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.