Brevetoxin, together with its functional analog Ciguatoxin, are the only known toxin ligands that interact with Site 5 on the sodium channel. We have devised experiments to investigate more completely the effects of brevetoxins, derivatized in the A-ring """"""""head-down"""""""" region and K-ring """"""""tail up"""""""" region, on Na channel binding and function. Using receptor binding, electrophysiology, and photoaffinity biochemistry, we employ Brevetoxin B (PbTx) derivatives to decipher the molecular toxicological mechanism: 1. Derivatize and chemically characterize 6 selected brevetoxins for use in electrophysiological protocols. Specific derivatives are proposed to modify the A-ring and the K-ring regions of the toxin molecular, for whole cell and single channel electrophysiology; 2. Produce three separate type of [3H] brevetoxin photoaffinity probes. Derivatives are at the A-ring end, on the K-ring side chain, and bidentate photoprobes for covalent modification of native Na channels in synaptosomes. Detailed examination of the conjugates should provide information on toxin channel stoichiometry, interactions at the two ends of a single toxin molecule, and specific loci of interaction; 3. Evaluate the effects of toxin derivatization on binding of [3H] brevetoxin PbTx-3 to ensure and quantify competitive binding and to ensure high affinity. Carry out complete Rosenthal analysis to characterize the nature of each inhibition, and determine Ki; 4. Investigate the electrophysiological characteristics of individual derivatives in the A-ring or K-ring regions from (1) using the whole-cell and on-cell patch versions of the patch clamp. These studies will be done on two neuronal-derived cell lines, B50 and B104, which express Na channel types I and II, and III, respectively. 5. Use [3H] photoaffinity probes synthesized in (2) to identify binding regions on the Na channel. Verify that the derivative photoprobes have binding affinity (3) and functionality in electrophysiology (4). Utilize enzymatic and/or chemical cleavage of channel-toxin photoprobe conjugates coupled with brevetoxin antibody affinity chemoatography to isolate and purify peptide conjugates. Assess purity by disc gel electrophoresis and antibrevetoxin-fluorescent conjugate visualization. Sequence the peptides and correlate the sequence with known primary sequences of the channel.
