Overturning circulation, nutrient limitation, and warming in the Glacial North Pacific.

Although the Pacific Ocean is a major reservoir of heat and CO2, and thus an important component of the global climate system, its circulation under different climatic conditions is poorly understood. Here, we present evidence that during the Last Glacial Maximum (LGM), the North Pacific was better ventilated at intermediate depths and had surface waters with lower nutrients, higher salinity, and warmer temperatures compared to today. Modeling shows that this pattern is well explained by enhanced Pacific meridional overturning circulation (PMOC), which brings warm, salty, and nutrient-poor subtropical waters to high latitudes. Enhanced PMOC at the LGM would have lowered atmospheric CO2-in part through synergy with the Southern Ocean-and supported an equable regional climate, which may have aided human habitability in Beringia, and migration from Asia to North America.

Solution conformation of alpha-conotoxin EI, a neuromuscular toxin specific for the alpha 1/delta subunit interface of torpedo nicotinic acetylcholine receptor.

A high resolution structure of alpha-conotoxin EI has been determined by (1)H NMR spectroscopy and molecular modeling. alpha-Conotoxin EI has the same disulfide framework as alpha 4/7 conotoxins targeting neuronal nicotinic acetylcholine receptors but antagonizes the neuromuscular receptor as do the alpha 3/5 and alpha A conotoxins. The unique binding preference of alpha-conotoxin EI to the alpha(1)/delta subunit interface of Torpedo neuromuscular receptor makes it a valuable structural template for superposition of various alpha-conotoxins possessing distinct receptor subtype specificities. Structural comparison of alpha-conotoxin EI with the gamma-subunit favoring alpha-conotoxin GI suggests that the Torpedo delta-subunit preference of the former originates from its second loop. Superposition of three-dimensional structures of seven alpha-conotoxins reveals that the estimated size of the toxin-binding pocket in nicotinic acetylcholine receptor is approximately 20 A (height) x 20 A (width) x 15 A (thickness).

A novel D-leucine-containing Conus peptide: diverse conformational dynamics in the contryphan family.

A Conus peptide family (the contryphans) is noteworthy because of the presence of a post-translationally modified D-amino acid in all members of the family. A new contryphan peptide, Leu-contryphan-P, was isolated from the venom of Conus purpurascens; the sequence of this peptide is: Gly-Cys-Val-D-Leu-Leu-Pro-Trp-Cys-OH. This is the first known occurrence of D-leucine in a Conus peptide. The discovery of Leu-contryphan-P suggests that there may be branches of the contryphan peptide family that diverge much more in sequence than previously anticipated. Several natural contryphans provide dramatic examples of interconversion between multiple conformational states in small constrained peptides. The contryphans that have 4-trans-hydroxyproline and D-tryptophan in positions 3 and 4, respectively, exhibit two peaks under reverse-phase HPLC conditions, indicating interconversion between two discrete conformations. However, [L-Trp4]contryphan-Sm (with L-Trp substituted for D-Trp) exhibits a single, broad peak that elutes later than the natural peptide, suggesting that D-Trp stabilizes a conformation in which hydrophobic residues are buried. Leucontryphan-P which has valine and D-leucine instead of 4-trans-hydroxyproline and D-tryptophan shows only a single peak that elutes much later than the other contryphans.

An O-glycosylated neuroexcitatory conus peptide.

We purified and characterized a novel peptide from the venom of the fish-hunting cone snail Conus striatus that inhibits voltage-gated K+ channels. The peptide, kappaA-conotoxin SIVA, causes characteristic spastic paralytic symptoms when injected into fish, and in frog nerve-muscle preparations exposed to the toxin, repetitive action potentials are seen in response to a single stimulus applied to the motor nerve. Other electrophysiological tests on diverse preparations provide evidence that is consistent with the peptide blocking K+ channels. The peptide has three disulfide bonds; the locations of Cys residues indicate that the spastic peptide may be the first and defining member of a new family of Conus peptides, the kappaA-conotoxins, which are structurally related to, but pharmacologically distinct from, the alphaA-conotoxins. This 30 AA tricyclic toxin has several characteristics not previously observed in Conus peptides. In addition to the distinctive biological and physiological activity, a novel biochemical feature is the unusually long linear N-terminal tail (11 residues) which contains one O-glycosylated serine at position 7. This is the first evidence for O-glycosylation as a posttranslational modification in a biologically active Conus peptide.

mu-Conotoxin PIIIA, a new peptide for discriminating among tetrodotoxin-sensitive Na channel subtypes.

We report the characterization of a new sodium channel blocker, mu-conotoxin PIIIA(mu-PIIIA). The peptide has been synthesized chemically and its disulfide bridging pattern determined. The structure of the new peptide is: [sequence: see text] where Z = pyroglutamate and O = 4-trans-hydroxyproline. We demonstrate that Arginine-14 (Arg14) is a key residue; substitution by alanine significantly decreases affinity and results in a toxin unable to block channel conductance completely. Thus, like all toxins that block at Site I, mu-PIIIA has a critical guanidinium group. This peptide is of exceptional interest because, unlike the previously characterized mu-conotoxin GIIIA (mu-GIIIA), it irreversibly blocks amphibian muscle Na channels, providing a useful tool for synaptic electrophysiology. Furthermore, the discovery of mu-PIIIA permits the resolution of tetrodotoxin-sensitive sodium channels into three categories: (1) sensitive to mu-PIIIA and mu-conotoxin GIIIA, (2) sensitive to mu-PIIIA but not to mu-GIIIA, and (3) resistant to mu-PIIIA and mu-GIIIA (examples in each category are skeletal muscle, rat brain Type II, and many mammalian CNS subtypes, respectively). Thus, mu-conotoxin PIIIA provides a key for further discriminating pharmacologically among different sodium channel subtypes.

kappa-Conotoxin PVIIA is a peptide inhibiting the shaker K+ channel.

kappa-Conotoxin PVIIA (kappa-PVIIA), a 27-amino acid toxin from Conus purpurascens venom that inhibits the Shaker potassium channel, was chemically synthesized in a biologically active form. The disulfide connectivity of the peptide was determined. kappa-Conotoxin PVIIA has the following structure. This is the first Conus peptide known to target K+ channels. [structure: see text] Although the Shaker K+ channel is sensitive to kappa-PVIIA, the rat brain Kv1.1 subtype is resistant. Chimeras between Shaker and the Kv1.1 K+ channels were constructed and expressed in Xenopus oocytes. Only channels containing the putative pore-forming region between the fifth and sixth transmembrane domains of Shaker retained toxin sensitivity, indicating that the toxin target site is in this region of the channel. Evidence is presented that kappa-PVIIA interacts with the external tetraethyl-ammonium binding site on the Shaker channel. Although both kappa-PVIIA and charybdotoxin inhibit the Shaker channel, they must interact differently. The F425G Shaker mutation increases charybdotoxin affinity by 3 orders of magnitude but abolishes kappa-PVIIA sensitivity. The precursor sequence of kappa-PVIIA was deduced from a cDNA clone, revealing a prepropeptide comprising 72 amino acids. The N-terminal region of the kappa-PVIIA prepropeptide exhibits striking homology to the omega-, muO-, and delta-conotoxins. Thus, at least four pharmacologically distinct superfamilies of Conus peptides belong to the same "O" superfamily, with the omega- and kappa-conotoxins forming one branch, and the delta- and muO-conotoxins forming a second major branch.

Differential targeting of nicotinic acetylcholine receptors by novel alphaA-conotoxins.

We describe the isolation and characterization of two peptide toxins from Conus ermineus venom targeted to nicotinic acetylcholine receptors (nAChRs). The peptide structures have been confirmed by mass spectrometry and chemical synthesis. In contrast to the 12-18 residue, 4 Cys-containing alpha-conotoxins, the new toxins have 30 residues and 6 Cys residues. The toxins, named alphaA-conotoxins EIVA and EIVB, block both Torpedo and mouse alpha1-containing muscle subtype nAChRs expressed in Xenopus oocytes at low nanomolar concentrations. In contrast to alpha-bungarotoxin, alphaA-EIVA is inactive at alpha7-containing nAChRs even at micromolar concentrations. In this regard, alphaA-EIVA is similar to the previously described alpha-conotoxins (e.g. alpha-MI and alpha-GI) which also selectively target alpha1- versus alpha7-containing nAChRs. However, alpha-MI and alpha-GI discriminate between the alpha/delta versus alpha/gamma subunit interfaces of the mouse muscle nAChR with 10,000-fold selectivity. In contrast, alphaA-conotoxin EIVA blocks both the alpha/gamma site and alpha/delta site with equally high affinity but with distinct kinetics. The alphaA-conotoxins thus represent novel probes for the alpha/gamma as well as the alpha/delta binding sites of the nAChR.

A noncompetitive peptide inhibitor of the nicotinic acetylcholine receptor from Conus purpurascens venom.

A paralytic peptide, psi-conotoxin Piiie has been purified and characterized from Conus purpurascens venom. Electrophysiological studies indicate that the peptide inhibits the nicotinic acetylcholine receptor (nAChR). However, the peptide does not block the binding of alpha-bungarotoxin, a competitive nAChR antagonist. Thus, psi-conotoxin Piiie appears to inhibit the receptor at a site other than the acetylcholine-binding site. As ascertained by sequence analysis, mass spectrometry, and chemical synthesis, the peptide has the following covalent structure: HOOCCLYGKCRRYOGCSSASCCQR* (O = 4-trans hydroxyproline; * indicates an amidated C-terminus). The disulfide connectivity of the toxin is unrelated to the alpha- or the alphaA-conotoxins, the Conus peptide families that are competitive inhibitors of the nAChR, but shows homology to the mu-conotoxins (which are Na+ channel blockers).

Determinants involved in the affinity of alpha-conotoxins GI and SI for the muscle subtype of nicotinic acetylcholine receptors.

Nicotinic acetylcholine receptors from muscle contain two functionally active and pharmacologically distinct acetylcholine-binding sites located at the alpha/gamma and alpha/delta subunit interfaces. The alpha-conotoxins are competitive antagonists of nicotinic receptors and can be highly site-selective, displaying greater than 10,000-fold differences in affinities for the two acetylcholine-binding sites on a single nicotinic receptor. The higher affinity site for alpha-conotoxins GI, MI, and SI is the alpha/delta site on mouse muscle-derived BC3H-1 receptors. However, alpha-conotoxins GI and MI exhibit higher affinity for the other site (alpha/gamma site) on nicotinic receptors from Torpedo californica electric organ. alpha-Conotoxin SI does not distinguish between the two acetylcholine-binding sites on Torpedo receptors. In this study, alpha-conotoxins [K10H]SI and [K10N]SI displayed wild-type affinity for the two acetylcholine-binding sites on BC3H-1 receptors but a 10-20-fold decrease in apparent affinity at one of the two acetylcholine-binding sites on Torpedo receptors. alpha-Conotoxin [P9K]SI displayed a selective and dramatic increase in the apparent affinity for the alpha/delta site of BC3H-1 receptors and for the alpha/gamma site of Torpedo receptors. alpha-Conotoxin [R9A]GI displayed a reduction in affinity for both acetylcholine-binding sites on BC3H-1 receptors, although the extent of its selectivity for the alpha/delta site was retained. alpha-Conotoxin [R9A]GI also displayed a loss of affinity for the two acetylcholine-binding sites on Torpedo receptors, but its site-selectivity was apparently abolished. These results indicate that positions 9 and 10 in alpha-conotoxins GI and SI are involved in complex species- and subunit-dependent interactions with nicotinic receptors.

Bromocontryphan: post-translational bromination of tryptophan.

We demonstrate that post-translational bromination of a tryptophan residue occurs in the biologically active octapeptide bromocontryphan, purified and characterized from Conus radiatus venom. Clones encoding bromocontryphan were identified from a cDNA library made from C. radiatus venom ducts. The mRNA sequence obtained predicts a prepropeptide which has the mature peptide sequence at the C-terminal end, with the L-6-bromotryptophan residue encoded by UGG, the Trp codon. These data provide the first direct evidence for post-translational bromination of a polypeptide which is translated through the normal cellular machinery. In addition to bromination, the peptide, which induces a "stiff tail" syndrome in mice, has several other modifications as shown by the sequence [Formula: See Text] in which Hyp = hydroxyproline. Asterisks indicate post-translational modifications (left to right): proteolytic cleavage at the N-terminus; hydroxylation of Pro3; epimerization of Trp4; bromination of Trp7, and C-terminal amidation. Bromocontryphan appears to have the highest density of post-translational modifications known among gene-encoded polypeptides. The overall result is a molecule which closely resembles marine natural products produced through specialized biosynthetic pathways comprising many enzyme-catalyzed steps.

NMR structure determination of a novel conotoxin, [Pro 7,13] alpha A-conotoxin PIVA.

A high-resolution solution conformation of a novel conotoxin, [Pro 7,13] alpha A-conotoxin PIVA, GCCGSYPNAACHPCSCKDROSYCGQ-NH2, has been determined by two-dimensional 1H NMR methods and distance geometry calculations. The total of 324 NOE-derived interproton distance restraints including 33 long-range NOE restraints as well as 11 phi and 7 chi 1 torsion angle restraints was used for computation of structures. Back-calculation from the experimental NOE spectrum has provided 49 new NOE restraints and yielded the final R-factors of Ra = 0.641 and Rb = 0.157. The final RMSD values are 0.90 and 1.16 A for the backbone and the heavy atoms, respectively. The C-terminal half of the molecule involving the residues 12-24 is extremely well-defined with a backbone RMSD value of 0.56 A, whereas the N-terminal 3-11 disulfide loop is relatively flexible, possessing a backbone RMSD value of 1.09 A. The [Pro 7,13] alpha A-conotoxin PIVA does not contain any significant secondary structure although the 21S-24G nearly completes one turn of a 3(10) helix. The overall protein fold is largely maintained by the three disulfide bridges of 2-16, 3-11, and 14-23. The presence of the three disulfide bridges imposes geometric constraints that force the molecule to form six continuous bends involving the following residues: 3C-5S, 7P-10A, 12H-14C, 15S-17K, 17K-19R, and 21S-25Q. The overall shape of the [Pro 7,13] alpha A-conotoxin PIVA can be described as an "iron". Residues 15S-19R form a loop that protrudes out of the "bottom plate" formed by the rest of the protein and constitute the handle of the iron. The N-terminal tip of the molecule is relatively immobile due to attractive electrostatic interactions between the gamma-hydroxyl group of 20 Hyp and the phenolic hydroxyl group of 22Y. The flexible 3-11 disulfide loop consists mostly of hydrophobic residues, while the best-defined 14-23 disulfide loop contains the highly charged hydrophilic 15S-19R "handle" domain exposed to the exterior of the protein. Binding to nicotinic acetylcholine receptor can be mediated through two different types of interactions: one involving the aromatic hydrophobic residues such as 6Y and 12H and the other involving the positively charged hydrophilic side chain of the 19R. The side chain of the 19R in the [Pro 7, 13] alpha A-conotoxin PIVA and that of the 9R of the alpha-conotoxin G1, and also the side chains of the 12H and 6Y in the former and those of 10H and 11Y in the latter can be aligned to point to the same direction when the corresponding backbone atoms are superimposed to an RMSD value of 2.5 A.

Folding of omega-conotoxins. 1. Efficient disulfide-coupled folding of mature sequences in vitro.

Disulfide-coupled refolding reactions of five omega-conotoxins, Ca2+ channel antagonists derived from marine snails of the genus Conus, were examined. These peptides are 23-26 amino acid residues long, and the native conformation of each is stabilized by three disulfide bonds. Although the primary structures of the peptides show only limited sequence similarity, the patterns of disulfides and three-dimensional conformations are very similar. Refolding of the reduced proteins was promoted by the disulfide form of glutathione (GSSG) in the presence of reduced glutathione (GSH). All five of the peptides examined were able to refold to the native conformation, as judged by reversed-phase HPLC behavior, with efficiencies of 16% for omega-MVIIC, 28% for omega-MVIID, and 50% for omega-MVIIA, omega-GVIA, and omega-SVIA. The refolded form of omega-MVIIA was further shown to have biological activity indistinguishable from that of the native form, as well as the same rate of reductive unfolding in the presence of dithiothreitol. The overall folding rate and efficiency of omega-MVIIA was found to be quite sensitive to the thiol-disulfide redox potential, with optimum rates and yields obtained in the presence of GSSG and GSH at concentrations similar to those believed to be present in the endoplasmic reticulum. The folding efficiency of this peptide was greatly reduced by the addition of 8 M urea, indicating that formation of the correct disulfides is determined largely by noncovalent interactions, as opposed to steric constraints arising from the spacing between Cys residues. These results demonstrate that the mature forms of at least some omega-conotoxins contain sufficient information to direct correct folding and disulfide formation, in spite of their small size and limited sequence conservation.

Folding of omega-conotoxins. 2. Influence of precursor sequences and protein disulfide isomerase.

The peptide Ca2+ channel antagonists found in the venoms of Conus snails, omega-conotoxins, are synthesized as precursors that include a leader peptide, presumed to direct the polypeptide to the endoplasmic reticulum, and a propeptide of unknown function. In addition, the precursors are synthesized with a C-terminal Gly residue that is posttranslationally converted to a terminal amide group. In order to determine whether the precursor sequences contain information that helps direct folding of the mature sequences, the disulfide-coupled folding of mature omega-conotoxin MVIIA was compared with that of two putative precursor forms: pro-omega-MVIIA-Gly, which contains the propeptide and the C-terminal Gly residue, and omega-MVIIA-Gly, which differs from the mature form only at the C-terminus. The three forms folded with similar kinetics, but the folding efficiency of omega-MVIIA-Gly was greater than 80%, versus approximately 50% for both mature omega-MVIIA and the form containing the propeptide. The enzyme protein disulfide isomerase was found to catalyze disulfide formation and folding of all three forms similarly. The affinity of omega-MVIIA-Gly for receptors in chick brain synaptosomes was approximately 10-fold lower than that of the mature peptide, and the N-terminal propeptide of pro-omega-MVIIA-Gly was found to decrease binding further, by approximately 100-fold. These results suggest that the omega-conotoxins do not rely on the propeptide region of their precursors to facilitate folding. Rather, the mature sequence contains most of the information required to specify the native disulfide pairings and three-dimensional conformation. The C-terminal Gly may enhance the folding efficiency by forming interactions that stabilize the native conformation with respect to other disulfide-bonded forms.

Contryphan is a D-tryptophan-containing Conus peptide.

In this report, we document for the first time the occurrence of D-tryptophan in a normally translated polypeptide, contryphan. The peptide, isolated from the venom of the fish-hunting marine snail Conus radiatus, produces the "stiff-tail" syndrome in mice. Characterization of the octapeptide gave the following sequence, Gly-Cys-Hyp-D-Trp-Glu-Pro-Trp-Cys-NH2 where Hyp = 4-trans-hydroxyproline. The presence of D-tryptophan in position 4 of contryphan was confirmed by chemical synthesis. The post-translational epimerization in all other D-amino acid-containing small peptides characterized previously from vertebrates and molluscan systems is in position 2.

Three-dimensional structure of the alpha-conotoxin GI at 1.2 A resolution.

Predatory marine snails of the genus Conus paralyze their fish prey by injecting a potent toxin. The alpha-conotoxin GI is a 13-residue peptide isolated from venom of Conus geographus. It functions by blocking the postsynaptic nicotinic acetylcholine receptor. After crystallization in deionized water, the three-dimensional structure of the GI neurotoxin was determined to 1.2 A resolution by X-ray crystallography. This structure, which can be described as a triangular slab, shows overall similarities to those derived by NMR, CD, and predictive methods. The principal framework of the molecule is provided by two disulfide bonds, one linking Cys 2 and Cys 7 and the other Cys 3 and Cys 13. Opposite ends of the sequence are drawn together even further by hydrogen bonds between Glu 1 and Cys 13 and between Cys 2 and Ser 12. Since the C-terminus is amidated, only one negative charge is present (carboxylate of Glu 1), and this is not implicated in receptor binding. Two positively charged regions (the alpha-amino group of Glu 1 and the guanido group of Arg 9) are situated 15 A apart at the corners of the triangular face of the molecule. phi, psi angles characteristic of a 3(10) helix were observed for residues 5-7. For residues 8-11, these angles were consistent with either a type I beta-turn or a distorted 3(10) helix.

A new alpha-conotoxin which targets alpha3beta2 nicotinic acetylcholine receptors.

We have isolated a 16-amino acid peptide from the venom of the marine snail Conus magus which potently blocks nicotinic acetylcholine receptors (nAChRs) composed of alpha3beta2 subunits. This peptide, named alpha-conotoxin MII, was identified by electrophysiologically screening venom fractions against cloned nicotinic receptors expressed in Xenopus oocytes. The peptide's structure, which has been confirmed by mass spectrometry and total chemical synthesis, differs significantly from those of all previously isolated alpha-conotoxins. Disulfide bridging, however, is conserved. The toxin blocks the response to acetylcholine in oocytes expressing alpha3beta2 nAChRs with an IC50 of 0.5 nM and is 2-4 orders of magnitude less potent on other nAChR subunit combinations. We have recently reported the isolation and characterization of alpha-conotoxin ImI, which selectively targets homomeric alpha7 neuronal nAChRs. Yet other alpha-conotoxins selectively block the muscle subtype of nAChR. Thus, it is increasingly apparent that alpha-conotoxins represent a significant resource for ligands with which to probe structure-function relationships of various nAChR subtypes.

alpha-Conotoxin EI, a new nicotinic acetylcholine receptor antagonist with novel selectivity.

We report the isolation and characterization of a novel nicotinic acetylcholine receptor (nAChR) ligand. The toxin is an 18 amino acid peptide and is the first reported alpha-conotoxin from an Atlantic fish-hunting Conus. The peptide was purified from the venom of Conus ermineus and is called alpha-conotoxin EI. The sequence diverges from that of previously isolated alpha-conotoxins. We demonstrate that this structural divergence has functional consequences. In Torpedo nAChRs, alpha-conotoxin EI selectively binds the agonist site near the alpha/delta subunit interface in contrast to alpha-conotoxin MI which selectively targets the alpha/gamma agonist binding site. In mammalian nAChRs alpha-conotoxin EI shows high affinity for both the alpha/delta and alpha/gamma subunit interfaces (with some preference for the alpha/delta site), whereas alpha-conotoxin MI is highly selective for the alpha/delta ligand binding site. The sequence of the peptide is: Arg-Asp-Hyp-Cys-Cys-Tyr-His-Pro-Thr-Cys-Asn-Met-Ser-Asn-Pro-Gln-Ile-Cys- NH2, with disulfide bridging between Cys4-Cys10 and Cys5-Cys18, analogous to those of previously described alpha-conotoxins. This sequence has been verified by total chemical synthesis. Thus, alpha-conotoxin EI is a newly-available tool with unique structure and function for characterization of nAChRs.

A new family of Conus peptides targeted to the nicotinic acetylcholine receptor.

In this work, a new family of Conus peptides, the alpha A-conotoxins, which target the nicotinic acetylcholine receptor, is defined. The first members of this family have been characterized from the eastern Pacific species, Conus purpurascens (the purple cone); three peptides that cause paralysis in fish were purified and characterized from milked venom. The sequence and disulfide bonding pattern of one of these, alpha A-conotoxin PIVA, is as follows: [formula: see text] where O represents trans-4-hydroxyproline. The two other peptides purified from C. purpurascens venom are the under-hydroxylated derivatives, [Pro13]alpha A-conotoxin PIVA and [Pro7,13]alpha A-conotoxin PIVA. The peptides have been chemically synthesized in a biologically active form. Both electrophysiological experiments and competition binding with alpha-bungarotoxin demonstrate that alpha A-PIVA acts as an antagonist of the nicotinic acetylcholine receptor at the postsynaptic membrane.

A new family of conotoxins that blocks voltage-gated sodium channels.

Conus peptides, including omega-conotoxins and alpha-conotoxins (targeting calcium channels and nicotinic acetylcholine receptors, respectively) have been useful ligands in neuroscience. In this report, we describe a new family of sodium channel ligands, the mu-O-conotoxins. The two peptides characterized, mu-O-conotoxins MrVIA and MrVIB from Conus marmoreus potently block the sodium conductance in Aplysia neurons. This is in marked contrast to standard sodium channel blockers that are relatively ineffective in this system. The sequences of the peptides are as follows. mu-O-conotoxin MrVIA: ACRKKWEYCIVPIIGFIYCCPGLICGPFVCV mu-O-conotoxin MrVIB: ACSKKWEYCIVPILGFVYCCPGLICGPFVCV mu-O-conotoxin MrVIA was chemically synthesized and proved indistinguishable from the natural product. Surprisingly, the mu-O-conotoxins show no sequence similarity to the mu-O-conotoxins. However, ananalysis of cDNA clones encoding the mu-O-conotoxin MrVIB demonstrated striking sequence similarity to omega- and delta-conotoxin precursors. Together, the omega-, delta-, and mu-O-conotoxins define the O-superfamily of Conus peptides. The probable biological role and evolutionary affinities of these peptides are discussed.

Purification, characterization, synthesis, and cloning of the lockjaw peptide from Conus purpurascens venom.

The major groups of Conus peptides previously characterized from fish-hunting cone snail venoms (the alpha-, mu-, and omega-conotoxins) all blocked neuromuscular transmission. A novel activity, the "lockjaw peptide", from the fish-hunting Conus purpurascens, caused a rigid (instead of flaccid) paralysis in fish and increased excitability at the neuromuscular junction (instead of a block). We report the purification, biological activity, biochemical and preliminary physiological characterization, and chemical synthesis of the lockjaw peptide and the sequence of a cDNA clone encoding its precursor. Taken together, the data lead us to conclude that the lockjaw peptide is a vertebrate-specific delta-conotoxin, which targets voltage-sensitive sodium channels. The sequence of the peptide, which we designate delta-conotoxin PVIA, is (O = 4-trans-hydroxyproline) EACYAOGTFCGIKOGLCCSEFCLPGVCFG-NH2. This is the first of a diverse spectrum of Conus peptides which are excitotoxins in vertebrate systems.