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J. Biol. Chem., Vol. 279, Issue 42, 44133-44140, October 15, 2004

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Scanning Mutagenesis of -Atracotoxin-Hv1a Reveals a Spatially Restricted Epitope That Confers Selective Activity against Insect Calcium Channels^*

Hugo W. Tedford, Nicolas Gilles, André Ménez, Clinton J. Doering¶||, Gerald W. Zamponi¶||, and Glenn F. King**

From the Department of Molecular, Microbial, and Structural Biology, University of Connecticut Health Center, Farmington, Connecticut 06032-3305, Departement d'Ingenierie et d'Etudes des Proteines, Commissariat a l'Energie Atomique, Saclay, 91191 Gif-sur-Yvette, France, and ^¶Department of Physiology & Biophysics, Cellular and Molecular Neurobiology Research Group, University of Calgary, 3330 Hospital Dr. NW, Calgary, Alberta T2N 4N1, Canada

We constructed a complete panel of alanine mutants of the insect-specific calcium channel blocker -atracotoxin-Hv1a. Lethality assays using these mutant toxins identified three spatially contiguous residues, Pro¹⁰, Asn²⁷, and Arg³⁵, that are critical for insecticidal activity against flies (Musca domestica) and crickets (Acheta domestica). Competitive binding assays using radiolabeled -atracotoxin-Hv1a and neuronal membranes prepared from the heads of American cockroaches (Periplaneta americana) confirmed the importance of these three residues for binding of the toxin to target calcium channels presumably expressed in the insect membranes. At concentrations up to 10 µM, -atracotoxin-Hv1a had no effect on heterologously expressed rat Ca_v2.1, Ca_v2.2, and Ca_v1.2 calcium channels, consistent with the previously reported insect selectivity of the toxin. 30 µM -atracotoxin-Hv1a inhibited rat Ca_v currents by 10-34%, depending on the channel subtype, and this low level of inhibition was essentially unchanged when Asn²⁷ and Arg³⁵, which appears to be critical for interaction of the toxin with insect Ca_v channels, were both mutated to alanine. We propose that the spatially contiguous epitope formed by Pro¹⁰, Asn²⁷, and Arg³⁵ confers specific binding to insect Ca_v channels and is largely responsible for the remarkable phyletic selectivity of -atracotoxin-Hv1a. This epitope provides a structural template for rational design of chemical insecticides that selectively target insect Ca_v channels.

Received for publication, April 12, 2004 , and in revised form, August 3, 2004.

^* This work was supported in part by grants (to G. F. K.) from the National Science Foundation (MCB9983242) and the National Institute of Allergy and Infectious Diseases (1-R41-AI51791) and a grant (to G. W. Z.) from the Heart and Stroke Foundation of Alberta and the Northwest Territories. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^|| Supported by salary awards from the Alberta Heritage Foundation for Medical Research.

^** To whom correspondence should be addressed: Dept. of Molecular, Microbial, and Structural Biology, University of Connecticut Health Center, 263 Farmington Ave., Farmington, CT 06032-3305. Tel.: 860-679-8364; Fax: 860-679-1652; E-mail: glenn{at}psel.uchc.edu.

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