IMPORTANCE OF THE UNIQUE CATIONIC RESIDUES ARGININE-12 AND LYSINE-49 IN THE ACTIVITY OF THE CARDIOTONIC POLYPEPTIDE ANTHOPLEURIN-B

Sea anemone toxins are potentially important tools for understanding t he pharmacology of voltage-sensitive sodium channels. We have previous ly 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 de scribe characterization of three mutants at each of two unique cationi c sites of ApB, Arg-12 and Lys-49. The activities of all mutants on ca rdiac and neuronal sodium channels have been compared with that of wil d-type ApB. Mutation of Lys-49 has relatively minor effects on toxicit y, whereas the mutant R12A, but not R12S or R12K, is severely impaired . These results indicate that cationic residues per se are not absolut ely required at either position, but that polar side chains at positio n 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.