The natural marine toxins Brevetoxin and Ciguatoxin represent xenobiotics present in seafood. Although the intoxication signs in animals and symptoms in humans have been quite well characterized, and the specific interaction sites have been described with respect to dissociation constant and binding maximum, very little is known about the exact biochemical nature of the toxin ligand receptor. No endogenous biological molecule has been identified that naturally binds to the polyether toxin receptor, and no endogenous function for the receptor is known. Hence, these receptors can be classified as """"""""orphan receptors"""""""". We propose to define the receptor by isolating and purifying it. Derivatization of specific portions of the toxin ligands will also be undertaken. Specifically, we propose to prepare and utilize synthetic brevetoxin photoaffinity probes to covalently label the specific binding site in rat brain synaptosomes; to solubilize the specific binding component(s) covalently bound to the toxin probes, and purify the conjugates using a sequence of molecular size and affinity chromatography; to cleave the isolated conjugate into smaller peptide fragments using immobilized peptidases and or cyanogen bromide cleavage; and to isolate the specific covalent brevetoxin-peptide fragments using a combination of reverse phase HPLC and antibrevetoxin antibody column affinity chromatography. Peptides generated which contain brevetonin photoaffinity probe bound to them will be sequenced to determine the primary amino acid sequence. Sequences will be compared to published sequence for sodium channel to determine specific brevetoxin binding sites in the primary sequence. We also intend to produce synthetically a number of brevetoxin derivatives in both labeled and unlabeled forms to explore the structural requirements for binding to the specific orphan receptor, and then to correlate toxin ligand structural requirements with receptor binding locale to refine models for interaction of polyether toxin xenobiotic with orphan receptor on sodium channel. The goal of the proposed research is to provide a molecular view of the orphan receptor. When our long-term research goal is complete, we expect to have defined the mechanisms by which these three unique natural environmental toxicants exert their toxic effects, simply by binding with high affinity and avidity to specific """"""""orphan receptors"""""""" associated with voltage-sensitive sodium channels. The results may lead to development of ligand or receptor-based therapeutic strategies, and overall will contribute to our understanding of how these highly potent human seafood intoxicants cause human illness.
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