title: Molecular analysis of the sea anemone toxin Av3 reveals selectivity to insects and demonstrates the heterogeneity of receptor site-3 on voltage-gated Na+ channels.
abstract: Av3 is a short peptide toxin from the sea anemone Anemonia viridis shown to be active on crustaceans and inactive on mammals. It inhibits inactivation of Na(v)s (voltage-gated Na+ channels) like the structurally dissimilar scorpion alpha-toxins and type I sea anemone toxins that bind to receptor site-3. To examine the potency and mode of interaction of Av3 with insect Na(v)s, we established a system for its expression, mutagenized it throughout, and analysed it in toxicity, binding and electrophysiological assays. The recombinant Av3 was found to be highly toxic to blowfly larvae (ED50=2.65+/-0.46 pmol/100 mg), to compete well with the site-3 toxin LqhalphaIT (from the scorpion Leiurus quinquestriatus) on binding to cockroach neuronal membranes (K(i)=21.4+/-7.1 nM), and to inhibit the inactivation of Drosophila melanogaster channel, DmNa(v)1, but not that of mammalian Na(v)s expressed in Xenopus oocytes. Moreover, like other site-3 toxins, the activity of Av3 was synergically enhanced by ligands of receptor site-4 (e.g. scorpion beta-toxins). The bioactive surface of Av3 was found to consist mainly of aromatic residues and did not resemble any of the bioactive surfaces of other site-3 toxins. These analyses have portrayed a toxin that might interact with receptor site-3 in a different fashion compared with other ligands of this site. This assumption was corroborated by a D1701R mutation in DmNa(v)1, which has been shown to abolish the activity of all other site-3 ligands, except Av3. All in all, the present study provides further evidence for the heterogeneity of receptor site-3, and raises Av3 as a unique model for design of selective anti-insect compounds.
authors.affiliation: Department of Plant Sciences, George S. Wise Faculty of Life Sciences, Tel-Aviv University, Ramat-Aviv 69978, Tel-Aviv, Israel.
authors.forename: Yehu
authors.initials: Y
authors.lastname: Moran
authors.forename: Roy
authors.initials: R
authors.lastname: Kahn
authors.forename: Lior
authors.initials: L
authors.lastname: Cohen
authors.forename: Maya
authors.initials: M
authors.lastname: Gur
authors.forename: Izhar
authors.initials: I
authors.lastname: Karbat
authors.forename: Dalia
authors.initials: D
authors.lastname: Gordon
authors.forename: Michael
authors.initials: M
authors.lastname: Gurevitz
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001120:Arginine / Q000235:genetics; D001224:Aspartic Acid / Q000235:genetics; D002942:Circular Dichroism; D003064:Cnidarian Venoms / Q000737:chemistry* / Q000378:metabolism / Q000494:pharmacology*; D003058:Cockroaches / Q000187:drug effects; D004331:Drosophila melanogaster / Q000378:metabolism; D007313:Insecta / Q000187:drug effects* / Q000378:metabolism; D015640:Ion Channel Gating / Q000187:drug effects; D007814:Larva / Q000187:drug effects; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009154:Mutation / Q000235:genetics; D009865:Oocytes / Q000187:drug effects; D011956:Receptors, Cell Surface / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry; D012615:Sea Anemones / Q000737:chemistry / Q000378:metabolism*; D015222:Sodium Channels / Q000378:metabolism*; D013045:Species Specificity; D014981:Xenopus
chemical_list: D003064:Cnidarian Venoms; D011956:Receptors, Cell Surface; D011994:Recombinant Proteins; D015222:Sodium Channels; D001224:Aspartic Acid; D001120:Arginine