Skip to main content
NCBI home page
Biochemical Journal logoLink to Biochemical Journal
. 1997 Nov 15;328(Pt 1):321–327. doi: 10.1042/bj3280321

A four-disulphide-bridged toxin, with high affinity towards voltage-gated K+ channels, isolated from Heterometrus spinnifer (Scorpionidae) venom.

B Lebrun 1, R Romi-Lebrun 1, M F Martin-Eauclaire 1, A Yasuda 1, M Ishiguro 1, Y Oyama 1, O Pongs 1, T Nakajima 1
PMCID: PMC1218924  PMID: 9359871

Abstract

A new toxin, named HsTX1, has been identified in the venom of Heterometrus spinnifer (Scorpionidae), on the basis of its ability to block the rat Kv1.3 channels expressed in Xenopus oocytes. HsTX1 has been purified and characterized as a 34-residue peptide reticulated by four disulphide bridges. HsTX1 shares 53% and 59% sequence identity with Pandinus imperator toxin1 (Pi1) and maurotoxin, two recently isolated four-disulphide-bridged toxins, whereas it is only 32-47% identical with the other scorpion K+ channel toxins, reticulated by three disulphide bridges. The amidated and carboxylated forms of HsTX1 were synthesized chemically, and identity between the natural and the synthetic amidated peptides was proved by mass spectrometry, co-elution on C18 HPLC and blocking activity on the rat Kv1.3 channels. The disulphide bridge pattern was studied by (1) limited reduction-alkylation at acidic pH and (2) enzymic cleavage on an immobilized trypsin cartridge, both followed by mass and sequence analyses. Three of the disulphide bonds are connected as in the three-disulphide-bridged scorpion toxins, and the two extra half-cystine residues of HsTX1 are cross-linked, as in Pi1. These results, together with those of CD analysis, suggest that HsTX1 probably adopts the same general folding as all scorpion K+ channel toxins. HsTX1 is a potent inhibitor of the rat Kv1.3 channels (IC50 approx. 12 pM). HsTX1 does not compete with 125I-apamin for binding to its receptor site on rat brain synaptosomal membranes, but competes efficiently with 125I-kaliotoxin for binding to the voltage-gated K+ channels on the same preparation (IC50 approx. 1 pM).

Full Text

The Full Text of this article is available as a PDF (611.5 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Aiyar J., Rizzi J. P., Gutman G. A., Chandy K. G. The signature sequence of voltage-gated potassium channels projects into the external vestibule. J Biol Chem. 1996 Dec 6;271(49):31013–31016. doi: 10.1074/jbc.271.49.31013. [DOI] [PubMed] [Google Scholar]
  2. Aiyar J., Withka J. M., Rizzi J. P., Singleton D. H., Andrews G. C., Lin W., Boyd J., Hanson D. C., Simon M., Dethlefs B. Topology of the pore-region of a K+ channel revealed by the NMR-derived structures of scorpion toxins. Neuron. 1995 Nov;15(5):1169–1181. doi: 10.1016/0896-6273(95)90104-3. [DOI] [PubMed] [Google Scholar]
  3. Auguste P., Hugues M., Gravé B., Gesquière J. C., Maes P., Tartar A., Romey G., Schweitz H., Lazdunski M. Leiurotoxin I (scyllatoxin), a peptide ligand for Ca2(+)-activated K+ channels. Chemical synthesis, radiolabeling, and receptor characterization. J Biol Chem. 1990 Mar 15;265(8):4753–4759. [PubMed] [Google Scholar]
  4. Bontems F., Roumestand C., Gilquin B., Ménez A., Toma F. Refined structure of charybdotoxin: common motifs in scorpion toxins and insect defensins. Science. 1991 Dec 6;254(5037):1521–1523. doi: 10.1126/science.1720574. [DOI] [PubMed] [Google Scholar]
  5. Campos-Olivas R., Bruix M., Santoro J., Lacadena J., Martinez del Pozo A., Gavilanes J. G., Rico M. NMR solution structure of the antifungal protein from Aspergillus giganteus: evidence for cysteine pairing isomerism. Biochemistry. 1995 Mar 7;34(9):3009–3021. doi: 10.1021/bi00009a032. [DOI] [PubMed] [Google Scholar]
  6. Compton L. A., Johnson W. C., Jr Analysis of protein circular dichroism spectra for secondary structure using a simple matrix multiplication. Anal Biochem. 1986 May 15;155(1):155–167. doi: 10.1016/0003-2697(86)90241-1. [DOI] [PubMed] [Google Scholar]
  7. Crest M., Jacquet G., Gola M., Zerrouk H., Benslimane A., Rochat H., Mansuelle P., Martin-Eauclaire M. F. Kaliotoxin, a novel peptidyl inhibitor of neuronal BK-type Ca(2+)-activated K+ channels characterized from Androctonus mauretanicus mauretanicus venom. J Biol Chem. 1992 Jan 25;267(3):1640–1647. [PubMed] [Google Scholar]
  8. Dauplais M., Gilquin B., Possani L. D., Gurrola-Briones G., Roumestand C., Ménez A. Determination of the three-dimensional solution structure of noxiustoxin: analysis of structural differences with related short-chain scorpion toxins. Biochemistry. 1995 Dec 26;34(51):16563–16573. doi: 10.1021/bi00051a004. [DOI] [PubMed] [Google Scholar]
  9. Delepierre M., Prochnicka-Chalufour A., Possani L. D. A novel potassium channel blocking toxin from the scorpion Pandinus imperator: A 1H NMR analysis using a nano-NMR probe. Biochemistry. 1997 Mar 4;36(9):2649–2658. doi: 10.1021/bi9617116. [DOI] [PubMed] [Google Scholar]
  10. GRAY E. G., WHITTAKER V. P. The isolation of nerve endings from brain: an electron-microscopic study of cell fragments derived by homogenization and centrifugation. J Anat. 1962 Jan;96:79–88. [PMC free article] [PubMed] [Google Scholar]
  11. Galvez A., Gimenez-Gallego G., Reuben J. P., Roy-Contancin L., Feigenbaum P., Kaczorowski G. J., Garcia M. L. Purification and characterization of a unique, potent, peptidyl probe for the high conductance calcium-activated potassium channel from venom of the scorpion Buthus tamulus. J Biol Chem. 1990 Jul 5;265(19):11083–11090. [PubMed] [Google Scholar]
  12. Garcia-Calvo M., Leonard R. J., Novick J., Stevens S. P., Schmalhofer W., Kaczorowski G. J., Garcia M. L. Purification, characterization, and biosynthesis of margatoxin, a component of Centruroides margaritatus venom that selectively inhibits voltage-dependent potassium channels. J Biol Chem. 1993 Sep 5;268(25):18866–18874. [PubMed] [Google Scholar]
  13. Garcia M. L., Garcia-Calvo M., Hidalgo P., Lee A., MacKinnon R. Purification and characterization of three inhibitors of voltage-dependent K+ channels from Leiurus quinquestriatus var. hebraeus venom. Biochemistry. 1994 Jun 7;33(22):6834–6839. doi: 10.1021/bi00188a012. [DOI] [PubMed] [Google Scholar]
  14. Giangiacomo K. M., Sugg E. E., Garcia-Calvo M., Leonard R. J., McManus O. B., Kaczorowski G. J., Garcia M. L. Synthetic charybdotoxin-iberiotoxin chimeric peptides define toxin binding sites on calcium-activated and voltage-dependent potassium channels. Biochemistry. 1993 Mar 9;32(9):2363–2370. doi: 10.1021/bi00060a030. [DOI] [PubMed] [Google Scholar]
  15. Grissmer S., Nguyen A. N., Aiyar J., Hanson D. C., Mather R. J., Gutman G. A., Karmilowicz M. J., Auperin D. D., Chandy K. G. Pharmacological characterization of five cloned voltage-gated K+ channels, types Kv1.1, 1.2, 1.3, 1.5, and 3.1, stably expressed in mammalian cell lines. Mol Pharmacol. 1994 Jun;45(6):1227–1234. [PubMed] [Google Scholar]
  16. Gómez-Lagunas F., Olamendi-Portugal T., Possani L. D. Block of ShakerB K+ channels by Pi1, a novel class of scorpion toxin. FEBS Lett. 1997 Jan 3;400(2):197–200. doi: 10.1016/s0014-5793(96)01387-7. [DOI] [PubMed] [Google Scholar]
  17. Jones A., Bingham J. P., Gehrmann J., Bond T., Loughnan M., Atkins A., Lewis R. J., Alewood P. F. Isolation and characterization of conopeptides by high-performance liquid chromatography combined with mass spectrometry and tandem mass spectrometry. Rapid Commun Mass Spectrom. 1996;10(1):138–143. doi: 10.1002/(SICI)1097-0231(19960115)10:1<138::AID-RCM442>3.0.CO;2-3. [DOI] [PubMed] [Google Scholar]
  18. Kharrat R., Mabrouk K., Crest M., Darbon H., Oughideni R., Martin-Eauclaire M. F., Jacquet G., el Ayeb M., Van Rietschoten J., Rochat H. Chemical synthesis and characterization of maurotoxin, a short scorpion toxin with four disulfide bridges that acts on K+ channels. Eur J Biochem. 1996 Dec 15;242(3):491–498. doi: 10.1111/j.1432-1033.1996.0491r.x. [DOI] [PubMed] [Google Scholar]
  19. Laraba-Djebari F., Legros C., Crest M., Céard B., Romi R., Mansuelle P., Jacquet G., van Rietschoten J., Gola M., Rochat H. The kaliotoxin family enlarged. Purification, characterization, and precursor nucleotide sequence of KTX2 from Androctonus australis venom. J Biol Chem. 1994 Dec 30;269(52):32835–32843. [PubMed] [Google Scholar]
  20. Lucchesi K., Ravindran A., Young H., Moczydlowski E. Analysis of the blocking activity of charybdotoxin homologs and iodinated derivatives against Ca2+-activated K+ channels. J Membr Biol. 1989 Aug;109(3):269–281. doi: 10.1007/BF01870284. [DOI] [PubMed] [Google Scholar]
  21. Martin B. M., Ramirez A. N., Gurrola G. B., Nobile M., Prestipino G., Possani L. D. Novel K(+)-channel-blocking toxins from the venom of the scorpion Centruroides limpidus limpidus Karsch. Biochem J. 1994 Nov 15;304(Pt 1):51–56. doi: 10.1042/bj3040051. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Miller C., Moczydlowski E., Latorre R., Phillips M. Charybdotoxin, a protein inhibitor of single Ca2+-activated K+ channels from mammalian skeletal muscle. Nature. 1985 Jan 24;313(6000):316–318. doi: 10.1038/313316a0. [DOI] [PubMed] [Google Scholar]
  23. Miller C. The charybdotoxin family of K+ channel-blocking peptides. Neuron. 1995 Jul;15(1):5–10. doi: 10.1016/0896-6273(95)90057-8. [DOI] [PubMed] [Google Scholar]
  24. Olamendi-Portugal T., Gómez-Lagunas F., Gurrola G. B., Possani L. D. A novel structural class of K+-channel blocking toxin from the scorpion Pandinus imperator. Biochem J. 1996 May 1;315(Pt 3):977–981. doi: 10.1042/bj3150977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Park C. S., Miller C. Mapping function to structure in a channel-blocking peptide: electrostatic mutants of charybdotoxin. Biochemistry. 1992 Sep 1;31(34):7749–7755. doi: 10.1021/bi00149a002. [DOI] [PubMed] [Google Scholar]
  26. Ranganathan R., Lewis J. H., MacKinnon R. Spatial localization of the K+ channel selectivity filter by mutant cycle-based structure analysis. Neuron. 1996 Jan;16(1):131–139. doi: 10.1016/s0896-6273(00)80030-6. [DOI] [PubMed] [Google Scholar]
  27. Rogowski R. S., Collins J. H., O'Neill T. J., Gustafson T. A., Werkman T. R., Rogawski M. A., Tenenholz T. C., Weber D. J., Blaustein M. P. Three new toxins from the scorpion Pandinus imperator selectively block certain voltage-gated K+ channels. Mol Pharmacol. 1996 Nov;50(5):1167–1177. [PubMed] [Google Scholar]
  28. Romi-Lebrun R., Martin-Eauclaire M. F., Escoubas P., Wu F. Q., Lebrun B., Hisada M., Nakajima T. Characterization of four toxins from Buthus martensi scorpion venom, which act on apamin-sensitive Ca2+-activated K+ channels. Eur J Biochem. 1997 Apr 15;245(2):457–464. doi: 10.1111/j.1432-1033.1997.00457.x. [DOI] [PubMed] [Google Scholar]
  29. Romi R., Crest M., Gola M., Sampieri F., Jacquet G., Zerrouk H., Mansuelle P., Sorokine O., Van Dorsselaer A., Rochat H. Synthesis and characterization of kaliotoxin. Is the 26-32 sequence essential for potassium channel recognition? J Biol Chem. 1993 Dec 15;268(35):26302–26309. [PubMed] [Google Scholar]
  30. Sabatier J. M., Zerrouk H., Darbon H., Mabrouk K., Benslimane A., Rochat H., Martin-Eauclaire M. F., Van Rietschoten J. P05, a new leiurotoxin I-like scorpion toxin: synthesis and structure-activity relationships of the alpha-amidated analog, a ligand of Ca(2+)-activated K+ channels with increased affinity. Biochemistry. 1993 Mar 23;32(11):2763–2770. doi: 10.1021/bi00062a005. [DOI] [PubMed] [Google Scholar]
  31. Seagar M. J., Granier C., Couraud F. Interactions of the neurotoxin apamin with a Ca2+-activated K+ channel in primary neuronal cultures. J Biol Chem. 1984 Feb 10;259(3):1491–1495. [PubMed] [Google Scholar]
  32. Stampe P., Kolmakova-Partensky L., Miller C. Intimations of K+ channel structure from a complete functional map of the molecular surface of charybdotoxin. Biochemistry. 1994 Jan 18;33(2):443–450. doi: 10.1021/bi00168a008. [DOI] [PubMed] [Google Scholar]
  33. Stühmer W., Ruppersberg J. P., Schröter K. H., Sakmann B., Stocker M., Giese K. P., Perschke A., Baumann A., Pongs O. Molecular basis of functional diversity of voltage-gated potassium channels in mammalian brain. EMBO J. 1989 Nov;8(11):3235–3244. doi: 10.1002/j.1460-2075.1989.tb08483.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Sugg E. E., Garcia M. L., Reuben J. P., Patchett A. A., Kaczorowski G. J. Synthesis and structural characterization of charybdotoxin, a potent peptidyl inhibitor of the high conductance Ca2(+)-activated K+ channel. J Biol Chem. 1990 Nov 5;265(31):18745–18748. [PubMed] [Google Scholar]
  35. Vincent J. P., Schweitz H., Lazdunski M. Structure-function relationships and site of action of apamin, a neurotoxic polypeptide of bee venom with an action on the central nervous system. Biochemistry. 1975 Jun 3;14(11):2521–2525. doi: 10.1021/bi00682a035. [DOI] [PubMed] [Google Scholar]
  36. Wemmer D., Kallenbach N. R. Structure of apamin in solution: a two-dimensional nuclear magnetic resonance study. Biochemistry. 1983 Apr 12;22(8):1901–1906. doi: 10.1021/bi00277a025. [DOI] [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES