title: A novel toxicity-based assay for the identification of modulators of voltage-gated Na+ channels.
abstract: Voltage-gated Na+ channels are promising drug targets. Screening of large numbers of putative modulators, however, can be demanding and expensive. In this study, a simple, cheap, and robust assay to test the pharmacological modulation of Na+ channel function is presented. The assay makes use of the fact that the intracellular accumulation of Na+ ions can be cytotoxic. The toxicity of the Na+ channel activator veratridine in the presence of an inhibitor of the Na+/K+ ATPase (ouabain) in a Nav1.2a (rat brain IIA alpha) expressing cell line is assessed. Na+ channel blockers should reduce toxicity in this model. CHO cells which recombinantly expressed rat Nav1.2a subunits were seeded in 96-well plates, and cell survival was tested after 24 h incubation in medium containing veratridine and ouabain in the presence or absence of Na+ channel blockers. Propidium iodide fluorescence was used as toxicity readout. Veratridine (100 microM) or ouabain alone (500 microM) were not toxic to the cells. In the presence of 500 microM ouabain, however, veratridine induced halfmaximal cell death with an EC50 value of 15.1 +/- 2.3 microM. Ouabain's EC50 was 215.3 +/- 16.7 microM (with 30 microM veratridine). The effects of a number of Na+ channel blockers were tested and compared with their Na+ channel blocking activity measured in voltage-clamp experiments. Blockers from various chemical classes reduced toxicity half maximally with IC50 values ranging from 11.7 +/- 1.4 nM (tetrodotoxin) to 280.5 +/- 48.0 microM (lamotrigine). There was a linear relationship between the log IC50 values obtained by the two methods (slope: 1.1 +/- 0.08; correlation coefficient: 0.93). In summary, these data show that this novel toxicity assay is well suited to test Na+ channel blockers.
authors.affiliation: Boehringer Ingelheim Pharma GmbH & Co KG, D-88397 Biberach, Germany. thomas.weiser@bc.boehringer-ingelheim.com
authors.forename: Thomas
authors.initials: T
authors.lastname: Weiser
mesh_terms: D000818:Animals; D001681:Biological Assay; D016466:CHO Cells; D002470:Cell Survival / Q000187:drug effects; D006224:Cricetinae; D004305:Dose-Response Relationship, Drug; D004353:Drug Evaluation, Preclinical / Q000191:economics / Q000379:methods*; D004347:Drug Interactions; D000077213:Lamotrigine; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D062551:NAV1.2 Voltage-Gated Sodium Channel; D009419:Nerve Tissue Proteins / Q000819:agonists* / Q000037:antagonists & inhibitors*; D010042:Ouabain / Q000037:antagonists & inhibitors / Q000633:toxicity; D018408:Patch-Clamp Techniques; D051381:Rats; D062687:Sodium Channel Agonists; D026941:Sodium Channel Blockers / Q000494:pharmacology*; D000254:Sodium-Potassium-Exchanging ATPase / Q000037:antagonists & inhibitors; D013779:Tetrodotoxin / Q000494:pharmacology; D018675:Toxicity Tests / Q000191:economics / Q000379:methods*; D014227:Triazines / Q000494:pharmacology; D014701:Veratridine / Q000037:antagonists & inhibitors / Q000633:toxicity*
chemical_list: D062551:NAV1.2 Voltage-Gated Sodium Channel; D009419:Nerve Tissue Proteins; C568253:Scn2A protein, rat; D062687:Sodium Channel Agonists; D026941:Sodium Channel Blockers; D014227:Triazines; D013779:Tetrodotoxin; D010042:Ouabain; D014701:Veratridine; D000254:Sodium-Potassium-Exchanging ATPase; D000077213:Lamotrigine