Novel conopeptides of the I-superfamily occur in several clades of cone snails

The I-superfamily of conotoxins represents a new class of peptides in the venom of some Conus species. These toxins are characterized by four disulfide bridges and inhibit or modify ion channels of nerve cells. When testing venoms from 11 Conus species for a functional characterization, blocking activity on potassium channels (like Kv1.1 and Kv1.3 channels, but not Kv1.2 channels) was detected in the venom of Conus capitaneus, Conus miles, Conus vexillum and Conus virgo. Analysis at the cDNA level of these venoms using primers designed according to the amino acid sequence of a potassium channel blocking toxin (ViTx) from C. virgo confirmed the presence of structurally homologous peptides in these venoms. Moreover, peptides belonging to the I-superfamily, but with divergent amino acid sequences, were found in Conus striatus and Conus imperialis. In all cases, the sequences of the precursors' prepro-regions exhibited high conservation, whereas the sequences of the mature peptides ranged from almost identical to highly divergent between species. We then performed phylogenetic analyses of new and published mitochondrial 16S rDNA sequences representing 104 haplotypes from these and numerous other Conus species, using Bayesian, maximum-likelihood, maximum-parsimony and neighbor-joining methods of inference. Cone snails known to possess I-superfamily toxins were assigned to five different major clades in all of the resulting gene trees. Moreover, I-superfamily conopeptides were detected both in vermivorous and piscivorous species of Conus, thus demonstrating the widespread presence of such toxins in this speciose genus beyond evolutionary and ecological groups

Characterization of a novel alpha-conotoxin TxID from Conus textile that potently blocks rat alpha3/beta4 nicotinic acetylcholine receptors

The alpha 3 beta 4 nAChRs are implicated in pain sensation in the PNS and addiction to nicotine in the CNS. We identified an alpha-4/6-conotoxin (CTx) TxID from Conus textile. The new toxin consists of 15 amino acid residues with two disulfide bonds. TxID was synthesized using solid phase methods, and the synthetic peptide was functionally tested on nAChRs heterologously expressed in Xenopus laevis oocytes. TxID blocked rat alpha 3 beta 4 nAChRs with a 12.5 nM IC50, which places it among the most potent alpha 3 beta 4 nAChR antagonists. TxID also blocked the closely related alpha 6/alpha 3 beta 4 with a 94 nM IC50 but showed little activity on other nAChR subtypes. NMR analysis showed that two major structural isomers exist in solution, one of which adopts a regular alpha-CTx fold but with different surface charge distribution to other 4/6 family members. alpha-CTx TxID is a novel tool with which to probe the structure and function of alpha 3 beta 4 nAChRs

Mutation in S6 domain of HCN4 channel in patient with suspected Brugada syndrome modifies channel function

Diseases such as the sick sinus and the Brugada syndrome are cardiac abnormalities, which can be caused by a number of genetic aberrances. Among them are mutations in HCN4, a gene, which encodes the hyperpolarization-activated, cyclic nucleotide-gated ion channel 4; this pacemaker channel is responsible for the spontaneous activity of the sinoatrial node. The present genetic screening of patients with suspected or diagnosed Brugada or sick sinus syndrome identified in 1 out of 62 samples the novel mutation V492F. It is located in a highly conserved site of hyperpolarization-activated cyclic nucleotide-gated (HCN)4 channel downstream of the filter at the start of the last transmembrane domain S6. Functional expression of mutant channels in HEK293 cells uncovered a profoundly reduced channel function but no appreciable impact on channel synthesis and trafficking compared to the wild type. The inward rectifying HCN4 current could be partially rescued by an expression of heteromeric channels comprising wt and mutant monomers. These heteromeric channels were responsive to cAMP but they required a more negative voltage for activation and they exhibited a lower current density than the wt channel. This suggests a dominant negative effect of the mutation in patients, which carry this heterozygous mutation. Such a modulation of HCN4 activity could be the cause of the diagnosed cardiac abnormality

Venomic study on cone snails (Conus spp.) from South Africa

From six Conus species (Conus coronatus, Conus lividus, Conus mozambicus f. lautus, Conus pictus, Conus sazanka, Con us tinianus) collected off the eastern coast of South Africa the venoms were analyzed using MALDI-TOF mass spectrometry. Between 56 and 151 molecular masses most in a range of 1000 to 2500 Da, were identified. Among the six venoms, between 0 and 27% (C coronatus versus C. sazanka) of the peptide masses were found to be similar. In a study on venoms from 6 Conus species collected in the Philippines, the percentage of identical masses was between none and 9% only. The venoms from the South African Conus species antagonized the rat neuronal nicotinic acetylcholine receptors (nAChRs) alpha 3 beta 2, alpha 4 beta 2, and alpha 7, except for C coronatus venom that blocked the alpha 4 beta 2 and alpha 7 nAChRs only. HPLC-fractionation of C tinianus venom led to the isolation of a peptide that is active on all three receptor subtypes. It consists of 16 amino acid residues crosslinked by two disulfide bridges as revealed by de novo sequencing using tandem mass spectrometry: GGCCSHPACQNNPDYC. Posttranslational modifications include C-terminal amidation and tyrosine sulfation. The new peptide is a member of the alpha-conotoxin family that are competitive antagonists of nAChRs. Phylogenetic analysis of the 16S RNA from numerous Conus species has clarified the evolutionary position of endemic South African Conus species and provided the first evidence for their close genetic relationships. (C) 2010 Elsevier Ltd. All rights reserved

Molecular phylogeny and evolution of the cone snails (Gastropoda, Conoidea)

We present a large-scale molecular phylogeny that includes 320 of the 761 recognized valid species of the cone snails (Conus), one of the most diverse groups of marine molluscs, based on three mitochondrial genes (COI, 16S rDNA and 12S rDNA). This is the first phylogeny of the taxon to employ concatenated sequences of several genes, and it includes more than twice as many species as the last published molecular phylogeny of the entire group nearly a decade ago. Most of the numerous molecular phylogenies published during the last 15 years are limited to rather small fractions of its species diversity. Bayesian and maximum likelihood analyses are mostly congruent and confirm the presence of three previously reported highly divergent lineages among cone snails, and one identified here using molecular data. About 85% of the species cluster in the single Large Major Cade; the others are divided between the Small Major Cade (similar to 12%), the Conus califomicus lineage (one species), and a newly defined clade (similar to 3%). We also define several subclades within the Large and Small major clades, but most of their relationships remain poorly supported. To illustrate the usefulness of molecular phylogenies in addressing specific evolutionary questions, we analyse the evolution of the diet, the biogeography and the toxins of cone snails. All cone snails whose feeding biology is known inject venom into large prey animals and swallow them whole. Predation on polychaete worms is inferred as the ancestral state, and diet shifts to molluscs and fishes occurred rarely. The ancestor of cone snails probably originated from the Indo-Pacific; rather few colonisations of other biogeographic provinces have probably occurred. A new classification of the Conidae, based on the molecular phylogeny, is published in an accompanying paper

Genotype-phenotype dilemma in a case of sudden cardiac death with the E1053K mutation and a deletion in the SCN5A gene

Mutations in the cardiac sodium channel gene SCN5A may result in various arrhythmia syndromes such as long QT syndrome type 3 (LQTS), Brugada syndrome (BrS), sick sinus syndrome (SSS), cardiac conduction diseases (CCD) and possibly dilated cardiomyopathy (DCM). In most of these inherited cardiac arrhythmia syndromes the phenotypical expression may range from asymptomatic phenotypes to sudden cardiac death (SCD). A 16-year-old female died during sleep. Autopsy did not reveal any explanation for her death and a genetic analysis was performed. A variant in the SCN5A gene (E1053K) that was previously described as disease causing was detected. Family members are carriers of the same E1053K variant, some even in a homozygous state, but surprisingly did not exhibit any pathological cardiac phenotype. Due to the lack of genotype-phenotype correlation further genetic studies were performed. A novel deletion in the promoter region of SCN5A was identified in the sudden death victim but was absent in other family members. These findings demonstrate the difficulties in interpreting the results of a family-based genetic screening and underline the phenotypic variability of SCN5A mutations. (C) 2017 Elsevier B.V. All rights reserve