Development and utilization of a fluorescence-based receptor-binding assay for the site 5 voltage-sensitive sodium channel ligands brevetoxin and ciguatoxin.
Brevetoxins are a family of ladder-frame polyether toxins produced during blooms of the marine dinoflagellate Karenia brevis. Consumption of fish 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 toxins has historically been measured using a radioligand competition assay that is fraught with difficulty. In this study, we developed a novel fluorescence-based binding assay for the brevetoxin receptor. Several fluorophores were conjugated to polyether brevetoxin-2 and used as the labeled ligand. Brevetoxin analogs were able to compete for binding with the fluorescent ligands. This assay was qualified against the standard radioligand receptor assay for the brevetoxin receptor. Furthermore, the fluorescence-based assay was used to determine relative concentrations of toxins in raw extracts of K. brevis culture, and to determine ciguatoxin affinity to site 5 of VSSCs. The fluorescence-based assay was quicker, safer, and far less expensive. As such, this assay can be used to replace the current radioligand assay and will be a vital tool for future experiments examining the binding affinity of various ligands for site 5 on sodium channels.
Jennifer R
JR
McCall
Henry M
HM
Jacocks
Susan C
SC
Niven
Mark A
MA
Poli
Daniel G
DG
Baden
Andrea J
AJ
Bourdelais
D000818:Animals; D001921:Brain; D002853:Chromatography, Liquid / Q000379:methods; D002922:Ciguatoxins / Q000737:chemistry*; D005456:Fluorescent Dyes; D015200:Fluoroimmunoassay / Q000379:methods*; D005504:Food Analysis; D008297:Male; D008387:Marine Toxins / Q000737:chemistry*; D013058:Mass Spectrometry / Q000379:methods; D035964:Oxocins / Q000737:chemistry*; D011485:Protein Binding; D011869:Radioligand Assay / Q000379:methods; D051381:Rats; D017207:Rats, Sprague-Dawley; D013574:Synaptosomes; D061585:Voltage-Gated Sodium Channel Agonists / Q000737:chemistry*; D000095702:Polyether Toxins
D002922:Ciguatoxins; D005456:Fluorescent Dyes; D008387:Marine Toxins; D035964:Oxocins; D061585:Voltage-Gated Sodium Channel Agonists; C053342:brevetoxin; D000095702:Polyether Toxins