Development of a competitive fluorescence-based synaptosome binding assay for brevetoxins

Abstract

Brevetoxins are a family of ladder-frame polyether toxins produced during blooms of the marine dinoflagellate Karenia brevis. Inhalation of brevetoxins aerosolized by wind and wave action can lead to asthma-like symptoms in beach goers. Consumption of either shellfish or finfish exposed to K. brevis blooms can lead to the development of neurotoxic shellfish poisoning. The toxic effects of brevetoxins are due to activation of voltage-sensitive sodium channels (VSSCs) in cell membranes. Binding of brevetoxin analogs and competitors to site 5 on these channels has historically been measured using a radioligand competition assay that is fraught with difficulty, including slow analysis time, production of radioactive waste, and cumbersome and expensive methods associated with the generation of radioactive labeled ligands. In this study, we describe the development of a novel fluorescent synaptosome binding assay for the brevetoxin receptor. BODIPY®-conjugated to PbTx-2 was used as the labeled ligand. The BODIPY®-PbTx-2 conjugate was found to displace [3H]-PbTx-3 from its binding site on VSSCs on rat brain synaptosomes with an equilibrium inhibition constant of 0.11 nM. We have shown that brevetoxin A and B analogs are all able to compete for binding with the fluorescent ligand. Most importantly, this assay was validated against the current site 5 receptor binding assay standard, the radioligand receptor assay for the brevetoxin receptor using [3H]-PbTx-3 as the labeled ligand. The fluorescence based assay yielded equilibrium inhibition constants comparable to the radioligand assay for all brevetoxin analogs. The fluorescence based assay was quicker, far less expensive, and did not generate radioactive waste or need radioactive facilities. As such, this fluorescence-based assay can be used to replace the current radioligand assay for site 5 on voltage-sensitive sodium channels and will be a vital tool for future experiments examining the binding affinity of various ligands for site 5 on sodium channels.

Flow Cytometric-Membrane Potential Detection of Sodium Channel Active Marine Toxins: Application to Ciguatoxins in Fish Muscle and Feasibility of Automating Saxitoxin Detection

“…Like receptor binding assays based on neurosynaptosomes (5)(6)(7)(8), neuroblastoma-based assays exploit these toxin binding sites. The first neuroblastoma assays for potential screening of toxins binding to site 1 and causing VGSC blockade were developed using a cell morphology end point (9).…”

Uptake and localization of fluorescently‐labeled Karenia brevis metabolites in non‐toxic marine microbial taxa

“…Primary stocks of lyophilized Karenia brevis BODIPY conjugated metabolites BODIPY‐PbTx‐2 (B‐PbTx), MW = 1,182.61 g · mol −1 ; BODIPY‐Brevenal (B‐Bn), MW = 944.56 g · mol −1 ; and BODIPY‐Brevisin (B‐Bs) MW = 994.56 g · mol −1 were prepared at the UNCW Center for Marine Sciences (McCall et al. , ) and each made up to a final stock concentration of 1 mM in dimethyl sulfoxide (DMSO). Unconjugated BODIPY (Life Technologies, Carlsbad, CA, USA; MW = 306.12 g · mol −1 ) was a negative control.…”

“…All three compounds possess terminal aldehyde groups that enable the conjugation of the fluorescent dye BODIPY resulting in effective markers for tracking uptake and localization in cells (McCall et al. ). The taxa we studied using these fluorescently labeled compounds included eukaryotic phytoplankton (i.e., diatoms, a cryptophyte, a haptophyte, and dinoflagellates) commonly found in the Gulf of Mexico and associated with K. brevis populations (Dixon et al.…”

Development of a Fluorescence Assay for the Characterization of Brevenal Binding to Rat Brain Synaptosomes

“…19 All buffers, reagents, and plasticware were at ice temperature throughout each experiment. All experiments were performed in standard binding medium with 1 mg/mL BSA and 0.02% Alkamuls detergent.…”