Leucine 18, a hydrophobic residue essential for high affinity binding of anthopleurin B to the voltage-sensitive sodium channel.

Anthopleurin B is a potent anemone toxin that binds with nanomolar affinity to the cardiac and neuronal isoforms of the voltage-gated sodium channel. A cationic cluster that includes Arg-12, Arg-14 and Lys-49 has been shown previously to be important in this interaction. In this study, we have used site-directed mutagenesis to determine the contribution to activity of two aliphatic residues, Leu-18 and Ile-43, that have previously been experimentally inaccessible. Leu-18, a residue proximal to the cationic cluster, plays a critical role in defining the high affinity of the toxin. In ion flux studies, this is exemplified by the several hundredfold loss in affinity (231-672-fold) observed for both L18A and L18V toxins on either isoform of the sodium channel. When analyzed electrophysiologically, L18A, the most severely compromised mutant, also displays a substantial loss in affinity (34-fold and 328-fold) for the neuronal and cardiac isoforms. This difference in affinities may reflect an increased preference of the L18A mutant for the closed state of the neuronal channel. In contrast, Ile-43, a residue distal to the cationic cluster, plays at most a very modest role in affinity toward both isoforms of the sodium channel. Only conservative substitutions are tolerated at this position, implying that it may contribute to an important structural component. Our results indicate that Leu-18 is the most significant single contributor to the high affinity of Anthopleurin B identified to date. These results have extended the binding site beyond the cationic cluster to include Leu-18 and broadened our emphasis from the basic residues to include the crucial role of hydrophobic residues in toxin-receptor interactions.