Brevenal, a brevetoxin antagonist from Karenia brevis, binds to a previously unreported site on mammalian sodium channels

• Brevenal competes with brevetoxin for binding at site 5.
• A new radiolabeled ligand [3H]-brevenol was produced.
• Brevetoxin cannot inhibit binding of [3H]-brevenol.
• Ligands selective for other sites do not inhibit [3H]-brevenol binding.
• Brevenal binds to a previously unreported site.

Brevetoxins are a family of ladder-frame polyether toxins produced by the marine dinoflagellate Karenia brevis. During blooms of K. brevis, inhalation of brevetoxins aerosolized by wind and wave action can lead to asthma-like symptoms in persons at the beach. Consumption of either shellfish or finfish contaminated by K. brevis blooms can lead to the development of neurotoxic shellfish poisoning. The toxic effects of brevetoxins are due to binding at a defined site on, and subsequent activation of, voltage-sensitive sodium channels (VSSCs) in cell membranes (site 5). In addition to brevetoxins, K. brevis produces several other ladder-frame compounds. One of these compounds, brevenal, has been shown to antagonize the effects of brevetoxin. In an effort to further characterize the effects of brevenal, a radioactive analog ([3H]-brevenol) was produced by reducing the terminal aldehyde moiety of brevenal to an alcohol using tritiated sodium borohydride. A KD of 67 nM and Bmax of 7.1 pmol/mg protein were obtained for [3H]-brevenol in rat brain synaptosomes, suggesting a 1:1 matching with VSSCs. Brevenal and brevenol competed for [3H]-brevenol binding with Ki values of 75 nM and 56 nM, respectively. However, although both brevenal and brevenol inhibited brevetoxin binding, brevetoxin was completely ineffective at competition for [3H]-brevenol binding. After examining other site-specific compounds, it was determined that [3H]-brevenol binds to a site that is distinct from the other known sites on the sodium channel, including the brevetoxin site, (site 5) although some interaction with site 5 is apparent.