Amino Acid Sequence Determination and Chemical Synthesis of CllErg1 (γ-KTx1.5), a K+ Channel Blocker Peptide Isolated from the Scorpion Centruroides limpidus limpidus
Keywords:
ERG, Centruroides limpidus limpidus, chemical synthesis, K -channel, scorpion toxinAbstract
Abstract. A novel toxin named CllErg1 (systematic nomenclature γ-KTx1.5) was purified from the venom of the scorpion Centruroides limpidus limpidus and its amino acid sequence was determined. It has 42 amino-acid residues cross-linked by four disulfide bridges and blocks specifically a potassium channel of the family ether-a-go-go (ERG). The full peptide was chemically synthesized and properly folded, showing that it blocks the human ERG-channels (HERG) with identical affinity to that of the native peptide. Synthetic CllErg1 can be produced in quantities enough to compensate its low concentration in the natural venom. It paves the way to conduct studies aimed at the identification of the structural motifs of HERG critical for proper channel function. Additionally, another analogous peptide CllErg2 (systematic name γ-KTx4.1) was purified and had its full amino acid sequence determined. It contained 43 amino acid residues, maintained closely packed by four disulfide bridge.
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References
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2. Hill, B.; Ionic Channels of Excitable Membranes, Sinauer Associates Inc.: USA, 1992.
3. Abbreviations: a.m.u., atomic mass units; C.l. limpidus, Centruroides limpidus limpidus; ERG, ether-a-go-go potassium channel family; HERG, human potassium ERG channel; IKr, rapid delayed rectifier potassium current; QT, time interval on the surface electrocardiogram; Boc, ter-butyloxycarbonyl; Bocamino acid-Pam-Resin, Boc-aminoacyl-4-(oxymethyl)-phenacetamidomethyl-resin; DCM, dichoromethane; DIEA, diisopropylethylamine; DMF, N,N-dimethyformamide; HBTU,
2-(1-H-benzotriazol-1-yl)-1,1,3,3-tetramethyl-uronium hexafluorophosphate; HF, hydrofluoric acid; HPLC, high performance liquid chromatography; TFA, trifluoroacetic acid.
4. QT: the duration of QT interval (reviewed in Keating, M. T.; Sanguinetti, M. C.; Cell 2001, 104, 569) is a measure of the time required for depolarization and repolarisation of the heart. Prolongation of the QT interval significantly increases the risk of ventricular arrhythmia and in particular an arrhythmia known as “torsade de pointes”, which causes syncope and, if it persists, sudden death.
5. Sanguinetti, M. C.; Jiang, C.; Curran, M. E.; Keating, M. T.; Cell 1995, 81, 299.
6. Trudeau, M. C.; Warmke, J. W.; Ganetzky, B.; Robertson, G. A.; Science 1995, 269, 92.
7. Curran, M. E.; Splawski, I.; Timothy, K. W.; Vincent, G. M.; Green, E. D.; Keating, M.T.; Cell 1995, 80, 795.
8. Roden, D. M.; Lazzara, R.; Rosen, M.; Schwartz, P. J.; Towbin, J.; Vincent, M. G.; Circulation 1996, 94, 1996.
9. Schwartz, P. J.; Priori, S. G.; Spazzolini, C.; Moss, A. J.; Vincent, G. M.; Napolitano, C.; Denjoy, I.; Guicheney, P.; Breithardt, G.; Keating, M. T.; Towbin, J. A.; Beggs, A. H.; Brink, P.; Wilde, A. A.; Toivonen, L.; Zareba, W.; Robinson, J. L.; Timothy, K. W.; Corfield, V.; Wattanasirichaigoon, D.; Corbett, C.; Haverkamp, W.; Schulze-Bahr, E.; Lehmann, M. H.; Schwartz, K.; Coumel, P.; Bloise, R.; Circulation 2001,103,89.
10. Emmi, A.; Wenzel, H. J.; Schwartzroin, P. A.; Taglialatela, M.; Castaldo, P.; Biandi, L.; Nerbonne, J.; Robertson, G. A.; J. Neurosci. 2001, 20, 3915.
11. Rosati, B.; Manchetti, P.; Crociani, O.; Lecchi, M.; Lapi, R.; Arcangeli, A.; Olivotto, M.; Wanke, E.; FASEB J. 2000, 14, 2601
12. Gullo, F.; Ales, E.; Rosati, B.; Lecchi, M.; Masi, A.; Guasti, L.; Cano-Abad, M. F.; Arcangeli, A.; López, M. G.; Wanke, E.; FASEB J. 2003, 17, 330.
13. Shoeb, F.; Malykhina, A. P.; Akbarali, H. I.; J. Biol. Chem. 2003, 278, 2503.
14. Tseng, G. N.; J. Mol. Cell Cardiol. 2001, 33, 835.
15. Vandenberg, J. I.; Torres, A.; Campbell, T. J.; Kuchel, P. W.; Eur. Biophys. J. 2004, 33, 89.
16. Possani, L. D.; Becerril, B.; Tytgat, J.; Delepierre, M. In Ion Channel Localization; Lopatin, A.N.; Nichols, C.G., eds., Human Press: Towa, 2001, p. 145.
17. Rodríguez de la Vega, R. C.; Possani, L. D.; Toxicon 2004, 43, 865.
18. Carbone, E.; Wanke, E.; Prestipino, G.; Possani, L. D.; Maelicke, A.; Nature 1982, 296, 90.
19. Park, C. S.; Miller, C.; Neuron 1992, 9, 307.
20. MacKinnon, R.; Aldrich, R. W.; Lee, A. W.; Science 1993, 262, 757.
21. Hidalgo, P.; MacKinnon, R.; Science 1995, 268, 307
22. Garcia, M. L.; Gao, Ying-Duo; McManus, O. B.; Kaczorowski, G. J.; Toxicon 2001, 39, 739.
23. Gurrola, G. B.; Rosati, B.; Rocchetti, M.; Pimienta, G.; Zaza, A.; Arcangeli, A.; Olivotto, M.; Possani, L. D.; Wanke, E.; FASEB J. 1999, 13, 953.
24. Scaloni, A.; Bottigleri, C.; Ferrara, L.; Corona, M.; Gurrola, G. B.; Batista, C.; Wanke, E.; Possani, L. D.; FEBS Lett. 2000, 479, 156.
25. Tytgat, J.; Chandy, K. G.; García, M. L.; Gutman, G. A.; Martin-Eauclaire, M. F.; van der Walt, J. J.; Possani, L. D.; Trends Pharmacol. Sci. 1999, 40, 444.
26. Corona, M.; Gurrola, G. B.; Merino, E.; Restano Cassulini, R.; Valdez-Cruz, N. A.; García, B.; Ramírez-Dominguez, M. E.; Coronas, F. I. V.; Zamudio, F. Z.; Wanke, E.; Possani, L. D.; FEBS Lett. 2002, 532, 121.
27. Korolkova, Y. V.; Kozlov, S. A.; Lipkin, A. V.; Pluzhnikov, K. A.; Hadley, J. K.; Filippov, A. K.; Brown, D. A.; Angelo, K.; Strobak, D.; Jespersen, T.; Olsen, S. P.; Jensen, B. S.; Grishin, E. V.; J. Biol. Chem. 2001, 276, 9868.
28. Nastainczyk, W.; Meves, H.; Watt, D. D.; Toxicon 2002, 40, 1053.
29. Lecchi, M.; Redaelli, E.; Rosati, B.; Gurrola, G. B.; Florio, T.; Crociani, O.; Curia, G.; Restano Cassulini, R.; Masi, A.; Arcangeli, A.; Olivotto, M.; Schettini, G.; Possani, L. D.; Wanke
E.; J. Neurosci. 2002, 22, 3414.
30. Pardo-López, L.; García-Valdez, J.; Gurrola, G. B.; Robertson, G. A.; Posssani, L.D.; FEBS Lett. 2002, 510, 45.
31. Pardo-López, L.; Zhang, M.; Liu, J.; Jiang, M.; Possani, L. D.; Tseng, G. N.; J. Biol. Chem. 2002, 277, 16403.
32. Torres, A. M.; Bansal, P.; Alewood, P. F.; Bursill, J. A.; Kuchel, P. W.; Vandenberg, J. L.; FEBS Lett. 2003, 539,38.
33. Frenal, K.; Xu, C-Q.; Wolff, N.; Wecker, K.; Gurrola, G. B.; Zhu, S-Y.; Possani, L. D.; Tytgat, J.; Delepierre, M.; Proteins: Struct. Funct. Genet. 2004, 56, 367.
34. Korolkova, Y. V.; Bocharov, E.V.; Angelo, K.; Maslennikov, I. V.; Grinenko, O. V.; Lipkin, A. V.; Nosyreva, E. D.; Pluzhnikov, K. A.; Olesen, S. P.; Arseniev, A. S.; Grishin, E. V.; J. Biol. Chem. 2002, 277, 43104.
35. Zhang, M.; Korolkova, Y. V.; Liu, J.; Jiang, M.; Grishing E. V.; Tseng, G. N.; Biophysical J. 2003, 84, 30.
36. Korolkova, Y.V.; Tseng, G. N.; Grishin, E. V.; J. Mol. Recogni. 2004, 17, 209.
37. Ramírez, A. N.; Martín, B. M.; Gurrola, G. B.; Possani, L. D.; Toxicon 1994, 32 479.
38. Torres-Larios, A.; Gurrola, G. B.; Zamudio, F. Z.; Possani, L. D.; Eur. J. Biochem. 2000, 267, 5023.
39. Andrews, P. C.; Dixon, J. E.; Anal. Biochem. 1987, 161, 524.
40. Merrifield, B. R.; J. Am. Chem. Soc. 1963, 85, 2144.
41. Schnölzer, M.; Alewood, P.; Jones, A.; Alewood, D.; Kent, S. B. H.; Int. J. Peptide Protein Res. 1992, 40, 180.
42. Mitchell, A. R.; Kent, S. B. H.; Engelhard, M.; Merrifield, B. R.; J. Org. Chem. 1978, 43, 2845.
43. Sarin, V. K; Kent, S. B. H.; Tam, J. P.; Merrifield, B. R.; Anal. Biochem. 1981, 117, 147.
44. Hesberg, I.M.; Trudeau, M.C.; Robertson, G. A.; J. Physiol. 1998, 511, 3.
45. Altamirano, M.; García, C.; Possani, L.D.; Fersht, A.R.; Nat. Biotechnol. 1999, 17, 187.
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