Scanning mutagenesis of ω-atracotoxin-Hv1a reveals a spatially restricted epitope that confers selective activity against insect calcium channels

We constructed a complete panel of alanine mutants of the insect-specific calcium channel blocker omega-atracotoxin-Hv1a. Lethality assays using these mutant toxins identified three spatially contiguous residues, Pro(10), Asn(27), and Arg(35), that are critical for insecticidal activity against flies (Musca domestica) and crickets (Acheta domestica). Competitive binding assays using radiolabeled omega-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 muM, omega-atracotoxin-Hv1a had no effect on heterologously expressed rat Ca(v)2.1, Ca(v)2.2, and Ca(v)1.2 calcium channels, consistent with the previously reported insect selectivity of the toxin. 30 muM omega-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 Asn(27) and Arg(35), 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 Pro(10), Asn(27), and Arg(35) confers specific binding to insect Ca-v channels and is largely responsible for the remarkable phyletic selectivity of omega-atracotoxin-Hv1a. This epitope provides a structural template for rational design of chemical insecticides that selectively target insect Ca-v channels.