Importance of the unique cationic residues arginine 12 and lysine 49 in the activity of the cardiotonic polypeptide anthopleurin B.

Abstract

Sea anemone toxins are potentially important tools for understanding the pharmacology of voltage-sensitive sodium channels. We have previously described a bacterial expression system capable of producing large amounts of one such toxin, anthopleurin B (ApB), which delays channel repolarization (Gallagher, M. J., and Blumenthal K. M. (1992) J. Biol. Chem. 267, 13958-13963). It has been suggested that cationic residues are a major determinant of anemone toxin binding. In this paper, we describe characterization of three mutants at each of two unique cationic sites of ApB, Arg-12 and Lys-49. The activities of all mutants on cardiac and neuronal sodium channels have been compared with that of wild-type ApB. Mutation of Lys-49 has relatively minor effects on toxicity, whereas the mutant R12A, but not R12S or R12K, is severely impaired. These results indicate that cationic residues per se are not absolutely required at either position, but that polar side chains at position 12 contribute significantly to binding affinity. Furthermore, Arg-12 appears to be involved in the toxin's ability to discriminate between neuronal and cardiac sodium channels.