Title

Structure/activity relationship studies and target identification methodologies for the omega-ACTX-1 neurotoxins

Date of Completion

January 2004

Keywords

Biology, Molecular|Biology, Neuroscience

Degree

Ph.D.

Abstract

The ω-atracotoxin-1 (ω-ACTX-1) neurotoxins are 36–37-residue peptides found in the venom of all species of Australian funnel-web spiders examined to date. The prototypic family member, ω-ACTX-Hv1a, is lethal to a wide range of insects and potently antagonizes high-voltage activated (HVA) calcium channels—a component of the insect nervous system never before targeted by a commercially available insecticide. Yet various lines of evidence suggest that ω-ACTX-Hv1a is nontoxic to vertebrate organisms and has little or no detectable activity against many, if not all, vertebrate nervous system components. Thus, continued characterization of the ω-ACTX-1's should enable better understanding of the insect nervous system and perhaps also facilitate development of selective agents which kill insect pests with minimal collateral damage to the environment. ^ Described herein is the development of a plasmid-based, bacterial expression system for ω-ACTX-Hv1a. Also described are procedures which yielded detailed characterizations of the structure and bioactive surface of ω-ACTX-Hv1a. These included heteronuclear NMR experiments which enabled the toxin structure to be determined with extremely high precision, and scanning mutagenesis which facilitated identification of several functionally critical regions in the toxin structure. ^ Also described is the development of methods and strategies which may enable definitive identification of the molecular target of the ω-ACTX-1's: First, stocks of Drosophila melanogaster carrying inducible transgenes encoding ω-ACTX-Hv1a were genetically engineered and propagated. The behavior of these stocks in response to transgene activity was consistent with antagonism of the Dmca1D subfamily of calcium channels, offering one of several lines of evidence presented and/or discussed herein that these channels represent the primary targets of the ω-ACTX-1 toxins. Second, facile methods were developed for labeling ω-ACTX-Hv1a with tracers and/or affinity tags without compromising its biological activity. Third, Anopheles gambiae cDNAs encoding subunits of a Dmca1D family calcium channel were subcloned. A heterologous calcium channel expression system based on these cDNAs could enable definitive confirmation that the ω-ACTX-1 family selectively targets Dmca1D channels and could provide a valuable resource both for toxin-target interaction studies and for discovery of novel small molecule insecticides. ^

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