κM-Conotoxin RIIIK, Structural and Functional Novelty in a K+ Channel Antagonist

Venomous organisms have evolved a variety of structurally diverse peptide neurotoxins that target ion channels. Despite the lack of any obvious structural homology, unrelated toxins that interact with voltage-activated K+ channels share a dyad motif composed of a lysine and a hydrophobic amino acid residue, usually a phenylalanine or a tyrosine. κM-Conotoxin RIIIK (κM-RIIIK), recently characterized from the cone snail Conus radiatus, blocks Shaker and TSha1 K+ channels. The functional and structural study presented here reveals that κM-conotoxin RIIIK blocks voltage-activated K+ channels with a novel pharmacophore that does not comprise a dyad motif. Despite the quite different amino acid sequence and no overlap in the pharmacological activity, we found that the NMR solution structure of κM-RIIIK in the C-terminal half is highly similar to that of μ-conotoxin GIIIA, a specific blocker of the skeletal muscle Na+ channel Nav1.4. Alanine substitutions of all non-cysteine residues indicated that four amino acids of κM-RIIIK (Leu1, Arg10, Lys18, and Arg19) are key determinants for interaction with K+ channels. Following the hypothesis that Leu1, the major hydrophobic amino acid determinant for binding, serves as the hydrophobic partner of a dyad motif, we investigated the effect of several mutations of Leu1 on the biological function of κM-RIIIK. Surprisingly, both the structural and mutational analysis suggested that, uniquely among well-characterized K+ channel-targeted toxins, κM-RIIIK blocks voltage-gated K+ channels with a pharmacophore that is not organized around a lysine−hydrophobic amino acid dyad motif.