To paralyze their more agile prey, the venomous fish-hunting cone snails (Conus) have developed a potent biochemical strategy. They produce several classes of toxic peptides (conotoxins) that attack a series of successive physiological targets in the neuromuscular system of the fish. The peptides include presynaptic omega-conotoxins that prevent the voltage-activated entry of calcium into the nerve terminal and release of acetylcholine, postsynaptic alpha-conotoxins that inhibit the acetylcholine receptor, and muscle sodium channel inhibitors, the mu-conotoxins, which directly abolish muscle action potentials. These distinct peptide toxins share several common features: they are relatively small (13 to 29 amino acids), are highly cross-linked by disulfide bonds, and strongly basic. The fact that they inhibit sequential steps in neuromuscular transmission suggests that their action is synergistic rather than additive. Five new omega-conotoxins that block presynaptic calcium channels are described. They vary in their activity against different vertebrate classes, and also in their actions against different synapses from the same animal. There are susceptible forms of the target molecule in peripheral synapses of fish and amphibians, but those of mice are resistant. However, the mammalian central nervous system is clearly affected, and these toxins are thus of potential significance for investigating the presynaptic calcium channels.

A novel toxin, omega-conotoxin (omega-CgTX), from the venom of the fish-eating marine mollusc Conus geographus has been purified and biochemically characterized. Recently, this omega-conotoxin has been shown to inhibit the voltage-activated entry of Ca2+, thus providing a potentially powerful probe for exploring the vertebrate presynaptic terminal [Kerr, L. M., & Yoshikami, D. (1984) Nature (London) 308, 282-284]. The toxin is a basic 27 amino acid peptide amide with three disulfide bridges. An unusual feature is a remarkable preponderance of hydroxylated amino acids. The sequence of omega-CgTx GVIA is Cys-Lys-Ser- Hyp-Gly5-Ser-Ser-Cys-Ser-Hyp10-Thr-Ser-Tyr-Asn-Cys15-C ys-Arg-Ser- Cys-Asn20-Hyp-Tyr-Thr-Lys-Arg25-Cys-Tyr-NH2.

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 micromethod involving the use of fluorescent derivatives of 1-dimethylaminonaphthalene-5-sulphonyl chloride (Gray & Hartley, 1963) has been used in a parallel study of the structure of peptide C8Pla (as defined by Ambler, 1963) of the Pseudomonas cytochrome c-551. The basis of the method was a modification of the phenyl isothiocyanate procedure for the removal of the N-terminal residue (Edman, 1950 a, b), followed by investigation of the free amino groups of the residual peptide The very high sensitivity of the fluorescent end-group reagent used enabled the subtractive procedure to be applied over six consecutive steps, commencing with approx. 0-02 timole of peptide. The partial sequence Gly(Pro2,Ileu)-Met-Pro-Pro-Asp(NH2)-Ala had already been determined by cyanogen bromide cleavage and partial acid hydrolysis (Ambler, 1963).The peptide (0-02 ,imole) was oxidized with performic acid, to convert methionine into its sulphone. The oxidized peptide is referred to below as CPO, and in subsequent stages of degradation as CPOb1, CPO,2, according to the convention adopted by Ambler (1963). Interaction of peptides with the sulphonyl chloride attaches the fluorescent dimethylaminonaphthalenesulphonyl residue to free amino groups.Abbreviations. The 1-dimethylaminonaphthalene-5-sulphonyl derivatives are indicated by the term DNS-.Phenyl isothiocyanate degradation. The oxidized peptide (CPO) was dissolved in 0 1 ml. of water in a 5 ml. stoppered tube, and 10 ul. was removed for end-group studies. To the remainder was added 0-2 ml. of a solution of phenyl isothiocyanate in pyridine (5 %, v/v). The resulting single-phase mixture was incubated overnight at room temperature, and, after the addition of 0-2 ml. of water, the pyridine and excess of phenyl isothiocyanate were removed by four extractions with 3 ml. of benzene.The aqueous solution was then freeze-dried in situ, and 0-2 ml. of acetic acid saturated with anhydrous hydrogen chloride was added. After 30 min. at room temperature, the acid was removed in vacuo and the residual peptide dissolved in 0-1 ml. of water. A portion corresponding to one-tenth of the original material was removed for end-group studies, and the remainder resubmitted to the degradation procedure. At each successive cycle a similar portion was removed.N-Terminal studies. Samples from each cycle of the phenyl isothiocyanate degradation, corresponding to approx. 2 ,um-moles, were placed in small fermentation tubes containing 10 ,u. of 0 1 Msodium hydrogen carbonate in ammonia-free water.To each of these was added 15 ,l. of a 1 % (w/v) solution of 1-dimethylaminonaphthalene-5-sulphonyl chloride in acetone, forming a single-phase reaction mixture. After 2 hr. at room temperature, during which time all excess of reagent had been hydrolysed to sulphonic acid, the mixture was applied as a 2 cm. band to a strip of WVhatman no. 3 MM paper. High-voltage ionophoresis was then carried out for 1-5 hr. at 120 v/cm., with the apparatus describedby Gross (1961). The buffer used was 10 % (v/v) ofpyridine in 0 4 % ace...

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.