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

Time Series of RBA_(F) Reaction.

<p>RBA_(F) standard curves of percent (%) binding versus P-CTX-3C (g mL^-1) where the standards were allowed to incubate with the synaptosomes for 1.5 (open circle), 3.0 (open triangle) and 4.0 h (open square).</p

CBA-N2a EC₅₀ Values.

<p>Literature and this study’s cytotoxicity EC₅₀ values of the ciguatoxins P-CTX-3C, P-CTX-1, and C-CTX-1 in association with CBA-N2a. Error is represented as ± standard deviations (SD).</p

RBA_(F) and RBA_(R) Linear Standard Curves.

<p>Lower ciguatoxin concentrations found in fish surveyed in this study were accurately measured using a linear standard curve consisting of 0.10, 0.25, 0.50 and 1.0 ppb P-CTX-3C. Results were equivalent for the RBA_(F) (open circle; solid line) and RBA_(R) (closed triangle; dashed line) (t-test, P = 0.982).</p

LC-MS/MS Confirmation of Ciguatoxins.

<p>LC-MS/MS chromatogram showing the retention time (4.76 min) and the three characteristic ion transitions (1123.6 > 1105.6, 1123.6 > 1087.9, and 1123.6 > 1069.6) of a C-CTX-1 standard and in a lionfish sample.</p

CBA-N2a Ciguatoxin Standard Curves.

<p>CBA-N2a standard curves for P-CTX-3C (closed circle) and P-CTX-1 (open triangle) with percent (%) viability of Neuro-2a plotted against toxin concentration (g mL^-1). Percent cell viability is defined as the survival ratio of cells treated with and without ouabain and veratridine. Error bars represent ± standard deviations of multiple standard curves. The number of curves included is given as (N).</p

RBA_(F) Sample Binding Curves.

<p>Representative RBA_(F) curves of 1:3 serially diluted of fish samples spiked with 0 (open diamond), 0.050 (open circle), 0.075 (open triangle), and 0.100 ppb (open square) P-CTX-3C with percent (%) binding versus fish extract concentration (g mL^-1). The detection limit (small dashed line) was 0.075 ppb P-CTX-3C and the limit of quantitation (larger dashed line) was 0.100 ppb P-CTX-3C.</p

RBA_(F) and RBA_(R) Ciguatoxin Standard Binding Curves.

<p>Comparison showing the similarity of the binding kinetics (percent [%] binding vs. toxin concentration [g mL^-1]) between the fluorescent (RBA_(F), open circle; solid line) and radioactive receptor binding (RBA_(R), open triangle; solid line) assays when P-CTX-3C was used as the standard. Also shown are the RBA_(F) binding kinetics when C-CTX-1 was used as the standard (open triangle; solid line). Error bars equal ± one standard deviation with solid lines representing RBA_(F) standard deviations and dashed lines for the RBA_(R) results.</p

RBA_(F) and RBA_(R) IC₅₀ Values.

<p>Literature and this study’s IC₅₀ values of ciguatoxins P-CTX-3C and C-CTX-1 for fluorescent and radiolabeled receptor binding assays (RBA_(F) and RBA_(R)). Error is represented as ± standard deviations (SD).</p

Solid-Phase Extraction Results.

<p>Separate 5 g fish extracts from a fish containing no measurable ciguatoxin were spiked to a final concentration of either 0.1 (open squares), 0.25 (open diamonds), or 0.5 (open circles) ppb P-CTX-3C. Comparable buffer samples were similarly spiked with of either 0.10 (solid squares), 0.25 (solid diamonds), or 0.50 (solid circles) ppb P-CTX-3C. The control was tissue extract with no ciguatoxin added. Spiked and control samples were serially diluted 1:3 and results plotted as % binding versus equivalents of fish tissue extracted (g mL^-1). Results indicated that matrix effects in the fish extracts did not interfere with binding of P-CTX-3C.</p