Intraspecific variation of venom injected by fish-hunting Conus snails. (1/96)

Venom peptides from two species of fish-hunting cone snails (Conus striatus and Conus catus) were characterized using microbore liquid chromatography coupled with matrix-assisted laser desorption/ionization-time of flight-mass spectrometry and electrospray ionization-ion trap-mass spectrometry. Both crude venom isolated from the venom duct and injected venom obtained by milking were studied. Based on analysis of injected venom samples from individual snails, significant intraspecific variation (i.e. between individuals) in the peptide complement is observed. The mixture of peptides in injected venom is simpler than that in the crude duct venom from the same snail, and the composition of crude venom is more consistent from snail to snail. While there is animal-to-animal variation in the peptides present in the injected venom, the composition of any individual's injected venom remains relatively constant over time in captivity. Most of the Conus striatus individuals tested injected predominantly a combination of two neuroexcitatory peptides (s4a and s4b), while a few individuals had unique injected-venom profiles consisting of a combination of peptides, including several previously characterized from the venom duct of this species. Seven novel peptides were also putatively identified based on matches of their empirically derived masses to those predicted by published cDNA sequences. Profiling injected venom of Conus catus individuals using matrix-assisted laser desorption/ionization-time of flight-mass spectrometry demonstrates that intraspecific variation in the mixture of peptides extends to other species of piscivorous cone snails. The results of this study imply that novel regulatory mechanisms exist to select specific venom peptides for injection into prey.  (+info)

Association/dissociation of a channel-kinase complex underlies state-dependent modulation. (2/96)

Although ion channels are regulated by protein kinases, it has yet to be established whether the behavioral state of an animal may dictate whether or not modulation by a kinase can occur. Here, we describe behaviorally relevant changes in the ability of a nonselective cation channel from Aplysia bag cell neurons to be regulated by protein kinase C (PKC). This channel drives a prolonged afterdischarge that triggers the release of egg-laying hormone and a series of reproductive behaviors. The afterdischarge is followed by a lengthy refractory period, during which additional bursting cannot be elicited. Previously, we reported that, in excised inside-out patches, the cation channel is closely associated with PKC, which increases channel activity. We now show that this channel-kinase association is plastic, because channels excised from certain neurons lack PKC-dependent modulation. Although direct application of PKC-activating phorbol ester to these patches had no effect, exposing the neurons themselves to phorbol ester reinstated modulation, suggesting that an absence of modulation was attributable to a lack of associated kinase. Furthermore, modulation was restored by pretreating neurons with either PP1 [4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine] or SU6656, inhibitors of Src tyrosine kinase, an enzyme whose Src homology 3 domain is required for channel-PKC association. Neurons that were stimulated to afterdischarge and had entered the prolonged refractory period were found to have more phosphotyrosine staining and less channel-PKC association than unstimulated neurons. These findings suggest that Src-dependent regulation of the association between the cation channel and PKC controls both the long-term excitability of these neurons and their ability to induce reproduction.  (+info)

Piscivorous behavior of a temperate cone snail, Conus californicus. (3/96)

Most of the more than 500 species of predatory marine snails in the genus Conus are tropical or semitropical, and nearly all are thought to be highly selective regarding type of prey. Conus californicus Hinds, 1844, is unusual in that it is endemic to the North American Pacific coast and preys on a large variety of benthic organisms, primarily worms and other molluscs, and also scavenges. We studied the feeding behavior of C. californicus in captivity and found that it regularly killed and consumed live prickleback fishes (Cebidichthys violaceus and Xiphister spp.). Predation involved two behavioral methods similar to those employed by strictly piscivorous relatives. One method utilized stings delivered by radular teeth; the other involved engulfing the prey without stinging. Both methods were commonly used in combination, and individual snails sometimes employed multiple stings to subdue a fish. During the course of the study, snails became aroused by the presence of live fish more quickly, as evidenced by more rapid initiation of hunting behavior. Despite this apparent adaptation, details of prey-capture techniques and effectiveness of stings remained similar over the same period.  (+info)

The muO-conotoxin MrVIA inhibits voltage-gated sodium channels by associating with domain-3. (4/96)

Several families of peptide toxins from cone snails affect voltage-gated sodium (Na(V)) channels: mu-conotoxins block the pore, delta-conotoxins inhibit channel inactivation, and muO-conotoxins inhibit Na(V) channels by an unknown mechanism. The only currently known muO-conotoxins MrVIA and MrVIB from Conus marmoreus were applied to cloned rat skeletal muscle (Na(V)1.4) and brain (Na(V)1.2) sodium channels in mammalian cells. A systematic domain-swapping strategy identified the C-terminal pore loop of domain-3 as the major determinant for Na(V)1.4 being more potently blocked than Na(V)1.2 channels. muO-conotoxins therefore show an interaction pattern with Na(V) channels that is clearly different from the related mu- and delta-conotoxins, indicative of a distinct molecular mechanism of channel inhibition.  (+info)

cDNA cloning of two novel T-superfamily conotoxins from Conus leopardus. (5/96)

The full-length cDNAs of two novel T-superfamily conotoxins, Lp5.1 and Lp5.2, were cloned from a vermivorous cone snail Conus leopardus using 3'/5'-rapid amplification of cDNA ends. The cDNA of Lp5.1 encodes a precursor of 65 residues, including a 22-residue signal peptide, a 28-residue propeptide and a 15-residue mature peptide. Lp5.1 is processed at the common signal site-X-Arg- immediately before the mature peptide sequences. In the case of Lp5.2, the precursor includes a 25-residue signal peptide and a 43-residue sequence comprising the propeptide and mature peptide, which is probably cleaved to yield a 29-residue propeptide and a 14-residue mature toxin. Although these two conotoxins share a similar signal sequence and a conserved disulfide pattern with the known T-superfamily, the pro-region and mature peptides are of low identity, especially Lp5.2 with an identity as low as 10.7% compared with the reference Mr5.1a. The elucidated cDNAs of these two toxins will facilitate a better understanding of the species distribution, the sequence diversity of T-superfamily conotoxins, the special gene structure and the evolution of these peptides.  (+info)

Isolation and characterisation of conomap-Vt, a D-amino acid containing excitatory peptide from the venom of a vermivorous cone snail. (6/96)

Cone snail venom is a rich source of bioactives, in particular small disulfide rich peptides that disrupt synaptic transmission. Here, we report the discovery of conomap-Vt (Conp-Vt), an unusual linear tetradecapeptide isolated from Conus vitulinus venom. The sequence displays no homology to known conopeptides, but displays significant homology to peptides of the MATP (myoactive tetradecapeptide) family, which are important endogenous neuromodulators in molluscs, annelids and insects. Conp-Vt showed potent excitatory activity in several snail isolated tissue preparations. Similar to ACh, repeated doses of Conp-Vt were tachyphylactic. Since nicotinic and muscarinic antagonists failed to block its effect and Conp-Vt desensitised tissue remained responsive to ACh, it appears that Conp-Vt contractions were non-cholinergic in origin. Finally, biochemical studies revealed that Conp-Vt is the first member of the MATP family with a d-amino acid. Interestingly, the isomerization of L-Phe to D-Phe enhanced biological activity, suggesting that this post-translational modified conopeptide may have evolved for prey capture.  (+info)

Diversity and evolution of conotoxins based on gene expression profiling of Conus litteratus. (7/96)

Cone snails are attracting increasing scientific attention due to their unprecedented diversity of invaluable channel-targeted peptides. As arguably the largest and most successful evolutionary genus of invertebrates, Conus also may become the model system to study the evolution of multigene families and biodiversity. Here, a set of 897 expressed sequence tags (ESTs) derived from a Conus litteratus venom duct was analyzed to illuminate the diversity and evolution mechanism of conotoxins. Nearly half of these ESTs represent the coding sequences of conotoxins, which were grouped into 42 novel conotoxin cDNA sequences (seven superfamilies), with T-superfamily conotoxins being the dominant component. The gene expression profile of conotoxin revealed that transcripts are expressed with order-of-magnitude differences, sequence divergence within a superfamily increases from the N to the C terminus of the open reading frame, and even multiple scaffold-different mature peptides exist in a conotoxin gene superfamily. Most excitingly, we identified a novel conotoxin superfamily and three novel cysteine scaffolds. These results give an initial insight into the C. litteratus transcriptome that will contribute to a better understanding of conotoxin evolution and the study of the cone snail genome in the near future.  (+info)

Two toxins from Conus striatus that individually induce tetanic paralysis. (8/96)

We describe structural properties and biological activities of two related O-glycosylated peptide toxins isolated from injected (milked) venom of Conus striatus, a piscivorous snail that captures prey by injecting a venom that induces a violent, spastic paralysis. One 30 amino acid toxin is identified as kappaA-SIVA (termed s4a here), and another 37 amino acid toxin, s4b, corresponds to a putative peptide encoded by a previously reported cDNA. We confirm the amino acid sequences and carry out structural analyses of both mature toxins using multiple mass spectrometric techniques. These include electrospray ionization ion-trap mass spectrometry and nanoelectrospray techniques for small volume samples, as well as matrix-assisted laser desorption/ionization time of flight mass spectrometric analysis as a complementary method to assist in the determination of posttranslational modifications, including O-linked glycosylation. Physiological experiments indicate that both s4a and s4b induce intense repetitive firing of the frog neuromuscular junction, leading to a tetanic contracture in muscle fiber. These effects apparently involve modification of voltage-gated sodium channels in motor axons. Notably, application of either s4a or s4b alone mimics the biological effects of the whole injected venom on fish prey.  (+info)

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