The design of animal toxins with high target selectivity has long been a goal in protein engineering. Based on evolutionary relationship between the Drosophila antifungal defensin (drosomycin) and scorpion depressant Na+ channel toxins, we exploited a strategy to create a novel chimeric molecule (named drosotoxin) with high selectivity for channel subtypes, which was achieved by using drosomycin to substitute the structural core of BmKITc, a depressant toxin acting on both insect and mammalian Na+ channels. Recombinant drosotoxin selectively inhibited tetrodotoxin-resistant (TTX-R) Na+ channels in rat dorsal root ganglion (DRG) neurons with a 50% inhibitory concentration (IC50) of 2.6 +/- 0.5 mu M. This chimeric peptide showed no activity on K+, Ca2+ and TTX-sensitive (TTX-S) Na+ channels in rat DRG neurons and Drosophila para/tipE channels at micromolar concentrations. Drosotoxin represents the first chimeric toxin and example of a non-toxic core scaffold with high selectivity on mammalian TTX-R Na+ channels. (C) 2010 Elsevier Inc. All rights reserved.