Replacement of individual residues of anthopleurin b with those native to anthopleurin A: A functional analysis

Anthopleurin A (ApA) and B (ApB) are 49 amino acid toxins which cause delayed inactivation of voltage-gated Na channels. ApA displays enhanced channel isoform selectivity when compared with ApB, acting preferentially on cardiac over neuronal Na channels. Two unique residues in ApB, R12 and K49, have previously been shown to affect this toxin's ability to discriminate between Na channel subtypes, and when combined as a double mutant (R12S/K49Q) display a greatly reduced affinity for neuronal channels. When the remaining residues unique to ApB are individually converted to those of ApA, only P13V shows a major effect, reducing the affinity about 10-fold in both channel isoforms. However, when combined with the double mutant, the new triple mutant (R12S/P13V/K49Q) displays an enhanced affinity for neuronal Na channels as compared to either the single (P13V) or double (R12S/K49Q) mutants. We suggest that the P13V mutation results in a shift in the relative orientation of the cationic residues within the large flexible loop between residues 9-18, thus strenathenino an electrostatic interaction with taraet seauences of the neuronal channel.