DOCSLIB.ORG

Disul¢De Bridge Reorganization Induced by Proline Mutations In

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Disul¢De Bridge Reorganization Induced by Proline Mutations In FEBS Letters 489 (2001) 202^207 FEBS 24520 View metadata, citation and similar papers at core.ac.uk brought to you by CORE Disul¢de bridge reorganization induced by proline mutationsprovided by Elsevier in - Publisher Connector maurotoxin E. Carliera, Z. Fajlounb, P. Mansuelleb, M. Fathallahb, A. Mosbahc, R. Oughidenib, G. Sandoza, E. Di Lucciob, S. Geiba, I. Regayab, J. Brocarda, H. Rochatb, H. Darbonc, C. Devauxb, J.M. Sabatierb, M. de Waarda;* aLaboratoire de Neurobiologie des Canaux Ioniques, INSERM U464, IFR Jean Roche, Faculte¨ de Me¨decine Nord, Bd Pierre Dramard, 13916 Marseille Cedex 20, France bLaboratoire de Biochimie, CNRS UMR 6560, IFR Jean Roche, Faculte¨ de Me¨decine Nord, Bd Pierre Dramard, 13916 Marseille Cedex 20, France cAFMB, CNRS UPR 9039, IFR1, 31 Chemin Joseph-Aiguier, 13402 Marseille Cedex 20, France Received 1 December 2000; accepted 21 December 2000 First published online 18 January 2001 Edited by Pierre Jolles channel subtypes (Kv and SK channels). Contrary to most Abstract Maurotoxin (MTX) is a 34-residue toxin that has been isolated from the venom of the chactidae scorpion Scorpio short-chain K channel-acting scorpion toxins, this family maurus palmatus, and characterized. Together with Pi1 and can be distinguished by the presence of an additional disul¢de HsTx1, MTX belongs to a family of short-chain four-disulfide- bridge (four instead of the three commonly present in such bridged scorpion toxins acting on potassium channels. However, toxins). This structural class also includes Pi1 and HsTx1 contrary to other members of this family, MTX exhibits an from the venoms of the scorpions Pandinus imperator [2] uncommon disulfide bridge organization of the type C1^C5, C2^ and Heterometrus spinnifer [3], respectively. These toxins share C6, C3^C4 and C7^C8, versus C1^C5, C2^C6, C3^C7 and C4^ from 53 to 68% sequence identity with MTX but display dif- C8 for both Pi1 and HsTx1. Here, we report that the substitution ferent pharmacological selectivities. For instance, MTX and of MTX proline residues located at positions 12 and/or 20, Pi1 are both active on apamin-sensitive SK channels, whereas adjacent to C3 (Cys ) and C4 (Cys ), results in conventional 13 19 HsTx1 is reportedly inactive on this channel type. Also, MTX Pi1- and HsTx1-like arrangement of the half-cystine pairings. In this case, this novel disulfide bridge arrangement is without was found to be active on rat Kv1.3 channels contrary to obvious incidence on the overall three-dimensional structure of synthetic Pi1 [4]. Interestingly, MTX structurally di¡ers the toxin. Pharmacological assays of this structural analog, from Pi1 and HsTx1, but also from other `classical' three- [A12,A20]MTX, reveal that the blocking activities on Shaker B disul¢de-bridged scorpion toxins, by its unique disul¢de and rat Kv1.2 channels remain potent whereas the peptide bridge pattern. In three-disul¢de-bridged toxins, the half-cys- becomes inactive on rat Kv1.3. These data indicate, for the first tine pairings are of the type C1^C4, C2^C5 and C3^C6 (e.g. time, that discrete point mutations in MTX can result in a charybdotoxin, P05, agitoxin 2, leiurotoxin 1). In short-chain marked reorganization of the half-cystine pairings, accompanied four-disul¢de-bridged toxins, this pattern is altered by the with a novel pharmacological profile for the analog. ß 2001 insertion of two additional half-cystines within the amino Federation of European Biochemical Societies. Published by acid sequence, one located after C3 and the other after C6. Elsevier Science B.V. All rights reserved. As a result, two novel patterns of the disul¢de bridges are Key words: Maurotoxin; Scorpion toxin; Potassium channel; experimentally found depending on the toxin: (i) a pattern Synthetic peptide; Half-cystine pairing; Xenopus oocyte of the type C1^C5, C2^C6, C3^C7 and C4^C8 in both Pi1 and HsTx1 (which corresponds to an organization similar to that observed in three-disul¢de-bridged toxins) and (ii) an uncommon pattern of the type C1^C5, C2^C6, C3^C4 and 1. Introduction C7^C8 in MTX. These two patterns possess in common the ¢rst two disul¢de bridges but di¡er in the two remaining Maurotoxin (MTX), a toxin from the venom of the Tuni- disul¢des. Though di¡erences in pharmacological properties sian chactidae scorpion Scorpio maurus palmatus, is a 34-mer between short-chain four-disul¢de-bridged toxins obviously peptide cross-linked by four disul¢de bridges [1]. MTX be- rely on their distinct amino acid sequences, it is also likely longs to a distinct family of short-chain scorpion toxins that changes in half-cystine pairings may contribute to dis- with less than 40 residues, that are active on several potassium crete conformational alterations and repositioning of key res- idues that are involved in toxin selectivity. In MTX, we no- ticed that the two proline residues in positions 12 and 20 are *Corresponding author. Fax: (33)-491-09 05 06. adjacent to the non-conventional disul¢de bridge C3^C4 E-mail: [email protected] (Cys13^Cys19). Though these residues are also present in ho- mologous positions in HsTx1, Pi1 and Pi4, we expected that Abbreviations: HPLC, high-pressure liquid chromatography; MTX, structural variations induced by the mutagenesis of these pro- maurotoxin, a scorpion toxin from the venom of Scorpio maurus line residues could favor the shift from a MTX-like to a Pi1/ palmatus; Pi1, a scorpion toxin from the venom of Pandinus impe- rator; HsTx1, toxin 1 from the venom of the scorpion Heterometrus HsTx1-like disul¢de bridge pattern. To test this hypothesis, spinnifer we chemically synthesized by the solid-phase method an MTX 0014-5793 / 01 / $20.00 ß 2001 Federation of European Biochemical Societies. Published by Elsevier Science B.V. All rights reserved. PII: S0014-5793(00)02433-9 FEBS 24520 25-1-01 Cyaan Magenta Geel Zwart E. Carlier et al./FEBS Letters 489 (2001) 202^207 203 scent method, then re¢ned by the conjugate gradient method until analog ([A12,A20]MTX) in which the two proline residues are substituted by alanine residues. Our data show that these the ¢nal root-mean-square derivative of the energy was less than 0.001 kcal mol31 Aî 31 in each case. substitutions are responsible for a Pi1- and HsTx1-like rear- rangement of the half-cystine pairings of MTX accompanied 2.6. Binding assay of [125I]apamin and competition by by subtle changes in pharmacological activity. [A12,A20]MTX onto rat brain synaptosomes Rat brain synaptosomes (P2 fraction) were prepared as described by Gray and Whittaker [6]. The protein content was determined by a 2. Materials and methods modi¢ed Lowry method. [125I]apamin (2000 Ci/mmol) was obtained according to Seagar et al. [7]. Aliquots of 50 Wl 0.1 nM [125I]apamin 2.1. Materials were added to 400 Wl synaptosome suspension (0.4 mg protein/ml). N-K-£uorenylmethyloxycarbonyl (N-K-Fmoc)-L-amino acids, Samples were incubated for 1 h at 4³C with 50 Wl of various concen- Fmoc-amide resin and reagents used for peptide synthesis were ob- trations of [A12,A20]MTX in 500 Wl ¢nal volume. The incubation tained from Perkin-Elmer. Solvents were analytical grade products bu¡er was 25 mM Tris^HCl, 10 mM KCl, pH 7.2. The samples from SDS. Enzymes (trypsin and chymotrypsin) were obtained from were centrifuged and the resulting pellets were washed three times Boehringer Mannheim. in 1 ml of the same bu¡er. Bound radioactivity was determined by Q counting (Packard Crystal II). The values expressed are the means of 2.2. Chemical synthesis and physicochemical characterization of triplicate experiments þ S.D. Non-speci¢c binding, less than 10% of [A12,A20]MTX the total binding, was determined in the presence of an excess [A12,A20]MTX was obtained by the solid-phase method [5] using a (10 nM) of unlabeled apamin. peptide synthesizer (Model 433A, Applied Biosystems). Peptide chains were assembled stepwise on 0.25 meq of Fmoc-amide resin (0.89 meq 2.7. Oocyte preparation and electrophysiological recordings of amino group/g) using 1 mmol of Fmoc-amino acid derivatives. N- Xenopus laevis oocytes were prepared for cRNA injection and elec- K-amino groups were deprotected by treatment with 18 and 20% (v/v) trophysiological recordings as described [8]. Plasmids coding for K piperidine/N-methylpyrrolidone for 3 and 8 min, respectively. The channels were linearized with SmaI(Shaker B), NotI (rat Kv1.1), XbaI Fmoc-amino acid derivatives were coupled (20 min) as their hydroxy- (rat Kv1.2) and EcoRI (rat Kv1.3), and transcribed into cRNA with benzotriazole active esters in N-methylpyrrolidone (four-fold excess). T7 or SP6 (mMessage mMachine kit, Ambion). The cRNA were The peptide resin (about 2 g) was treated for 2.5 h at room temper- stored frozen in H2Oat380³C at 1 Wg/Wl. Cells were micro-injected ature with a mixture of tri£uoroacetic acid (TFA)/H2O/thioanisole/ 2 days later with 50 nl of cRNA (0.1^0.2 Wg/Wl Shaker B, rat Kv1.1, ethanedithiol (88:5:5:2, v/v) in the presence of crystalline phenol rat Kv1.2 or rat Kv1.3 channels) and incubated 2^6 days at 16³C into (2.25 g). After lyophilization, the crude reduced peptide (2 mM) was a de¢ned nutrient oocyte medium [9] before current recordings. Both oxidized in 200 mM Tris^HCl bu¡er, pH 8.3. [A12,A20]MTX was current and voltage electrodes were ¢lled with 140 mM KCl and had puri¢ed by reversed-phase high-pressure liquid chromatography resistances ranging from 0.5 to 1 M6.
Load more

Recommended publications
  • Animal Venom Derived Toxins Are Novel Analgesics for Treatment Of
    Short Communication iMedPub Journals 2018 www.imedpub.com Journal of Molecular Sciences Vol.2 No.1:6 Animal Venom Derived Toxins are Novel Upadhyay RK* Analgesics for Treatment of Arthritis Department of Zoology, DDU Gorakhpur University, Gorakhpur, UP, India Abstract *Corresponding authors: Ravi Kant Upadhyay Present review article explains use of animal venom derived toxins as analgesics of the treatment of chronic pain and inflammation occurs in arthritis. It is a [email protected] progressive degenerative joint disease that put major impact on joint function and quality of life. Patients face prolonged inappropriate inflammatory responses and bone erosion. Longer persistent chronic pain is a complex and debilitating Department of Zoology, DDU Gorakhpur condition associated with a large personal, mental, physical and socioeconomic University, Gorakhpur, UttarPradesh, India. burden. However, for mitigation of inflammation and sever pain in joints synthetic analgesics are used to provide quick relief from pain but they impose many long Tel: 9838448495 term side effects. Venom toxins showed high affinity to voltage gated channels, and pain receptors. These are strong inhibitors of ion channels which enable them as potential therapeutic agents for the treatment of pain. Present article Citation: Upadhyay RK (2018) Animal Venom emphasizes development of a new class of analgesic agents in form of venom Derived Toxins are Novel Analgesics for derived toxins for the treatment of arthritis. Treatment of Arthritis. J Mol Sci. Vol.2 No.1:6 Keywords: Analgesics; Venom toxins; Ion channels; Channel inhibitors; Pain; Inflammation Received: February 04, 2018; Accepted: March 12, 2018; Published: March 19, 2018 Introduction such as the back, spine, and pelvis.
    [Show full text]
  • Slow Inactivation in Voltage Gated Potassium Channels Is Insensitive to the Binding of Pore Occluding Peptide Toxins
    Biophysical Journal Volume 89 August 2005 1009–1019 1009 Slow Inactivation in Voltage Gated Potassium Channels Is Insensitive to the Binding of Pore Occluding Peptide Toxins Carolina Oliva, Vivian Gonza´lez, and David Naranjo Centro de Neurociencias de Valparaı´so, Facultad de Ciencias, Universidad de Valparaı´so, Valparaı´so, Chile ABSTRACT Voltage gated potassium channels open and inactivate in response to changes of the voltage across the membrane. After removal of the fast N-type inactivation, voltage gated Shaker K-channels (Shaker-IR) are still able to inactivate through a poorly understood closure of the ion conduction pore. This, usually slower, inactivation shares with binding of pore occluding peptide toxin two important features: i), both are sensitive to the occupancy of the pore by permeant ions or tetraethylammonium, and ii), both are critically affected by point mutations in the external vestibule. Thus, mutual interference between these two processes is expected. To explore the extent of the conformational change involved in Shaker slow inactivation, we estimated the energetic impact of such interference. We used kÿconotoxin-PVIIA (kÿPVIIA) and charybdotoxin (CTX) peptides that occlude the pore of Shaker K-channels with a simple 1:1 stoichiometry and with kinetics 100-fold faster than that of slow inactivation. Because inactivation appears functionally different between outside-out patches and whole oocytes, we also compared the toxin effect on inactivation with these two techniques. Surprisingly, the rate of macroscopic inactivation and the rate of recovery, regardless of the technique used, were toxin insensitive. We also found that the fraction of inactivated channels at equilibrium remained unchanged at saturating kÿPVIIA.
    [Show full text]
  • Ion Channels
    UC Davis UC Davis Previously Published Works Title THE CONCISE GUIDE TO PHARMACOLOGY 2019/20: Ion channels. Permalink https://escholarship.org/uc/item/1442g5hg Journal British journal of pharmacology, 176 Suppl 1(S1) ISSN 0007-1188 Authors Alexander, Stephen PH Mathie, Alistair Peters, John A et al. Publication Date 2019-12-01 DOI 10.1111/bph.14749 License https://creativecommons.org/licenses/by/4.0/ 4.0 Peer reviewed eScholarship.org Powered by the California Digital Library University of California S.P.H. Alexander et al. The Concise Guide to PHARMACOLOGY 2019/20: Ion channels. British Journal of Pharmacology (2019) 176, S142–S228 THE CONCISE GUIDE TO PHARMACOLOGY 2019/20: Ion channels Stephen PH Alexander1 , Alistair Mathie2 ,JohnAPeters3 , Emma L Veale2 , Jörg Striessnig4 , Eamonn Kelly5, Jane F Armstrong6 , Elena Faccenda6 ,SimonDHarding6 ,AdamJPawson6 , Joanna L Sharman6 , Christopher Southan6 , Jamie A Davies6 and CGTP Collaborators 1School of Life Sciences, University of Nottingham Medical School, Nottingham, NG7 2UH, UK 2Medway School of Pharmacy, The Universities of Greenwich and Kent at Medway, Anson Building, Central Avenue, Chatham Maritime, Chatham, Kent, ME4 4TB, UK 3Neuroscience Division, Medical Education Institute, Ninewells Hospital and Medical School, University of Dundee, Dundee, DD1 9SY, UK 4Pharmacology and Toxicology, Institute of Pharmacy, University of Innsbruck, A-6020 Innsbruck, Austria 5School of Physiology, Pharmacology and Neuroscience, University of Bristol, Bristol, BS8 1TD, UK 6Centre for Discovery Brain Science, University of Edinburgh, Edinburgh, EH8 9XD, UK Abstract The Concise Guide to PHARMACOLOGY 2019/20 is the fourth in this series of biennial publications. The Concise Guide provides concise overviews of the key properties of nearly 1800 human drug targets with an emphasis on selective pharmacology (where available), plus links to the open access knowledgebase source of drug targets and their ligands (www.guidetopharmacology.org), which provides more detailed views of target and ligand properties.
    [Show full text]
  • CDNA Cloning and Sequence Analysis of an Alpha Neurotoxin-Like Gene BMK from the Scorpion Mesobuthus Eupeus Venom Glands Using Semi-Nested RT-PCR
    American-Eurasian Journal of Toxicological Sciences 3 (4): 219-223, 2011 ISSN 2079-2050 © IDOSI Publications, 2011 CDNA Cloning and Sequence Analysis of an Alpha Neurotoxin-Like Gene BMK from the Scorpion Mesobuthus eupeus Venom Glands Using Semi-Nested RT-PCR Ghafar Eskandari Young Researchers Club, Izeh Branch, Islamic Azad University, Izeh, Iran Abstract: This work aimed to study the characterization and isolation of a alpha neurotoxin- BmK, from the venom glands of Scorpion Buthida Mesobuthus eupeus Kuzestan (Iran). Scorpions Mesobuthus eupeus were collected from the Khuzestan province. Using RNXTM solution, the total RNA was extracted from the twenty separated venom glands. cDNA was synthesized with extracted total RNA as template and modified oligo(dT) as primer. In order to amplify cDNA encoding a BMK neurotoxin, Semi-Nested RT-PCR was done with the specific primers. Follow amplification, the fragment was sequenced. The length of the coding region was 255 bp, encoding a polypeptide of 85 amino acid residues with a calculated molecular weight of 9.565 kDa and theoretical isoelectric point of 4.69.The open reading frame encoded 85 amino acid residues corresponding to the BMK precursor Alpha neurotoxin from M. eupeus belongs to the Toxin_3 super family. The size of peptide is close to the "long chain neurotoxin" peptide family. Key words: Alpha Neurotoxin Semi-Nested RT-PCR Scorpion Venom INTRODUCTION linked by four disulfide bridges. These peptides are mainly active on sodium channels [2, 4]. Classical -toxins Scorpion venom is contains a mixture of various toxic are also mainly found in the venoms of Androctonus proteins with different functions.
    [Show full text]
  • DISSERTACAO 2008 Edelyncr
    Universidade de Brasília Programa de Pós-Graduação em Biologia Animal Construção da biblioteca de cDNA da glândula de peçonha do escorpião Opisthacanthus cayaporum e clonagem de genes que codificam para componentes da peçonha Édelyn Cristina Nunes Silva Orientadora: Profª Drª Elisabeth N. Ferroni Schwartz Dissertação apresentada ao Programa de Pós-Graduação em Biologia Animal da Universidade de Brasília como parte dos requisitos para a obtenção do título de Mestre. Brasília, 2008 1 UNIVERSIDADE DE BRASÍLIA INSTITUTO DE CIÊNCIAS BIOLÓGICAS PROGRAMA DE PÓS-GRADUAÇÃO EM BIOLOGIA ANIMAL Dissertação de Mestrado Édelyn Cristina Nunes Silva Título: “Construção da biblioteca de cDNA da glândula de peçonha do escorpião Opisthacanthus cayaporum e clonagem de genes que codificam para componentes da peçonha” Comissão examinadora: Profa.Dra. Elisabeth N. Ferroni Schwartz Presidente/Orientadora CFS/UnB Prof. Dr. Luciano Paulino da Silva Profa. Dra. Ildinete Silva Pereira Embrapa/Cenargen CEL/UnB Profa. Dra. Márcia Renata Mortari Suplente, CFS/UnB Brasília, julho de 2008 2 DEDICATÓRIA A Deus Ao meu noivo Flávio A minha amada Mãe, Matilde A minha irmã Évelyn A Dra. Elisabeth Schwartz e ao Dr. Lourival Possani 3 AGRADECIMENTOS Esta dissertação de Mestrado só teve êxito porque ela é fruto do trabalho de muitas pessoas, de dois países, vários laboratórios, duas universidades... Assim tentarei agradecer a todos por essa vitória! Meus sinceros agradecimentos ao Prof. Dr. Lourival Possani do Departamento de Medicina Molecular y Bioprocesos, Instituto de Biotecnología da Universidad Autônoma do México, em Cuernavaca / Morelos, pela oportunidade de viajar ao México, conhecer esse maravilhoso país e trabalhar em seu renomado laboratório onde pude realizar grande parte dessa dissertação.
    [Show full text]
  • Mapping of Maurotoxin Binding Sites on Hkv1.2, Hkv1.3, and Hikca1 Channels
    0026-895X/04/6605-1103–1112$20.00 MOLECULAR PHARMACOLOGY Vol. 66, No. 5 Copyright © 2004 The American Society for Pharmacology and Experimental Therapeutics 2774/1178283 Mol Pharmacol 66:1103–1112, 2004 Printed in U.S.A. Mapping of Maurotoxin Binding Sites on hKv1.2, hKv1.3, and hIKCa1 Channels Violeta Visan, Ziad Fajloun, Jean-Marc Sabatier, and Stephan Grissmer Department of Applied Physiology, University of Ulm, Ulm, Germany (V.V., S.G.); Laboratoire International Associe´ d’Inge´nierie Biomole´culaire, Centre National de la Recherche Scientifique Unite´ Mixte Recherche 6560, Marseille, France (Z.F., J.-M.S.) Received May 17, 2004; accepted July 27, 2004 Downloaded from ABSTRACT Maurotoxin (MTX) is a potent blocker of human voltage-acti- interactions. Lys23 from MTX protrudes into the channel pore vated Kv1.2 and intermediate-conductance calcium-activated interacting with the GYGD motif, whereas Tyr32 and Lys7 inter- potassium channels, hIKCa1. Because its blocking affinity on act with Val381, Asp363, and Glu355, stabilizing the toxin onto the both channels is similar, although the pore region of these channel pore. Because only Val381, Asp363, and the GYGD motif channels show only few conserved amino acids, we aimed to are conserved in hIKCa1 channels, and the replacement of His399 characterize the binding sites of MTX in these channels. Inves- from hKv1.3 channels with a threonine makes this channel MTX- molpharm.aspetjournals.org tigating the pHo dependence of MTX block on current through sensitive, we concluded that MTX binds to all three channels 355 hKv1.2 channels, we concluded that the block is less pHo- through the same amino acids.
    [Show full text]
  • Distribution and Kinetics of the Kv1.3-Blocking Peptide Hstx1[R14A]
    www.nature.com/scientificreports OPEN Distribution and kinetics of the Kv1.3-blocking peptide HsTX1[R14A] in experimental rats Received: 19 January 2017 Ralf Bergmann1, Manja Kubeil1,2, Kristof Zarschler1, Sandeep Chhabra3, Rajeev B. Tajhya4, Accepted: 9 May 2017 Christine Beeton 4, Michael W. Pennington5, Michael Bachmann1, Raymond S. Norton 3 & Published: xx xx xxxx Holger Stephan 1 The peptide HsTX1[R14A] is a potent and selective blocker of the voltage-gated potassium channel Kv1.3, which is a highly promising target for the treatment of autoimmune diseases and other conditions. In order to assess the biodistribution of this peptide, it was conjugated with NOTA and radiolabelled with copper-64. [64Cu]Cu-NOTA-HsTX1[R14A] was synthesised in high radiochemical purity and yield. The radiotracer was evaluated in vitro and in vivo. The biodistribution and PET studies after intravenous and subcutaneous injections showed similar patterns and kinetics. The hydrophilic peptide was rapidly distributed, showed low accumulation in most of the organs and tissues, and demonstrated high molecular stability in vitro and in vivo. The most prominent accumulation occurred in the epiphyseal plates of trabecular bones. The high stability and bioavailability, low normal-tissue uptake of [64Cu]Cu-NOTA-HsTX1[R14A], and accumulation in regions of up-regulated Kv channels both in vitro and in vivo demonstrate that HsTX1[R14A] represents a valuable lead for conditions treatable by blockade of the voltage-gated potassium channel Kv1.3. The pharmacokinetics shows that both intravenous and subcutaneous applications are viable routes for the delivery of this potent peptide. Voltage-gated potassium (Kv) channels are integral membrane proteins that regulate cell membrane potential and are involved in a variety of cellular functions including apoptosis and cell volume regulation1.
    [Show full text]
  • (K+) Channels: a Historical Overview of Peptide Bioengineering
    Toxins 2012, 4, 1082-1119; doi:10.3390/toxins4111082 OPEN ACCESS toxins ISSN 2072-6651 www.mdpi.com/journal/toxins Review Scorpion Toxins Specific for Potassium (K+) Channels: A Historical Overview of Peptide Bioengineering Zachary L. Bergeron and Jon-Paul Bingham * Department of Molecular Biosciences and Bioengineering, College of Tropical Agriculture and Human Resources, University of Hawaii at Manoa, Honolulu, HI 96822, USA; E-Mail: [email protected] * Author to whom correspondence should be addressed; E-Mail: [email protected]; Tel.: +1-808-956-4864; Fax: +1-808-956-3542. Received: 14 September 2012; in revised form: 22 October 2012 / Accepted: 23 October 2012 / Published: 1 November 2012 Abstract: Scorpion toxins have been central to the investigation and understanding of the physiological role of potassium (K+) channels and their expansive function in membrane biophysics. As highly specific probes, toxins have revealed a great deal about channel structure and the correlation between mutations, altered regulation and a number of human pathologies. Radio- and fluorescently-labeled toxin isoforms have contributed to localization studies of channel subtypes in expressing cells, and have been further used in competitive displacement assays for the identification of additional novel ligands for use in research and medicine. Chimeric toxins have been designed from multiple peptide scaffolds to probe channel isoform specificity, while advanced epitope chimerization has aided in the development of novel molecular therapeutics. Peptide backbone cyclization has been utilized to enhance therapeutic efficiency by augmenting serum stability and toxin half-life in vivo as a number of K+-channel isoforms have been identified with essential roles in disease states ranging from HIV, T-cell mediated autoimmune disease and hypertension to various cardiac arrhythmias and Malaria.
    [Show full text]
  • Modeling of the Binding of Peptide Blockers to Voltage-Gated Potassium Channels: Approaches and Evidence
    REVIEWS Modeling of the Binding of Peptide Blockers to Voltage-Gated Potassium Channels: Approaches and Evidence V. N. Novoseletsky1*, A. D. Volyntseva1, K. V. Shaitan1, M. P. Kirpichnikov1,2, A. V. Feofanov1,2 1M.V.Lomonosov Moscow State University, Faculty of Biology, Leninskie Gory 1, bldg. 12, 119992, Moscow, Russia 2Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Miklukho- Maklaya str. 16/10, 117997, Moscow, Russia *Email: [email protected] Received: 06.07.2015 Copyright © 2016 Park-media, Ltd. This is an open access article distributed under the Creative Commons Attribution License,which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. ABSTRACT Modeling of the structure of voltage-gated potassium (KV) channels bound to peptide blockers aims to identify the key amino acid residues dictating affinity and provide insights into the toxin-channel interface. Computational approaches open up possibilities for in silico rational design of selective blockers, new molecular tools to study the cellular distribution and functional roles of potassium channels. It is anticipated that optimized blockers will advance the development of drugs that reduce over activation of potassium channels and attenuate the associated malfunction. Starting with an overview of the recent advances in computational simulation strat- egies to predict the bound state orientations of peptide pore blockers relative to KV-channels, we go on to review algorithms
    [Show full text]
  • A Four-Disulphide-Bridged Toxin, with High Affinity Towards Voltage-Gated
    Biochem. J. (1997) 328, 321–327 (Printed in Great Britain) 321 A four-disulphide-bridged toxin, with high affinity towards voltage-gated K+ channels, isolated from Heterometrus spinnifer (Scorpionidae) venom ! Bruno LEBRUN*1,Regine ROMI-LEBRUN*, Marie-France MARTIN-EAUCLAIRE†, Akikazu YASUDA*, Masaji ISHIGURO*, Yoshiaki OYAMA‡, Olaf PONGS§ and Terumi NAKAJIMA* *Suntory Institute for Bioorganic Research, Mishima-Gun, Shimamoto-Cho, Wakayamadai 1-1-1, 618 Osaka, Japan, †Laboratoire de Biochimie, CNRS UMR 6560, Faculte! de Me! decine Nord, 13916 Marseille Cedex 20, France, ‡Suntory Ltd Institute for Biomedical Research, Mishima-Gun, Shimamoto-Cho, Wakayamadai 1-1-1, 618 Osaka, Japan, and §Zentrum fu$ r Molekulare Neurobiologie, Institute fu$ r Neurale Signalverarbeitung, D-20246 Hamburg, Federal Republic of Germany A new toxin, named HsTX1, has been identified in the venom of limited reduction–alkylation at acidic pH and (2) enzymic Heterometrus spinnifer (Scorpionidae), on the basis of its ability cleavage on an immobilized trypsin cartridge, both followed by to block the rat Kv1.3 channels expressed in Xenopus oocytes. mass and sequence analyses. Three of the disulphide bonds are HsTX1 has been purified and characterized as a 34-residue connected as in the three-disulphide-bridged scorpion toxins, peptide reticulated by four disulphide bridges. HsTX1 shares and the two extra half-cystine residues of HsTX1 are cross- 53% and 59% sequence identity with Pandinus imperator toxin1 linked, as in Pi1. These results, together with those of CD (Pi1) and maurotoxin, two recently isolated four-disulphide- analysis, suggest that HsTX1 probably adopts the same general bridged toxins, whereas it is only 32–47% identical with the folding as all scorpion K+ channel toxins.
    [Show full text]
  • Identification of Aethina Tumida Kir Channels As Putative Targets of The
    toxins Article Identification of Aethina tumida Kir Channels as Putative Targets of the Bee Venom Peptide Tertiapin Using Structure-Based Virtual Screening Methods Craig A. Doupnik Department of Molecular Pharmacology & Physiology, University of South Florida College of Medicine, Tampa, FL 33612, USA; [email protected] Received: 20 August 2019; Accepted: 18 September 2019; Published: 19 September 2019 Abstract: Venoms are comprised of diverse mixtures of proteins, peptides, and small molecules. Identifying individual venom components and their target(s) with mechanism of action is now attainable to understand comprehensively the effectiveness of venom cocktails and how they collectively function in the defense and predation of an organism. Here, structure-based computational methods were used with bioinformatics tools to screen and identify potential biological targets of tertiapin (TPN), a venom peptide from Apis mellifera (European honey bee). The small hive beetle (Aethina tumida (A. tumida)) is a natural predator of the honey bee colony and was found to possess multiple inwardly rectifying K+ (Kir) channel subunit genes from a genomic BLAST search analysis. Structure-based virtual screening of homology modelled A. tumida Kir (atKir) channels found TPN to interact with a docking profile and interface “footprint” equivalent to known TPN-sensitive mammalian Kir channels. The results support the hypothesis that atKir channels, and perhaps other insect Kir channels, are natural biological targets of TPN that help defend the bee colony from infestations by blocking K+ transport via atKir channels. From these in silico findings, this hypothesis can now be subsequently tested in vitro by validating atKir channel block as well as in vivo TPN toxicity towards A.
    [Show full text]
  • Mechanisms of Maurotoxin Action on Shaker Potassium Channels
    CORE Metadata, citation and similar papers at core.ac.uk Provided by Elsevier - Publisher Connector 776 Biophysical Journal Volume 79 August 2000 776–787 Mechanisms of Maurotoxin Action on Shaker Potassium Channels Vladimir Avdonin,* Brian Nolan,* Jean-Marc Sabatier,† Michel De Waard,‡ and Toshinori Hoshi* *Department of Physiology and Biophysics, The University of Iowa, Iowa City, Iowa 52242 USA; and ‡Laboratoire de Neurobiologie des Canaux Ioniques and †Laboratoire de Biochimie, UMR 6560, Faculte´deMe´ decine Nord, Institut Fe´de´ ratif Jean Roche, Institut National de la Sante´ et de la Recherche Me´dicale U464, 13916 Marseille Cedex 20, France ABSTRACT Maurotoxin (␣-KTx6.2) is a toxin derived from the Tunisian chactoid scorpion Scorpio maurus palmatus, and it is a member of a new family of toxins that contain four disulfide bridges (Selisko et al., 1998, Eur. J. Biochem. 254:468–479). We investigated the mechanism of the maurotoxin action on voltage-gated Kϩ channels expressed in Xenopus oocytes. Maurotoxin blocks the channels in a voltage-dependent manner, with its efficacy increasing with greater hyperpolarization. We show that an amino acid residue in the external mouth of the channel pore segment that is known to be involved in the actions of other peptide toxins is also involved in maurotoxin’s interaction with the channel. We conclude that, despite the unusual disulfide bridge pattern, the mechanism of the maurotoxin action is similar to those of other Kϩ channel toxins with only three disulfide bridges. INTRODUCTION Scorpion toxins constitute the largest group of potassium tion pore (MacKinnon and Miller, 1988). The toxin associ- channel toxins.
    [Show full text]