1 research outputs found
Development of a Fluorescence Assay for the Characterization of Brevenal Binding to Rat Brain Synaptosomes
The marine dinoflagellate <i>Karenia brevis</i> produces
a family of neurotoxins known as brevetoxins. Brevetoxins elicit their
effects by binding to and activating voltage-sensitive sodium channels
(VSSCs) in cell membranes. <i>K. brevis</i> also produces
brevenal, a brevetoxin antagonist, which is able to inhibit and/or
negate many of the detrimental effects of brevetoxins. Brevenal binding
to VSSCs has yet to be fully characterized, in part due to the difficulty
and expense of current techniques. In this study, we have developed
a novel fluorescence binding assay for the brevenal binding site.
Several fluorescent compounds were conjugated to brevenal to assess
their effects on brevenal binding. The assay was validated against
the radioligand assay for the brevenal binding site and yielded comparable
equilibrium inhibition constants. The fluorescence-based assay was
shown to be quicker and far less expensive and did not generate radioactive
waste or need facilities for handling radioactive materials. In-depth
studies using the brevenal conjugates showed that, while brevenal
conjugates do bind to a binding site in the VSSC protein complex,
they are not displaced by known VSSC site specific ligands. As such,
brevenal elicits its action through a novel mechanism and/or currently
unknown receptor site on VSSCs