title: Domain 2 of Drosophila para voltage-gated sodium channel confers insect properties to a rat brain channel.
abstract: The ability of the excitatory anti-insect-selective scorpion toxin AahIT (Androctonus australis hector) to exclusively bind to and modify the insect voltage-gated sodium channel (NaCh) makes it a unique tool to unravel the structural differences between mammalian and insect channels, a prerequisite in the design of selective pesticides. To localize the insect NaCh domain that binds AahIT, we constructed a chimeric channel composed of rat brain NaCh alpha-subunit (rBIIA) in which domain-2 (D2) was replaced by that of Drosophila Para (paralytic temperature-sensitive). The choice of D2 was dictated by the similarity between AahIT and scorpion beta-toxins pertaining to both their binding and action and the essential role of D2 in the beta-toxins binding site on mammalian channels. Expression of the chimera rBIIA-ParaD2 in Xenopus oocytes gave rise to voltage-gated and TTX-sensitive NaChs that, like rBIIA, were sensitive to scorpion alpha-toxins and regulated by the auxiliary subunit beta(1) but not by the insect TipE. Notably, like Drosophila Para/TipE, but unlike rBIIA/beta(1), the chimera gained sensitivity to AahIT, indicating that the phyletic selectivity of AahIT is conferred by the insect NaCh D2. Furthermore, the chimera acquired additional insect channel properties; its activation was shifted to more positive potentials, and the effect of alpha-toxins was potentiated. Our results highlight the key role of D2 in the selective recognition of anti-insect excitatory toxins and in the modulation of NaCh gating. We also provide a methodological approach to the study of ion channels that are difficult to express in model expression systems.
authors.affiliation: Department of Physiology, Sackler School of Medicine and Plant Sciences, George S. Wise Faculty of Life Sciences, Tel-Aviv University, 69978 Ramat-Aviv, Israel.
authors.forename: Iris
authors.initials: I
authors.lastname: Shichor
authors.forename: Eliahu
authors.initials: E
authors.lastname: Zlotkin
authors.forename: Nitza
authors.initials: N
authors.lastname: Ilan
authors.forename: Dodo
authors.initials: D
authors.lastname: Chikashvili
authors.forename: Walter
authors.initials: W
authors.lastname: Stuhmer
authors.forename: Dalia
authors.initials: D
authors.lastname: Gordon
authors.forename: Ilana
authors.initials: I
authors.lastname: Lotan
mesh_terms: D000818:Animals; D004330:Drosophila; D015870:Gene Expression; D019476:Insect Proteins / Q000187:drug effects / Q000235:genetics / Q000502:physiology*; D015640:Ion Channel Gating / Q000187:drug effects; D008564:Membrane Potentials / Q000187:drug effects; D009498:Neurotoxins / Q000494:pharmacology; D009865:Oocytes / Q000187:drug effects / Q000378:metabolism; D018408:Patch-Clamp Techniques; D017434:Protein Structure, Tertiary / Q000502:physiology; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000187:drug effects / Q000235:genetics / Q000502:physiology*; D012604:Scorpion Venoms / Q000494:pharmacology; D015222:Sodium Channels / Q000187:drug effects / Q000235:genetics / Q000502:physiology*; D013045:Species Specificity; D013329:Structure-Activity Relationship; D013779:Tetrodotoxin / Q000494:pharmacology; D014162:Transfection; D014981:Xenopus
chemical_list: D019476:Insect Proteins; D009498:Neurotoxins; D011993:Recombinant Fusion Proteins; D012604:Scorpion Venoms; D015222:Sodium Channels; C065314:scorpion toxin I, Androctonus; D013779:Tetrodotoxin