Isolation, structure, and activity of GID, a novel alpha 4/7-conotoxin with an extended N-terminal sequence.
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.
Nicke
Annette
A
Institute for Molecular Bioscience and School of Biomedical Sciences, University of Queensland, Brisbane, Queensland 4072, Australia.
Loughnan
Marion L
ML
Millard
Emma L
EL
Alewood
Paul F
PF
Adams
David J
DJ
Daly
Norelle L
NL
Craik
David J
DJ
Lewis
Richard J
RJ
D000595:Amino Acid Sequence; D000818:Animals; D020916:Conotoxins / Q000737:chemistry* / Q000302:isolation & purification / Q000494:pharmacology*; D005260:Female; D008958:Models, Molecular; D008969:Molecular Sequence Data; D008974:Mollusca; D018733:Nicotinic Antagonists / Q000494:pharmacology*; D009865:Oocytes / Q000187:drug effects / Q000502:physiology; D018408:Patch-Clamp Techniques; D010446:Peptide Fragments / Q000737:chemistry; D011487:Protein Conformation; D021122:Protein Subunits; D011978:Receptors, Nicotinic / Q000187:drug effects / Q000502:physiology*; D014982:Xenopus laevis
D020916:Conotoxins; D018733:Nicotinic Antagonists; D010446:Peptide Fragments; D021122:Protein Subunits; D011978:Receptors, Nicotinic; C472561:alpha-conotoxin GID, Conus geographus