Polypeptide neurotoxins have historically provided invaluable reagents for both purification and structural and pharmacologic characterization of voltage-sensitive cation channels. One such family of toxins, elaborated by sea anemones and exemplified in this proposal by Anthopleurin-B (ApB) from Anthopleura xanthogrammica has been demonstrated to greatly delay inactivation of voltage-dependent Na+ channel isoforms from heart, skeletal muscle, and brain (in order of binding affinity) by binding to toxin site III. ApB has been cloned and expressed in bacteria in the p.i.'s lab, and a number of functionally essential residues, including Arg-12, Leu-18, and Trp-33, identified by electrophysiologic and biochemical analyses of designed mutant toxins. By applying thermodynamic mutant cycle analysis to mutated forms of each protein, we have also demonstrated a direct interaction between Lys-37 of ApB and Asp-1612 of the cardiac Na-channel, thus confirming the S3-S4 extracellular loop of channel domain IV as a primary component of site III. Among the anemone toxins, ApB has the highest known affinity for cardiac Na-channels, and we have shown it to display an approximate 100fold preference for the cardiac over the neuronal isoform. In this application, we propose to (1) identify additional ApB residues essential for defining a high-affinity binding epitope, as well as residues important for determining channel isoform specificity of ApB; (2) fill out the terra incognita of the channel binding site, using both chimeric channels and thermodynamic mutant cycle analysis of mutated toxin:channel pairs; (3) characterize the role played by orientation of a flexible loop containing Residues 9-19 of ApB in defining isoform selectivity; and (4) solve the solution structure of a complex containing recombinant ApB and a synthetic peptide which recapitulates the sequence of the S3-S4 linker of channel domain IV. The results of these studies will provide important information on the 3-dimensional structure of the voltage-sensitive Na-channel. In addition, since ApB has been demonstrated in a number of whole animal studies to serve as a powerful positive inotropic agent, identification of additional atomic details regarding its interaction with the sodium channel should open the door to development of new cardiotonic peptidomimetics.
