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

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.

A Specific Interaction between the Cardiac Sodium Channel and Site-3 Toxin Anthopleurin B

“…Residues Arg-14, His-34, His-39, and Lys-48 have produced only small effects on toxin affinity when neutralized (25, 39) and so were relatively poor candidates for interacting with Asp-1612; similarly, short Nterminal extensions of the toxin have no measurable effect on its affinity (21,40). We tested toxin ApB H39A as a representative of this group.…”

“…Residues Arg-12 and Lys-49 are known to have a significant and cooperative effect on toxin affinity (23) but are not conserved among all anemone toxins; in particular, they are neutral in ATX-II, the toxin tested by Rogers and colleagues (18). We tested these residues by using toxin ApA, which has neutral residues at both positions (in addition to several more conservative differences which have been shown to have only minor effects on toxin affinity) (22,40). The remaining residue, Lys-37, significantly decreased the affinity of ApB when neutralized (25) and is also conserved among a number of anemone toxins.…”

Sea anemone toxins affecting voltage-gated sodium channels – molecular and evolutionary features

“…All ApB mutants were assayed either on cardiac rNa v 1.5 and brain rNa v 1.2a channels transiently expressed in HEK cells, or mammalian cell lines that constitutively express these channels among a minority of other channel subtypes. These studies have suggested a bioactive role for Arg-12, Asn-16, Leu-18, Ser-19, Trp-33, Lys-37 and Lys-49, of which most bioactive residues were associated with the Arg-14 loop and with the substitutions of Leu-18 having the strongest effect on ApB activity (Blumenthal and Seibert, 2003; Khera and Blumenthal, 1996; Dias-Kadambi et al, 1996a; Dias-Kadambi et al, 1996b; Kelso et al, 1996; Seibert et al, 2004; Fig. 2 and Fig.…”

“…However, these results were contradicted in later elecrophysiological studies showing preference of both ApA and ApB for cardiac channels (Khera et al, 1995). Attempts to ameliorate improve by site-directed mutagenesis ApB selectivity to the rat cardiac sodium channel rNa v 1.5, while retaining the original potency, had limited success (Khera and Blumenthal, 1996; Dias-Kadambi et al, 1996a; Dias-Kadambi et al, 1996b; Kelso et al, 1996). Characterization of Av2 effects on various heterologously expressed mammalian Na v s revealed that it was five times more active at hNa v 1.5 (human cardiac channel subtype) than at rNa v 1.4 (rat skeletal muscle channel subtype) (Chahine et al, 1996).…”

Developmental Biology of Neoplastic Growth

“…At low concentrations (< 40 μg/kg) these toxins, after in vivo intravenous, intracisternal (mammals) or intramuscular (crustaceans) injections, induce severe toxic syndromes including paralysis, general hyperexcitability, cardiac disorders, convulsions and death (Alsen et al 1978;Schweitz 1984). These toxins interact with a large variety of excitable cells including neurons, cardiac and skeletal muscle cells (Abita et al 1977;Bergman et al 1976;Kelso et al 1996;Renaud et al 1986;Romey et al 1976;Shibata et al 1976). In cardiac tissues they induce positive inotropic effects, arrhythmias or cell fibrillation (Hanck and Sheets 1995;Khera et al 1995;Reimer et al 1985;Renaud et al 1986;Shibata et al 1976).…”