Abstract
Many venomous organisms produce toxins that disrupt neuromuscular communication to paralyze their prey. One common class of such toxins comprises nicotinic acetylcholine receptor antagonists (nAChRs). Thus, most toxins that act on nAChRs are targeted to the neuromuscular subtype. The toxin characterized in this report, alpha-conotoxin GIC, is a most striking exception. The 16-amino acid peptide was identified from a genomic DNA clone from Conus geographus. The predicted mature toxin was synthesized, and synthetic toxin was used in all studies described. alpha-Conotoxin GIC shows no paralytic activity in fish or mice. Furthermore, even at concentrations up to 100 microm, the peptide has no detectable effect on the human muscle nicotinic receptor subtype heterologously expressed in Xenopus oocytes. In contrast, the toxin has high affinity (IC(50) approximately 1.1 nm) for the human alpha3beta2 subunit combination, making it the most neuronally selective nicotinic antagonist characterized thus far. Although alpha-conotoxin GIC shares some sequence similarity with alpha-conotoxin MII, which is also a potent alpha3beta2 nicotinic antagonist, it is much less hydrophobic, and the kinetics of channel block are substantially different. It is noteworthy that the nicotinic ligands in C. geographus venom fit an emerging pattern in venomous predators, with one nicotinic antagonist targeted to the muscle subtype (thereby causing paralysis) and a second nicotinic antagonist targeted to the alpha3beta2 nAChR subtype (possibly inhibiting the fight-or-flight response).
Highlights
Many organisms employ toxins that act on nAChRs1 to defend against predators or to facilitate prey capture
It is noteworthy that the nicotinic ligands in C. geographus venom fit an emerging pattern in venomous predators, with one nicotinic antagonist targeted to the muscle subtype and a second nicotinic antagonist targeted to the ␣32 nicotinic acetylcholine receptor antagonists (nAChRs) subtype
In the case of the ␣-conotoxins, this prepropeptide is ϳ60 amino acids, with the mature ␣-conotoxin sequence of 13–18 amino acids located at the C terminus of the precursor
Summary
Identification and Sequencing of a Genomic DNA Clone Encoding ␣-Conotoxin GIC—DNA from C. geographus hepatopancreas was isolated using the Puregene DNA Isolation Kit (Gentra Systems, Minneapolis, MN). The nucleic acid sequences of the resulting clones were determined according to the standard protocol for the Sequenase version 2.0 DNA sequencing kit as described previously [13]. The monocyclic peptide and HPLC eluent was dripped into an equal volume of iodine (10 mM) in H2O: trifluoroacetic acid:acetonitrile (78:2:20 by volume) and allowed to react for 10 min. To apply a pulse of ACh to the oocytes, the perfusion fluid was switched to one containing ACh for 1 s. This was done automatically at intervals of 1–5 min. All ACh pulses contain no toxin, for it was assumed that little if any bound toxin washed away in the brief time (Ͻ2 s for the responses to peak). Mice were observed for symptoms of paralysis, and death time was determined by monitoring heartbeat with a stethoscope
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