Conotoxin GIIIA: selective inhibition of 22Na influx via voltage-dependent Na channels in adrenal medullary cells

Summary Conotoxin GIIIA and GIIIB from the marine snail Conus geographus have been reported to inhibit voltage-dependent Na channels in skeletal muscle and postganglionic sympathetic neuron, but have no effect on Na channels in brain, giant axon and heart. In eel electroplax, conotoxins were also shown to share the common binding sites with saxitoxin (see review Gray et al. 1988).In bovine adrenal medullary cells, conotoxin GIIIA inhibited veratridine-induced influx of ²²Na, ⁴⁵Ca and secretion of catecholamines with an IC₅₀ of 6 mol/l, while saxitoxin suppressed veratridine-induced responses with an IC₅₀ of 6.3 nmol/l. [³H]Saxitoxin binding to the cells was inhibited by unlabeled saxitoxin with an IC₅₀ of 5.1 nmol/l, but was slightly reduced by 10 mol/l conotoxin GIIIA. Conotoxin GIIIA, at 10 mol/l, did not alter carbachol-induced influx of ²²Na, ⁴⁵Ca and secretion of catecholamines as well as high K-induced ⁴⁵Ca influx and catecholamine secretion.These results indicate that conotoxin GIIIA, at concentrations 950 fold higher than saxitoxin, inhibits Na influx via voltage-dependent Na channels, but has no effect on the nicotinic receptor-ion channel complex or the voltage-dependent Ca channels. Conotoxin GIIIA seems to bind at the sites which are distinct from saxitoxin, but are functionally linked to the voltage-dependent Na channels. Conotoxins may be useful for the classification of Na channels in excitable cell membranes. Send offprint requests to A. Wada at the above address     

Conopeptide characterization and classifications: An analysis using ConoServer

Cone snails are carnivorous marine gastropods that have evolved potent venoms to capture their prey. These venoms comprise a rich and diverse cocktail of peptide toxins, or conopeptides, whose high diversity has arisen from an efficient hypermutation mechanism, combined with a high frequency of post-translational modifications. Conopeptides bind with high specificity to distinct membrane receptors, ion channels, and transporters of the central and muscular nervous system. As well as serving their natural function in prey capture, conopeptides have been utilized as versatile tools in neuroscience and have proven valuable as drug leads that target the nervous system in humans. This paper examines current knowledge on conopeptide sequences based on an analysis of gene and peptide sequences in ConoServer (http://www.conoserver.org), a specialized database of conopeptide sequences and three-dimensional structures. We describe updates to the content and organization of ConoServer and discuss correlations between gene superfamilies, cysteine frameworks, pharmacological families targeted by conopeptides, and the phylogeny, habitat, and diet of cone snails. The study identifies gaps in current knowledge of conopeptides and points to potential directions for future research.     

Mass spectrometric and high performance liquid chromatography profiling of the venom of the Brazilian vermivorous mollusk Conus regius: feeding behavior and identification of one novel conotoxin.

Carnivorous mollusks belonging to the genus Conus paralyze their prey by injecting a rich mixture of biologically active peptides. Conus regius is a vermivorous member of this genus that inhabits Brazilian tropical waters. Inter-, intra-species and individual variations of cone snail venom have been previously reported. In order to investigate intra-specific differences in C. regius venom, its feeding behavior and the correlation between these two factors, animals were pooled according to gender, size and season of collection, and their venom composition was compared by high performance liquid chromatography (HPLC). Both the whole venom and one specific peak were monitored by HPLC. Chromatographic profiles revealed no significant differences in their peak areas, indicating that the venom composition, based solely in the presence or absence of the major peaks, is stable regardless of season, gender and size. Therefore, analysis of one given toxin, eluting in one of the major peaks, is representative among the population. Moreover, this work presents the identification of one novel conotoxin (rg11a), which amino acid sequence was deduced by mass spectrometry.     

The calcium channel antagonist, ω-conotoxin, and electric organ nerve terminals: binding and inhibition of transmitter release and calcium influx

We have previously shown that the calcium channel antagonist ω-conotoxin M-VII-A blocks neurotransmitter release from isolated nerve terminals (synaptosomes) from the electric organ of the electric ray (Yeager et al., J. Neurosci., 7 (1987) 2390–2396). We now demonstrate that a related but more readily available peptide, ω-conotoxin G-VI-A (CgTx), also blocks the release of transmitter from these terminals and, in addition, inhibits depolarization-dependent uptake of Ca²⁺ into these terminals. The half-maximal inhibitory concentration (IC₅₀ for block of depolarization-evoked release and for depolarization-dependent uptake of Ca²⁺ are approximately 3 and 2 μM, respectively. These results suggest the inhibitory effects of CgTx are due to inhibition of Ca²⁺ entry into synaptosomes through voltage-sensitive calcium channels. Assays of radioiodinated CgTx binding to electric organ synaptosomal membranes and synaptosomes appear to show a single binding site with a apparent dissociation constant (K_d of 3–5 μM and toxin receptor densities of 290 and 52 pmol/mg protein, respectively. These CgTx receptor densities are equivalent to 6% of the total synaptosomal membrane protein and 1% of the total synaptosomal protein (assuming a molecular weight of 200 kDa for the toxin receptor). If the observed CgTx receptor densities reflect the actual densities of voltage-sensitive calcium channels in electric organ synaptosomal membranes and synaptosomes, these preparations would be the richest source of these channels yet described.     

A novel conotoxin inhibiting vertebrate voltage-sensitive potassium channels.

Toxins from cone snail (Conus species) venoms are multiple disulfide bonded peptides. Based on their pharmacological target (ion channels, receptors) and their disulfide pattern, they have been classified into several toxin families and superfamilies. Here, we report a new conotoxin, which is the first member of a structurally new superfamily of Conus peptides and the first conotoxin affecting vertebrate K+ channels. The new toxin, designated conotoxin ViTx, has been isolated from the venom of Conus virgo and comprises a single chain of 35 amino acids cross-linked by four disulfide bridges. Its amino acid sequence (SRCFPPGIYCTSYLPCCWGICCSTCRNVCHLRIGK) was partially determined by Edman degradation and deduced from the nucleotide sequence of the toxin cDNA. Nucleic acid sequencing also revealed a prepropeptide comprising 67 amino acid residues and demonstrated a posttranslational modification of the protein by releasing a six-residue peptide from the C-terminal. Voltage clamp studies on various ion channels indicated that the toxin inhibits the vertebrate K+ channels Kv1.1 and Kv1.3 but not Kv1.2. The chemically synthesized product exhibited the same physiological activity and identical molecular mass (3933.7 Da) as the native toxin.     

Discovery and characterization of the short kappaA-conotoxins: a novel subfamily of excitatory conotoxins.

We have characterized the defining members of a novel subfamily of excitatory conotoxins, the short kappaA-conotoxins (kappaA(S)-conotoxins). kappaA-conotoxins PIVE and PIVF (kappaA-PIVE and kappaA-PIVF) were purified from Conus purpurascens venom. Both peptides elicited excitatory activity upon injection into fish. kappaA-PIVE was synthesized for further characterization. The excitatory effects of kappaA-PIVE in vivo were dose dependent, causing hyperactivity at low doses and rapid immobilization at high doses, symptomatic of a type of excitotoxic shock. Consistent with these observations, kappaA-PIVE caused repetitive action potentials in frog motor axons in vitro. Similar results have been reported for other structurally distinct conotoxin families; such peptides appear to be required by most fish-hunting cone snails for the rapid immobilization of prey. Unexpected structure-function relationships were revealed between these peptides and two families of homologous conotoxins: the alphaA-conotoxins (muscle nAChR antagonists) and kappaA-conotoxins (excitotoxins), which all share a common arrangement of cysteine residues (CC-C-C-C-C). Biochemically, the kappaA(S)-conotoxins more closely resemble the alphaA(S)-conotoxins than the other kappaA-conotoxin subfamily, the long kappaA-conotoxins (kappaA(L)-conotoxins); however, kappaA(S)- and alphaA(S)-conotoxins produce different physiological effects. In contrast, the kappaA(S)-and kappaA(L)-conotoxins that diverge in several biochemical characteristics are clearly more similar in their physiological effects.     

Gating modifier toxins of voltage-gated calcium channels.

Some of the most potent and specific inhibitors of voltage-gated calcium channels are peptide toxins that inhibit channel function not by occlusion of the channel pore, but rather by interfering with the voltage dependence and kinetics of channel opening and closing. Many such gating modifier toxins conform to the inhibitor cystine knot structural family and have primary sequence or functional mechanism similar to toxins that target voltage-gated sodium or potassium channels. This review introduces known gating modifiers of calcium channels, discusses the selectivity, binding sites, and mechanism of the toxin-channel interaction, and reviews the usefulness of these toxins as research tools and as the basis for novel calcium channel pharmacology and therapeutics.     

Venomous cone snails: molecular phylogeny and the generation of toxin diversity

In order to investigate the generation of conotoxin diversity, δ-conotoxin sequences from nine Conus species were analyzed in the context of their phylogeny. Using a standard molecular marker, mitochondrial 16S RNA, we determined that the δ-conotoxins were derived from three distinct species clades based on the phylogenetic reconstruction of a large set (>80) of Conus species and other toxoglossate molluscs. Four different mechanisms appear to have contributed to the diversity of the δ-conotoxins analyzed: (1) Speciation: δ-Conotoxins in different species diverge from each other (the prepro regions of orthologous genes somewhat more slowly than the reference rRNA rate, the mature toxin regions significantly faster). (2) Duplication: Intraspecific δ-conotoxin divergence is initiated by gene duplication events, some of which may have predated the species itself. (3) Recombination: A novel δ-conotoxin may arise through recombination of two parental δ-contoxin genes. (4) ‘Focal hypermutation’: This sudden, almost saltatory change in sequence is always restricted to the mature toxin region.

The first three have been recognized previously as mechanisms important for the evolution of gene families in other phylogenetic systems; the last is a remarkable, mechanistically unexplained and specialized feature of Conus peptide diversification.     

Fourier transform mass spectrometry: a powerful tool for toxin analysis.

The crude venom of Conus virgo was analyzed by Fourier transform mass spectrometry (FTMS) using both nano-electrospray ionization and MALDI. The analyses were performed directly on the crude venom, without chromatographic separation. The mass fingerprinting of the venom yielded 64 distinct molecular masses in the range 500-4500 Da with two major components at 1328.5142 and 1358.5592 Da. To facilitate the de novo sequencing of these compounds, the disulfide bonds of all components were reduced for the whole venom. The mass accuracy, resolution and sensitivity provided by FTMS were necessary to complete the sequencing of the two new peptides named ViVA and ViVB, that turned out to be conotoxins belonging to the T-superfamily, with the disulfide framework V. The peptides shared 80% similarity and as often observed for this class of compound, they were highly post-translationally modified: amidated C-terminus, pyroglutamic acid residue at the N-terminus and two disulfide bonds. Complementary online nano-LC-nano-ESI-FTMS experiments were undertaken. Among the 130 molecular masses found in the coupling experiments, only 45 were common with those obtained in the direct approach, which means that 21 compounds observed by nano-ESI-FTMS were not detected. This clearly shows that some discriminations against some classes of compounds occur when a chromatographic step is used before mass spectrometry.     

Leu of α-conotoxin PnIB confers potency for neuronal nicotinic responses in bovine chromaffin cells

Two α-conotoxins PnIA and PnIB (previously reported as being “mollusc specific”) which differ in only two amino acid residues (AN versus LS at residues 10 and 11, respectively), show markedly different inhibition of the neuronal nicotinic acetylcholine receptor response in bovine chromaffin cells, a mammalian preparation. Whereas α-conotoxin PnIB completely inhibits the nicotine-evoked catecholamine release at 10 μM, with IC₅₀=0.7 μM, α-conotoxin PnIA is some 30–40 times less potent. Two peptide analogues, [A10L]PnIA and [N11S]PnIA were synthesized to investigate the extent to which each residue contributes to activity. [A10L]PnIA (IC₅₀=2.0 μM) completely inhibits catecholamine release at 10 μM whereas [N11S]PnIA shows little inhibition. In contrast, none of the peptides inhibit muscle-type nicotinic responses in the rat hemi-diaphragm preparation. We conclude that the enhanced potency of α-conotoxin PnIB over α-conotoxin PnIA in the neuronal-type nicotinic response is principally determined by the larger, more hydrophobic leucine residue at position 10 in α-conotoxin PnIB.