Abstract
A novel, membrane potential sensitive dye and a fluorescence imaging plate reader (FLIPRR) have been used to characterize the pharmacological properties of rat Nav1.8 voltage-gated sodium channels (VGSC) in parallel with rat Nav1.2a and human Nav1.5 VGSC subtypes, respectively. The sensitivity of recombinant Nav1.2a-CHO, Nav1.5-293-EBNA, and Nav1.8-F-11 cells to VGSC activators was subtype dependent. Veratridine evoked depolarization of Nav1.2a-CHO and Nav1.5-293-EBNA cells with pEC50 values of 4.78 ± 0.13 and 4.84 ± 0.12, respectively (n = 3), but had negligible effect on Nav1.8-F-11 cells (pEC50 < 4.5). Type I pyrethroids were without significant effect at all subtypes. In contrast, the type II pyrethroids deltamethrin and fenvalerate evoked direct depolarization of Nav1.8-F-11 and Nav1.5-293-EBNA cells. Deltamethrin potentiated the veratridine-evoked response in Nav1.8-F-11 cells by ≥ 20-fold, in contrast to a ≤ 3-fold potentiation of the response in Nav1.2a, and Nav1.5 cells. Tetrodotoxin (TTX) inhibited VGSC activator–evoked depolarization of Nav1.8-F-11 cells with a biphasic concentration-response curve. The calculated pIC50 values were 8.05 ± 0.25 (n = 4) and 4.32 ± 0.21 (n = 4), corresponding to TTX inhibition of endogenous TTX-sensitive (TTX-S), and recombinant Nav1.8 TTX-resistant (TTX-R) VGSCs, respectively. With the exception of TTX, the potencies of a number of ion channel blockers for the Nav1.8, Nav1.2a, and Nav1.5 VGSC subtypes were similar. In summary, these high-throughput FLIPRR assays represent a valuable tool for the determination of the relative potencies of compounds at different VGSC subtypes and may prove useful for the identification of novel subtype-selective inhibitors.