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An impressive biodiversity (>10,000 species) of marine snails (suborder Toxoglossa or superfamily Conoidea) have complex venoms, containing ca. 100 biologically active, disulfide-rich peptides. In the genus Conus, the most intensively investigated toxoglossan lineage (~500 species), a small set of venom gene superfamilies undergo rapid sequence hyperdiversification within their mature toxin regions. Each major lineage of Toxoglossa has its own distinct set of venom gene superfamilies. Two recently identified venom gene superfamilies are expressed in the large Turridae clade, but not in Conus. Thus, as major venomous molluscan clades expand, a small set of lineage specific venom gene superfamilies undergo accelerated evolution. The juxtaposition of extremely conserved signal sequences with hypervariable mature peptide regions is unprecedented and raises the possibility that in these gene superfamilies, the signal sequences are conserved as a result of an essential role they play in enabling rapid sequence evolution of the region of the gene that encodes the active toxin.

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Conotoxins are small, disulfide-rich peptides that have been isolated from Conus, a large genus of predatory marine snails. The primary structures of more than 100 conotoxins have been determined and classified into gene superfamilies on the basis of the amino acid sequences of the signal peptides of their a-Conotoxins from marine snails are known to be selective and potent competitive antagonists of nicotinic acetylcholine receptors. Here we describe the purification, structural features and activity of two novel toxins, SrIA and SrIB, isolated from Conus spurius collected in the Yucatan Channel, Mexico. As determined by direct amino acid and cDNA nucleotide sequencing, the toxins are peptides containing 18 amino acid residues with the typical 4 ⁄ 7-type framework but with completely novel sequences. Therefore, their actions (and that of a synthetic analog, [c15E]SrIB) were compared to those exerted by the a4 ⁄ 7-conotoxin EI from Conus ermineus, used as a control. Their target specificity was evaluated by the patch-clamp technique in mammalian cells expressing a 1 b 1 cd, a 4 b 2 and a 3 b 4 nicotinic acetylcholine receptors. At high concentrations (10 lm), the peptides SrIA, SrIB and [c15E]SrIB showed weak blocking effects only on a 4 b 2 and a 1 b 1 cd subtypes, but EI also strongly blocked a 3 b 4 receptors. In contrast to this blocking effect, the new peptides and EI showed a remarkable potentiation of a 1 b 1 cd and a 4 b 2 nicotinic acetylcholine receptors if briefly (2-15 s) applied at concentrations several orders of magnitude lower (EC 50 , 1.78 and 0.37 nm, respectively). These results suggest not only that the novel a-conotoxins and EI can operate as nicotinic acetylcholine receptor inhibitors, but also that they bind both a 1 b 1 cd and a 4 b 2 nicotinic acetylcholine receptors with very high affinity and increase their intrinsic cholinergic response. Their unique properties make them excellent tools for studying the toxin-receptor interaction, as well as models with which to design highly specific therapeutic drugs.Abbreviations a 1 b 1 cd, muscular nicotinic acetylcholine receptor; a 3 b 4 , peripheral nervous system nicotinic acetylcholine receptor; a 4 b 2 , central nervous system nicotinic acetylcholine receptor; Acm, S-acetamidomethyl; ACN, acetonitrile; [c15E]SrIB, synthetic a-conotoxin from Conus spurius; nAChR, nicotinic acetylcholine receptor; PTH, phenylthiohydantoin; SrIA, a-conotoxin IA from Conus spurius; SrIB, a-conotoxin IB from Conus spurius.

A novel peptide, conorfamide-Sr2 (CNF-Sr2), was purified from the venom extract of Conus spurius, collected in the Caribbean Sea off the Yucatan Peninsula. Its primary structure was determined by automated Edman degradation and amino acid analysis, and confirmed by electrospray ionization mass spectrometry. Conorfamide-Sr2 contains 12 amino acids and no Cys residues, and it is only the second FMRFamide-related peptide isolated from a venom. Its primary structure GPM gammaDPLgammaIIRI-nh2, (gamma, gamma-carboxyglutamate; -nh2, amidated C-terminus; calculated monoisotopic mass, 1468.72Da; experimental monoisotopic mass, 1468.70Da) shows two features that are unusual among FMRFamide-related peptides (FaRPs, also known as RFamide peptides), namely the novel presence of gamma-carboxyglutamate, and a rather uncommon C-terminal residue, Ile. CNF-Sr2 exhibits paralytic activity in the limpet Patella opea and causes hyperactivity in the freshwater snail Pomacea paludosa and in the mouse. The sequence similarities of CNF-Sr2 with FaRPs from marine and freshwater mollusks and mice might explain its biological effects in these organisms. It also resembles FaRPs from polychaetes (the prey of C. spurius), which suggests a natural biological role. Based on these similarities, CNF-Sr2 might interact with receptors of these three distinct types of FaRPs, G-protein-coupled receptors, Na+ channels activated by FMRFamide (FaNaCs), and acid-sensing ion channels (ASICs). The biological activities of CNF-Sr2 in mollusks and mice make it a potential tool to study molecular targets in these and other organisms.

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