Differences in the binding sites of two site-3 sodium channel toxins

Abstract

Site-3 toxins from scorpion and sea anemone bind to Na channels and selectively inhibit current decay. Anthopleurins A and B (ApA and ApB, respectively), toxins found in the venom of the sea anemone Anthopleura xanthogrammica, bind to closed states of mammalian skeletal and cardiac Na channels with differing affinities which arise from differences in first-order toxin/channel dissociation rate constants, koff. Using chimera comprising domain interchanges between channel isoforms, we examined the structural basis of this differential affinity. Toxin/channel association rates, kon, were similar for both toxins and both parental channels. Domain 4 determined koff for ApA, while ApB dissociated from all tested chimera in a cardiac-like manner. To probe this surprising difference between two such closely related toxins, we examined the interaction of chimeric channels with a form of ApB in which the two nonconserved basic residues, Arg-12 and Lys-49, were converted to the corresponding neutral amino acids from ApA. In the chimera comprising domain 1 from the cardiac muscle isoform and domains 2-4 from the skeletal muscle isoform, toxin dissociated at a rate intermediate between those of the parental channels. We conclude that the differential component of ApA binding is controlled by domain 4 and that some component of ApB binding is not shared by ApA. This additional component probably binds to an interface between channel domains and is partly mediated by toxin residues Arg-12 and Lys-49.

Sea anemone venom as a source of insecticidal peptides acting on voltage-gated Na+ channels

“…These conspicuous disparities in bioactive surfaces imply that despite similarities in the Arg 14 loop, the interaction site of ATX-II with site 3 is different from that of AP-B. Support for this conclusion can be found in the report that the binding sites of AP-A and AP-B on Na v 1.4 and Na v 1.5 are slightly different (Benzinger et al, 1997).…”

Potent Modulation of the Voltage-Gated Sodium Channel Nav1.7 by OD1, a Toxin from the Scorpion Odonthobuthus doriae

“…This slowing, combined with a possible augmentation of the open-state recovery rate I 3 O, produces the observed plateau current. Finally, destabilization of the final inactivated state enhances the recovery from closed state inactivation (Benzinger et al, 1997). We assume the effects of OD1 on Na v 1.7 could be explained in a similar way.…”

“…See Tables 2 and 3 for V 1/2 values and slope factors. tion and increases the rate of whole-cell recovery of cardiac and neuronal VGSCs (Benzinger et al, 1997). They propose that the noninactivating current in the presence of toxin arises from an O % I equilibrium that partially favors the open state but that the overall rate of I 3 O recovery is still not sufficiently large to cause appreciable numbers of channels to recover through the open state during repolarization.…”

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

“…Cell Culture-Channels from the rH1-pREP9 constructs were expressed in tsA201 cells (33) and maintained as described previously (17). Two to three days prior to recording, cells were seeded onto 60-mm culture dishes and transfected with 10 g of plasmid pREP9-rH1 using a calcium phosphate coprecipitate.…”

“…Studies involving covalent attachment of scorpion toxin derivatives (15) and protection against such labeling by site-specific antibodies (16) have implicated regions in the first channel domain in toxin binding, while other studies involving antibodies (16) and chimeric channels (17) have pointed to regions in domain 4. Most recently, mutation of a negative amino acid at the outer end of the third helix in the fourth domain of rat brain sodium channels (Glu-1613) was shown to inhibit the binding of the sea anemone toxin ATX-II (from Anemonia sulcata) by 80-fold (18).…”