine Radioisotopes , Kv1.2 Potassium Channel , Kv1.3 Potassium Channel , Magnetic Resonance Spectroscopy , Membrane Potentials/drug effects , Molecular Sequence Data , Mutation , Oocytes/drug effects , Oocytes/metabolism , Oocytes/physiology , Peptides/antagonists & inhibitors , Peptides/genetics , Peptides/physiology , Potassium Channel Blockers , Potassium Channels/genetics , Potassium Channels/physiology , Proline/genetics , Protein Conformation , Rats , Scorpion Venoms/metabolism , Scorpion Venoms/pharmacology , Sequence Analysis, Protein , Shaker Superfamily of Potassium Channels , Synaptosomes/metabolism , Xenopus
7.
J Biol Chem ; 275(50): 39394-402, 2000 Dec 15.
Article in English | MEDLINE | ID: mdl-10970898

ABSTRACT

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


Subject(s)
Disulfides , Scorpion Venoms/chemistry , Amino Acid Sequence , Animals , Brain/metabolism , Chromatography, High Pressure Liquid , Cysteine/chemistry , Dose-Response Relationship, Drug , Electrophysiology , Kinetics , Lethal Dose 50 , Ligands , Magnetic Resonance Spectroscopy , Mice , Mice, Inbred C57BL , Models, Molecular , Molecular Sequence Data , Mutation , Oocytes/metabolism , Peptide Biosynthesis , Point Mutation , Potassium Channels/chemistry , Protein Conformation , Protein Structure, Secondary , Rats , Scorpion Venoms/genetics , Sequence Homology, Amino Acid , Synaptosomes/metabolism , Time Factors , Xenopus
8.
Eur J Biochem ; 267(16): 5149-55, 2000 Aug.
Article in English | MEDLINE | ID: mdl-10931199

ABSTRACT

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


Subject(s)
Potassium Channel Blockers , Potassium Channels, Voltage-Gated , Scorpion Venoms/chemical synthesis , Scorpion Venoms/pharmacology , Amino Acid Sequence , Animals , Cerebral Ventricles/drug effects , Cerebral Ventricles/physiology , Cystine , Disulfides/analysis , Humans , Injections, Intraventricular , Kv1.1 Potassium Channel , Kv1.2 Potassium Channel , Kv1.3 Potassium Channel , Lethal Dose 50 , Mice , Molecular Sequence Data , Potassium Channels/physiology , Rats , Scorpion Venoms/chemistry , Sequence Alignment , Sequence Homology, Amino Acid , Xenopus laevis
9.
J Pept Res ; 55(6): 419-27, 2000 Jun.
Article in English | MEDLINE | ID: mdl-10888198

ABSTRACT

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.


Subject(s)
Potassium Channel Blockers , Potassium Channels/metabolism , Scorpion Venoms/pharmacology , Amino Acid Motifs , Amino Acid Sequence , Animals , CHO Cells , Cricetinae , Disulfides , Dose-Response Relationship, Drug , Gene Expression , Microinjections , Molecular Sequence Data , Oocytes , Patch-Clamp Techniques , Protein Conformation , RNA, Complementary/metabolism , Scorpion Venoms/chemistry , Scorpion Venoms/metabolism , Shaker Superfamily of Potassium Channels , Sodium Channels/metabolism , Xenopus laevis
10.
Proteins ; 40(3): 436-42, 2000 Aug 15.
Article in English | MEDLINE | ID: mdl-10861934

ABSTRACT

We determined the structure in solution by (1)H two-dimensional NMR of Maurocalcine from the venom of Scorpio maurus. This toxin has been demonstrated to be a potent effector of ryanodyne-sensitive calcium channel from skeletal muscles. This is the first description of a scorpion toxin which folds following the Inhibitor Cystine Knot fold (ICK) already described for numerous toxic and inhibitory peptides, as well as for various protease inhibitors. Its three dimensional structure consists of a compact disulfide-bonded core from which emerge loops and the N-terminus. A double-stranded antiparallel beta-sheet comprises residues 20-23 and 30-33. A third extended strand (residues 9-11) is perpendicular to the beta-sheet. Maurocalcine structure mimics the activating segment of the dihydropyridine receptor II-III loop and is therefore potentially useful for dihydropyridine receptor/ryanodine receptor interaction studies. Proteins 2000;40:436-442.


Subject(s)
Calcium Channel Agonists/chemistry , Scorpion Venoms/chemistry , Amino Acid Sequence , Calcium Channel Agonists/toxicity , Calcium Channels, L-Type/metabolism , Computer Simulation , Models, Molecular , Molecular Mimicry , Molecular Sequence Data , Nuclear Magnetic Resonance, Biomolecular , Ryanodine Receptor Calcium Release Channel/metabolism , Scorpion Venoms/toxicity , Sequence Homology, Amino Acid
11.
J Biol Chem ; 275(18): 13605-12, 2000 May 05.
Article in English | MEDLINE | ID: mdl-10788477

ABSTRACT

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


Subject(s)
Drug Design , Scorpion Venoms/chemistry , Toxins, Biological/chemistry , Amino Acid Sequence , Animals , Disulfides , Magnetic Resonance Spectroscopy , Mice , Molecular Sequence Data , Protein Conformation , Scorpion Venoms/genetics , Scorpions , Toxins, Biological/chemical synthesis , Toxins, Biological/genetics
12.
FEBS Lett ; 469(2-3): 179-85, 2000 Mar 10.
Article in English | MEDLINE | ID: mdl-10713267

ABSTRACT

Maurocalcine is a novel toxin isolated from the venom of the chactid scorpion Scorpio maurus palmatus. It is a 33-mer basic peptide cross-linked by three disulfide bridges, which shares 82% sequence identity with imperatoxin A, a scorpion toxin from the venom of Pandinus imperator. Maurocalcine is peculiar in terms of structural properties since it does not possess any consensus motif reported so far in other scorpion toxins. Due to its low concentration in venom (0.5% of the proteins), maurocalcine was chemically synthesized by means of an optimized solid-phase method, and purified after folding/oxidation by using both C18 reversed-phase and ion exchange high-pressure liquid chromatographies. The synthetic product (sMCa) was characterized. The half-cystine pairing pattern of sMCa was identified by enzyme-based cleavage and Edman sequencing. The pairings were Cys3-Cys17, Cys10-Cys21, and Cys16-Cys32. In vivo, the sMCa was lethal to mice following intracerebroventricular inoculation (LD(50), 20 microg/mouse). In vitro, electrophysiological experiments based on recordings of single channels incorporated into planar lipid bilayers showed that sMCa potently and reversibly modifies channel gating behavior of the type 1 ryanodine receptor by inducing prominent subconductance behavior.


Subject(s)
Ryanodine Receptor Calcium Release Channel/chemistry , Scorpion Venoms/chemical synthesis , Amino Acid Sequence , Animals , Chromatography, High Pressure Liquid , Cystine/chemistry , Electrophysiology , Injections, Intraventricular , Ion Channel Gating , Lethal Dose 50 , Lipid Bilayers , Mice , Molecular Sequence Data , Scorpion Venoms/chemistry , Scorpion Venoms/toxicity , Sequence Analysis, Protein , Sequence Homology, Amino Acid
13.
J Pept Res ; 56(6): 427-37, 2000 Dec.
Article in English | MEDLINE | ID: mdl-11152302

ABSTRACT

SPC3 is a multibranched peptide containing eight identical GPGRAF motifs which are derived from the human immunodeficiency virus (HIV)-1 gp120 V3 loop consensus sequence. This molecule was reported to prevent the infection of CD4+ cells by various HIV-1 and HIV-2 strains. However, the molecular mode of action of SPC3 remains unclear. Here, we investigated the possibility that SPC3 could interact with alpha/beta-chemokine receptors following observations that, first, the V3 loop is likely to be involved in alpha/beta-chemokine receptor-dependent HIV entry and, second, natural ligands of these receptors are potent inhibitors of cell infection. To address this point, we examined the effects of SPC3 on Xenopus oocytes either uninjected or expressing exogenous human CXCR4 alpha-chemokine receptors. Extracellular applications of micromolar concentrations of SPC3 onto Xenopus oocytes trigger potent inward chloride currents which can be inhibited by increasing extracellular Ca2+ concentration. This effect can be blocked by chloride channel antagonists and is highly specific to SPC3 as it is not triggered by structural analogs of SPC3. The SPC3-induced chloride conductance in oocytes is alpha/beta-chemokine receptor dependent because: (i) SPC3 alters the sensitivity of this channel to external applications of human recombinant MIP-1alpha, a natural ligand of human CCR5 receptor, and (ii) the amplitude of the inward current could be increased by the expression of exogenous human CXCR4 chemokine receptor. The effect of SPC3 appears to rely on the activation of a phospholipase A2 signaling pathway, but is not affected by changes in cytosolic Ca2+ concentration, or by alterations in Gi/Go protein, adenylate cyclase, phospholipase C or protein kinase C activity. Altogether, the data indicate that SPC3 is capable of activating a surface alpha/beta-chemokine-like receptor-mediated signaling pathway in competent cells, thereby triggering, either directly or indirectly, a Ca2+-inactivated chloride conductance.


Subject(s)
Chemokines/chemistry , Fungal Proteins/chemistry , HIV Envelope Protein gp120/chemistry , Ion Channels/chemistry , Ions/chemistry , Peptides/chemistry , Saccharomyces cerevisiae Proteins , Animals , Calcium/chemistry , Calcium/pharmacology , Chelating Agents/pharmacology , Dose-Response Relationship, Drug , Electrophysiology , Enzyme Inhibitors/pharmacology , Humans , Oocytes/chemistry , Peptide Biosynthesis , Phospholipases A/metabolism , Phospholipases A2 , Potassium/chemistry , Potassium/pharmacology , RNA, Complementary/chemistry , Receptors, CXCR4/chemistry , Signal Transduction , Time Factors , Xenopus
14.
J Pept Res ; 54(5): 369-76, 1999 Nov.
Article in English | MEDLINE | ID: mdl-10563502

ABSTRACT

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.


Subject(s)
Potassium Channels, Calcium-Activated , Potassium Channels/drug effects , Scorpion Venoms/chemical synthesis , Amino Acid Sequence , Amino Acids/analysis , Animals , Apamin , Binding, Competitive , Brain/metabolism , Chromatography, High Pressure Liquid , Disulfides/chemistry , Endopeptidases , Magnetic Resonance Spectroscopy , Models, Molecular , Molecular Sequence Data , Mutation , Peptide Fragments/chemistry , Protein Binding , Rats , Scorpion Venoms/pharmacology , Small-Conductance Calcium-Activated Potassium Channels , Structure-Activity Relationship , Synaptosomes/metabolism
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