Delta-conotoxins are Conus peptides that inhibit inactivation of voltage-gated sodium channels. The suggestion that delta-conotoxins might be an essential component of the venoms of fish-hunting cone snails which rapidly immobilize their prey [Terlau, H., Shon, K., Grilley, M., Stocker, M., Stühmer, W., and Olivera, B. M. (1996) Nature 381, 148-151] has not been tested. On the basis of cDNA cloning, all of the fish-hunting Conus analyzed yielded at least one delta-conotoxin sequence. In addition, one delta-conotoxin isolated from the venom of Conus striatus had an amino acid sequence identical to that predicted from cDNA cloning. This new peptide exhibited properties of delta-conotoxins: it targeted sodium channels and potentiated action potentials by slowing channel inactivation. Homologous sequences of delta-conotoxins from two groups (clades) of related fish-hunting Conus species share consensus features but differ significantly from the two known delta-conotoxins from mollusc-hunting Conus venoms. Three large hydrophobic amino acids were conserved; analogues of the previously described delta-conotoxin PVIA with alanine substituted for the conserved amino acids F9 and I12 lost substantial biological activity. In contrast, both the T8A and K13A delta-conotoxin PVIA analogues, where substitutions were at nonconserved loci, proved to be biologically active. Taken together, our results indicate that a cladistic approach can identify amino acids critical for the activity of conotoxins and provide extensive information as to which amino acid substitutions can be made without significant functional consequences.

Voltage-gated ion channels determine the membrane excitability of cells. Although many Conus peptides that interact with voltage-gated Na ؉ and Ca 2؉ channels have been characterized, relatively few have been identified that interact with K ؉ channels. We describe a novel Conus peptide that interacts with the Shaker K ؉ channel, M-conotoxin RIIIK from Conus radiatus. The peptide was chemically synthesized. Although M-conotoxin RIIIK is structurally similar to the -conotoxins that are sodium channel blockers, it does not affect any of the sodium channels tested, but blocks Shaker K ؉ channels. Studies using Shaker K ؉ channel mutants with single residue substitutions reveal that the peptide interacts with the pore region of the channel. Introduction of a negative charge at residue 427 (K427D) greatly increases the affinity of the toxin, whereas the substitutions at two other residues, Phe 425 Basic research on voltage-gated ion channels advances on two broad fronts: first, the identification and characterization of the numerous molecular isoforms that comprise each voltage-gated ion channel family. A different stream of research focuses on a few model systems intensively to uncover basic mechanistic insights. For both of these contrasting facets of ion channel research, the small peptides made by predatory cone snails (conotoxins) (1, 2) have considerable potential. Thus, the -conotoxins, one family of conotoxins, have become standard reagents for discriminating among Ca 2ϩ channel subtypes (3), and characterizing the functional role of each subtype. The -conotoxins that block voltage-gated channels are used as probes for the outer vestibule of the channel pore. The subject of this report is a novel conotoxin that has promising properties to be an important reagent for structure/function studies of the Shaker K ϩ channel, arguably the most intensively studied of all voltage-gated ion channels. However, the characterization of the peptide also defines a new family of conotoxins, the M family, that should provide a set of new ligands specific for different K ϩ channel isoforms.The first Conus peptide shown to target a voltage-gated ion channel was -conotoxin GIIIA, which was discovered and characterized two decades ago (4 -8). A characteristic feature of all -conotoxins is the arrangement of disulfide cross-links in the primary sequence, the -conotoxin pattern can be recognized by the following pattern of Cys residues: -CC-C-C-CC-, now defined as a class III (or M-1) conotoxin scaffold (9). After the discovery of the -conotoxins, other groups of Conus peptides with three disulfide bonds (the -conotoxins, ␦-conotoxins, O-conotoxins, and the spasmodic peptides) were characterized, but these had a different arrangement of Cys residues. Only one other family of conopeptides with a class III disulfide framework has been characterized, the -conotoxins (10). The latter are noncompetitive antagonists of nicotinic acetylcholine receptors.In this report, a peptide from Conus radiatus venom ducts that has the same class III...

The mammalian skeletal muscle acetylcholine receptor contains two nonequivalent acetylcholine binding sites, one each at the alpha/delta and alpha/gamma subunit interfaces. Alpha-Conotoxin MI, a 14-amino acid competitive antagonist, binds at both interfaces but has approximately 10(4) higher affinity for the alpha/delta site. We performed an "alanine walk" to identify the residues in alpha-MI that contribute to this selective interaction with the alpha/delta site. Electrophysiological measurements with Xenopus oocytes expressing normal receptors or receptors lacking either the gamma or delta subunit were made to assay toxin-receptor interaction. Alanine substitutions in most amino acid positions had only modest effects on toxin potency at either binding site. However, substitutions in two positions, proline-6 and tyrosine-12, dramatically reduced toxin potency at the high-affinity alpha/delta site while having comparatively little effect on low-affinity alpha/gamma binding. When tyrosine-12 was replaced by alanine, the toxin's selectivity for the high-affinity site (relative to that for the low-affinity site) was reduced from 45,000- to 30-fold. A series of additional amino acid substitutions in this position showed that increasing side chain size/hydrophobicity increases toxin potency at the alpha/delta site without affecting alpha/gamma binding. In contrast, when tyrosine-12 is diiodinated, toxin binding is nearly irreversible at the alpha/delta site but also increases by approximately 500-fold at the alpha/gamma site. The effects of position 12 substitutions are accounted for almost entirely by changes in the rate of toxin dissociation from the high-affinity alpha/delta binding site.

Elucidating signaling pathways that regulate cellular metabolism is essential for a better understanding of normal development and tumorigenesis. Recent studies have shown that mitochondrial pyruvate carrier 1 (MPC1), a crucial player in pyruvate metabolism, is downregulated in colon adenocarcinomas. Utilizing zebrafish to examine the genetic relationship between MPC1 and Adenomatous polyposis coli (APC), a key tumor suppressor in colorectal cancer, we found that apc controls the levels of mpc1 and that knock down of mpc1 recapitulates phenotypes of impaired apc function including failed intestinal differentiation. Exogenous human MPC1 RNA rescued failed intestinal differentiation in zebrafish models of apc deficiency. Our data demonstrate a novel role for apc in pyruvate metabolism and that pyruvate metabolism dictates intestinal cell fate and differentiation decisions downstream of apc.DOI: http://dx.doi.org/10.7554/eLife.22706.001