Role for Pro-13 in Directing High-Affinity Binding of Anthopleurin B to the Voltage-Sensitive Sodium Channel

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Abstract
Anthopleurin A (ApA) and B (ApB) are 49-amino acid polypeptide toxins from the Pacific sea anemone Anthopleura xanthogrammica that interfere with inactivation of voltage-gated sodium channels. ApA, which differs from ApB in seven of the 49 amino acids, displays markedly enhanced isoform selectivity compared with ApB, acting preferentially on cardiac over neuronal sodium channels. Previous studies in this lab have indicated the importance of two unique charged residues in ApB, Arg-12 and Lys-49, in this toxin's ability to discriminate between neuronal and cardiac sodium channels. Likewise, a double mutant (R12S/K49Q) recently characterized in this lab (Khera et al., 1995) displays a greatly reduced affinity for neuronal channels, essentially restoring the discriminatory ability of ApA. When the remaining five residues unique to ApB are individually converted to those of ApA, only ApB (Pro-13) shows a major effect, reducing the affinity of the new mutant toxin (P13V) against both channel isoforms approximately 10-fold. This effect is most likely the result of a conformational rearrangement within the surrounding cationic cluster which includes Arg-12 and -14, as well as Lys-49. However, when placed into the context of the double mutant R12S/K49Q a unique effect is observed: the new triple mutant (R12S/P13V/K49Q) is no longer able to discriminate effectively between channel isoforms. Its affinity for the neuronal sodium channel is significantly enhanced compared to either P13V or to the double mutant R12S/K49Q. These results are consistent both with our proposed model (Khera et al., 1995) and with the recently reported solution structure of ApB, which implicate the cationic cluster in both affinity and channel isoform selectivity. We suggest that the P13V mutation results in a shift in the relative orientation of cationic residues within the large flexible loop between residues 9−18, thus strengthening their interactions with target sequences of the neuronal sodium channel.