Modification of the cell based assay for brevetoxins using human cardiac voltage dependent sodium channels expressed in HEK-293 cells.
Assays using living cells provide an effective means to generate activity measurements of toxins, especially in situations where the toxins are part of a complex mixture or in an unfamiliar form such as natural or synthetic derivatives or bioactive metabolites. An important step in the refinement of cell based assays is to simplify the cellular reactions needed or required to generate the functional response of interest. Advances in the engineering of functional responses in cells provide a means to direct the response to given toxins. In this report, we describe the homogeneous high level expression of the initial target for brevetoxin, the voltage dependent sodium channel in human embryonic kidney cells (HEK-293). HEK cells stably transfected with a 6.208 kb cDNA of human heart voltage-dependent Na(+) channel (hH1a) were examined as an alternative to mouse neuroblastoma cells (N2A). The HEK-hH1a cells showed a reduced dependence on cofactors, increased sensitivity to brevetoxin and a useful means to assure absolute selectivity to the sodium channel. We next assessed the assay in a reporter gene format. Expression of a panel of minimal response elements as well as the c-fos promoter failed to provide a response to brevetoxin, indicating that the HEK cells lack a necessary intermediate signaling component. The expression of voltage dependent sodium channels in HEK cells is anticipated to provide enhanced performance for cell-based detection of toxins for drug and natural product discovery, biomonitoring and environmental monitoring.
Fairey
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ER
Marine Biotoxins Program, Center for Coastal Environmental Health and Biomolecular Research, NOAA-National Ocean Service, 219 Fort Johnson Road, Charleston, SC 29412, USA.
Bottein Dechraoui
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MY
Sheets
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MF
Ramsdell
J S
JS
D000818:Animals; D015374:Biosensing Techniques / Q000379:methods*; D002460:Cell Line; D017930:Genes, Reporter; D016762:Genes, fos; D006801:Humans; D008387:Marine Toxins / Q000032:analysis* / Q000633:toxicity; D051379:Mice; D009206:Myocardium / Q000378:metabolism; D035964:Oxocins; D011401:Promoter Regions, Genetic; D015222:Sodium Channels / Q000235:genetics*; D014162:Transfection; D000095702:Polyether Toxins
D008387:Marine Toxins; D035964:Oxocins; D015222:Sodium Channels; C053342:brevetoxin; D000095702:Polyether Toxins