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 α) 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 μM) or ouabain alone (500 μM) were not toxic to the cells. In the presence of 500 μM ouabain, however, veratridine induced halfmaximal cell death with an EC 50 value of 15.1±2.3 μM. Ouabain’s EC 50 was 215.3±16.7 μM (with 30 μM 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 IC 50 values ranging from 11.7±1.4 nM (tetrodotoxin) to 280.5±48.0 μM (lamotrigine). There was a linear relationship between the log IC 50 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.