The venoms of predatory cone snails represent a rich combinatorial-like library of evolutionarily selected, neuropharmacologically active peptides. A major fraction of the venom components are conotoxins--small, disulfide-rich peptides that potently and specifically target components of the neuromuscular system, particularly ligand- and voltage-gated ion channels. This review focuses on Conus peptides, which act at nicotinic acetylcholine receptors. These nicotinic antagonist peptides from Conus are broadly divided into two groups: those that act at the neuromuscular junction and those that act at subtypes of neuronal nicotinic acetylcholine receptors. The latter include peptides specific for the alpha 7, alpha 3 beta 2, and alpha 3 beta 4 nicotinic receptor subtypes. The degree of specificity exhibited by these peptides is remarkable, particularly given their relatively small size. As a group the nicotinic acetylcholine receptor-targeted Conus peptides represent an increasingly well-defined set of tools for probing the structure, function, and physiological role of nicotinic acetylcholine receptors.

All 500 species of cone snails (Conus) are venomous predators. From a biochemical/genetic perspective, differences among Conus species may be based on the 50-200 different peptides in the venom of each species. Venom is used for prey capture as well as for interactions with predators and competitors. The venom of every species has its own distinct complement of peptides. Some of the interspecific divergence observed in venom peptides can be explained by differential expression of venom peptide superfamilies in different species and of peptide superfamily branching in various Conus lineages into pharmacologic groups with different targeting specificity. However, the striking interspecific divergence of peptide sequences is the dominant factor in the differences observed between venoms. The small venom peptides (typically 10-35 amino acids in length) are processed from larger prepropeptide precursors (ca. 100 amino acids). If interspecific comparisons are made between homologous prepropeptides, the three different regions of a Conus peptide precursor (signal sequence, pro-region, mature peptide) are found to have diverged at remarkably different rates. Analysis of synonymous and nonsynonymous substitution rates for the different segments of a prepropeptide suggests that mutation frequency varies by over an order of magnitude across the segments, with the mature toxin region undergoing the highest rate. The three sections of the prepropeptide which exhibit apparently different mutation rates are separated by introns. This striking segment-specific rate of divergence of Conus prepropeptides suggests a role for introns in evolution: exons separated by introns have the potential to evolve very different mutation rates. Plausible mechanisms that could underlie differing mutational frequency in the different exons of a gene are discussed.

The generation of functional novelty in proteins encoded by a gene superfamily is seldom well documented. In this report, we define the A-conotoxin superfamily, which is widely expressed in venoms of the predatory cone snails (Conus), and show how gene products that diverge considerably in structure and function have arisen within the same superfamily. A cDNA clone encoding ␣-conotoxin GI, the first conotoxin characterized, provided initial data that identified the A-superfamily. Conotoxin precursors in the A-superfamily were identified from six Conus species: most (11/16) encoded ␣-conotoxins, but some (5/16) belong to a family of excitatory peptides, the A-conotoxins that target voltagegated ion channels. ␣-Conotoxins are two-disulfidebridged nicotinic antagonists, 13-19 amino acids in length; A-conotoxins are larger (31-36 amino acids) with three disulfide bridges. Purification and biochemical characterization of one peptide, A-conotoxin MIVA is reported; five of the other predicted conotoxins were previously venom-purified. A comparative analysis of conotoxins purified from venom, and their precursors reveal novel post-translational processing, as well as mutational events leading to polymorphism. Patterns of sequence divergence and Cys codon usage define the major superfamily branches and suggest how these separate branches arose.