Isolation, structure, and activity of GID, a novel alpha 4/7-conotoxin with an extended N-terminal sequence

• Published in: The Journal of biological chemistry Vol. 278; no. 5; p. 3137
• Main Authors: Nicke, Annette, Loughnan, Marion L, Millard, Emma L, Alewood, Paul F, Adams, David J, Daly, Norelle L, Craik, David J, Lewis, Richard J
• Format: Journal Article
• Language: English
• Published: United States 31.01.2003
• Subjects: Amino Acid Sequence; Animals; Conotoxins - chemistry; Conotoxins - isolation & purification; Conotoxins - pharmacology; Female; Models, Molecular; Molecular Sequence Data; Mollusca; Nicotinic Antagonists - pharmacology; Oocytes - drug effects; Oocytes - physiology; Patch-Clamp Techniques; Peptide Fragments - chemistry; Protein Conformation; Protein Subunits; Receptors, Nicotinic - drug effects; Receptors, Nicotinic - physiology; Xenopus laevis
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M210280200

Using assay-directed fractionation of Conus geographus crude venom, we isolated alpha-conotoxin GID, which acts selectively at neuronal nicotinic acetylcholine receptors (nAChRs). Unlike other neuronally selective alpha-conotoxins, alpha-GID has a four amino acid N-terminal tail, gamma-carboxyglutamate (Gla), and hydroxyproline (O) residues, and lacks an amidated C terminus. GID inhibits alpha 7 and alpha 3 beta 2 nAChRs with IC(50) values of 5 and 3 nm, respectively and is at least 1000-fold less potent at the alpha 1 beta 1 gamma delta, alpha 3 beta 4, and alpha 4 beta 4 combinations. GID also potently inhibits the alpha 4 beta 2 subtype (IC(50) of 150 nm). Deletion of the N-terminal sequence (GID Delta 1-4) significantly decreased activity at the alpha 4 beta 2 nAChR but hardly affected potency at alpha 3 beta 2 and alpha 7 nAChRs, despite enhancing the off-rates at these receptors. In contrast, Arg(12) contributed to alpha 4 beta 2 and alpha 7 activity but not to alpha 3 beta 2 activity. The three-dimensional structure of GID is well defined over residues 4-19 with a similar motif to other alpha-conotoxins. However, despite its influence on activity, the tail appears to be disordered in solution. Comparison of GID with other alpha 4/7-conotoxins which possess an NN(P/O) motif in loop II, revealed a correlation between increasing length of the aliphatic side-chain in position 10 (equivalent to 13 in GID) and greater alpha 7 versus alpha 3 beta 2 selectivity.