IMPORTANCE OF HIGHLY CONSERVED ANIONIC RESIDUES AND ELECTROSTATIC INTERACTIONS IN THE ACTIVITY AND STRUCTURE OF THE CARDIOTONIC POLYPEPTIDEANTHOPLEURIN-B

Several polypeptide toxins from sea anemones cause delayed inactivatio n of mammalian voltage dependent sodium channels, resulting in a posit ive inotropic effect on the heart. Anthopleurin B (ApB), a toxin produ ced by the sea anemone Anthopleura xanthogrammica, is the most potent of all known anemone toxins. Previous studies in this laboratory have both defined and revealed an important role for the cationic cluster o f Arg-12, Arg-14, and Lys-49 in the expression of ApB's biological act ivity. In the present investigation, we explore the role of all remain ing charged residues by producing and characterizing mutants of ApB at Asp-7, Asp-9, Lys-37, His-39, and His-34. Recombinant toxins have bee n purified to homogeneity and their abilities to enhance veratridine-d ependent sodium uptake in eel lines expressing either the neuronal or cardiac isoform of the sodium channel evaluated. Replacement of Asp-7 results in a product that fails to fold, while muteins H39A and H34A h ave activities very similar or identical to wild-type ApB. In contrast , the D9N and K37A muteins are 7-12-fold less active than wild-type Ap B, and truncation of the side chain in D9A results in a further decrea se in activity, especially in the cardiac model. We conclude that alth ough a negative charge per se is not essential at position 9, the pres ence of a hydrogen-bond forming side chain is critical both for approp riate folding and for interaction with the sodium channel. Because the K37A and H39A mutant toxins can fold normally, neither Lys-37 nor His -39 seem to participate in an intramolecular salt bridge, in contrast to suggestions arising from NMR studies of ApA and ApB. However, Lys-3 7 may play a role in channel interaction.