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The venom of marine cone snails contains a variety of conformationally constrained peptides utilized by the animal to capture prey. Besides numerous conotoxins, which are characterized by complex disulfide patterns, other peptides with only a single disulfide bridge were isolated from different conus species. Here, we report the synthesis, structure elucidation and biological evaluation of the novel C-terminally amidated decapeptide CCAP-vil, PFc[CNSFGC]YN-NH 2 , from Conus villepinii . The linear precursor peptide was generated by standard solid phase synthesis. Oxidation of the cysteine residues to yield the disulfide-bridged peptide was investigated under different conditions, including several ionic liquids (ILs) as new biocompatible reaction media. Among the examined ILs, 1-ethyl-3-methylimidazolium tosylate ([C 2 mim][OTs]) was most efficient for CCAP-vil oxidative folding, since oxidation occurred without any byproduct formation. The structure of CCAP-vil was determined by NMR methods in aqueous solution and revealed a loop structure adopting a type(I) β-turn between residues 4–7 imposed by the flanking disulfide bridge. The amino acid side chains of Pro 1 , Phe 2 , Phe 6 and Tyr 9 point in three directions away from the cyclic core into the solvent creating a rather hydrophobic surface of the molecule. Based on sequence homology to cardioactive peptides (CAPs) from gastropods and arthropods, such as PFc[CNAFTGC]-NH 2 (CCAP), the influence of CCAP-vil on heart rate using zebrafish embryos was investigated. CCAP-vil reduced the heart rate immediately upon injection into the heart as well as upon indirect application indicating an opposite effect to the cardioaccelerating CCAP.

The physiological role of KCNH1 channels has not been clarified in detail yet, but the predominant neuronal expression in mammals indicates a role in electrical signaling. The only described physiological function of KCNH1 so far is the promotion of myoblast fusion. Moreover, KCNH1 channels apparently enhance proliferation of cancer cells, thus exhibiting oncogenic potential. Many genes involved in cancerogenesis play a physiological role during embryonic development. To study KCNH1 channel function in this respect, we surveyed genomic databases of Danio rerio, a widely used vertebrate-development model organism, and found two putative kcnh1 paralogs located on chromosomes 17 (kcnh1a) and 22 (kcnh1b). The corresponding amino acid sequences show >80% identity to that of human KCNH1. We observed both kcnh1 genes to be endogenously expressed in different organs of adult fish and at early developmental stages, most prominently in neuronal tissues. We electrophysiologically analyzed the encoded channels by two-electrode voltage-clamp and patch-clamp techniques on Xenopus oocytes, showing the gene products to form functional potassium channels. The Danio rerio channels exhibited typical characteristics known from other species, such as dependence of activation kinetics on prepulse potential and extracellular Mg2+ concentration, as well as current inhibition by intracellular Ca2+/calmodulin. After morpholino-mediated knockdown of the individual kcnh1 paralogs in zebrafish embryos we observed severe effects on development. The morphants were retarded in growth, showed abnormal body curvature, exhibited complete body malformation or even died within less than 24 hrs after fertilization, depending on the degree of knockdown. The most remarkable phenotypes were alterations of brain structures like head edemas, incomplete brain growth and necrotic degeneration. Given the electrophysiological similarity of Kcnh1 channels to the human homolog, we suggest that human KCNH1 potassium channels play a similar role during early embryonic development in humans.