Inactivation of a serotonin-gated ion channel by a polypeptide toxin from marine snails

Science

Volumn 281, Issue 5376, 1998, Pages 575-578

  England, Laura J ^a   Imperial, Julita ^a   Jacobsen, Richard ^a   Craig, A Grey ^a   Gulyas, Joseph ^a   Akhtar, Mateen ^a   Rivier, Jean ^a   Julius, David ^a   Olivera, Baldomero M ^a  

^a NONE

Author keywords

[No Author keywords available]

Indexed keywords

1 METHYL 3 INDOLECARBOXYLIC ACID [1 [2 (METHYLSULFONYLAMINO)ETHYL] 4 PIPERIDINYLMETHYL] ESTER; CONOTOXIN; GUANINE NUCLEOTIDE BINDING PROTEIN; ION CHANNEL; MARINE TOXIN; POLYPEPTIDE; SEROTONIN 3 RECEPTOR; SEROTONIN ANTAGONIST; SIGMA CONOTOXIN GVIIA; TRITIUM; UNCLASSIFIED DRUG; ZACOPRIDE;

ANIMAL CELL; ARTICLE; CONTROLLED STUDY; MARINE ENVIRONMENT; NEUROMUSCULAR BLOCKING; NEUROTOXICITY; NEUROTRANSMITTER RELEASE; NONHUMAN; OOCYTE; PRIORITY JOURNAL; SEROTONINERGIC TRANSMISSION; SNAIL; TOXIN ANALYSIS; XENOPUS;

AMINO ACID SEQUENCE; AMINO ACIDS; ANIMALS; BENZAMIDES; BICYCLO COMPOUNDS, HETEROCYCLIC; BINDING SITES; CELL LINE; CLONING, MOLECULAR; CONOTOXINS; DNA, COMPLEMENTARY; ION CHANNEL GATING; ION CHANNELS; MOLECULAR SEQUENCE DATA; MOLLUSK VENOMS; PEPTIDES, CYCLIC; RECEPTORS, SEROTONIN; RECEPTORS, SEROTONIN, 5-HT3; RECEPTORS, SEROTONIN, 5-HT4; RECOMBINANT FUSION PROTEINS; RECOMBINANT PROTEINS; SEROTONIN; SEROTONIN ANTAGONISTS; SNAILS; TRYPTOPHAN;

ANIMALIA; GASTROPODA; MAMMALIA; TRITIUM;

EID: 0032563252     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.281.5376.575     Document Type: Article

References (32)

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13. Crude C. geographus venom was collected from dissected ducts, lyophilized immediately, and stored at -70°C. Lyophilized crude venom (360 mg) was resuspended in 30 ml of 40% acetonitrile with 0.5% trifluoroacetic acid (TFA), vortexed, and sonicated four times for 30 s, with brief cooling intervals. The extract was clarified by centrifugation before HPLC purification. Venom extract was diluted 20-fold with 0.1% TFA and fractionated on a Vydac C18 preparative column (2.5 cm by 25 cm), with a guard column (22 mm by 50.8 mm) using 1000 ml of 0.1% TFA in a linear gradient from 0% to approximately 90% acetonitrile (flow rate of 20 ml/min). The active fraction was further fractionated on a Vydac C18 semipreparative column (10 mm by 250 mm) using 150 ml of 0.1% TFA with a linear gradient of 4.5 to 31.0% acetonitrile (a 0.9% increase in acetonitrile per minute at a flow rate of 5 ml/min). The final purification step was done on a Vydac C18 analytical column (4.6 mm by 250 mm) using 50 ml of 0.1% TFA with a linear gradient of 13.5 to 27.0% acetonitrile (a 0.45% increase in acetonitrile per min at a flow rate of 1 ml/min). For functional studies, purified (σ-conotoxin was stored in 50 mM tris-Cl (pH 7.6) with bovine gamma globulin (0.2 mg/ml), which had no effect on electrophysiological or radioligand binding assays.
14. Approximately 1 nmol of peptide in 70 μl of HPLC elution buffer was treated with dithiothreitol (DTT) (9 mM final concentration) after the pH was raised to 7.5 with 1.0 M tris-base. Argon was bubbled through the solution, and the reaction was incubated at 65°C for 15 min. After cooling to room temperature, 4 μl of 20% 4-vinyl pyridine in ethanol was added, and the reaction was incubated in the dark for 25 min at room temperature. The reaction was diluted fivefold with 0.1% TFA, and the modified peptide was isolated by HPLC using a Vydac C18 analytical column (4.6 mm by 250 mm) with a linear gradient of 2 to 42% acetonitrile in the presence of 0.1% TFA. The sequence was determined with Edman chemistry (Applied Biosystems model 477A Protein Sequencer, Protein/DNA Core Facility, University of Utah Cancer Center), with the exception of a blank cycle at position 34.
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19. 3CN + 0.05% TFA; flow rate, 200 ml/min).
20. L J. England et al., data not shown.
21. 3 chimera, n = 16).
22. 3 chimera, n = 16).
23. 3 chimera, n = 16).
24. 3 chimera, n = 16).
25. 3 chimera, n = 16).
26. 3 chimera, n = 16).
27. 3 chimera, n = 16).
28. i value of 4.8 ± 0.3 nM as described [Y. C. Cheng and W. H. Prusoff, Biochem. Pharmacol. 22, 3099
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32. 4 binding assays; J. Boulter for providing cDNA clones; and A. Brake, W. Lim, R. Nicoll, and K. Yamamoto for critical reading of the manuscript. This work was supported by NIH grants GM44298 (D.J.) and GM48677 (B.M.O.); NSF Major Research Instrumentation Program grant DDBT-972450 (A.G.C.); the Foundation for Medical Research (A.G.C.); the McKnight Foundation for Neuroscience (D.J.); and postdoctoral fellowships from the American Heart Association, California Affiliate, and the California Tobacco-Related Disease Program, grant number 6FT-0103 (L.J.E.).