FIGURE 6.

Assessing metabolic response of mutant K_ATP channels rescued by CBZ using ⁸⁶Rb⁺ efflux assays. A, schematic of experimental design. COSm6 cells transfected with WT or various mutant channels (human clones) were treated with 0.1% DMSO (vehicle control) or 10 μm CBZ overnight in the presence of ⁸⁶Rb⁺. Before efflux measurements, cells were incubated with metabolic inhibitors (1 mm deoxyglucose and 2.5 μg/ml oligomycin) to activate channels at the cell surface; ⁸⁶Rb⁺ was included during this time to maintain ⁸⁶Rb⁺ loading. During the 30-min metabolic inhibition (MI) period, CBZ was not included to wash out CBZ bound to rescued channels, whereas 200 μm diazoxide or 25 μm VU063 was included to test their effects on facilitating CBZ removal. Efflux was then measured for 40 min in the same solutions as during metabolic inhibition but in the absence of ⁸⁶Rb⁺. B, efflux over a 40-min period is expressed as a percentage of total ⁸⁶Rb⁺ counts. Untreated untransfected cells and cells transfected with WT channels were included as controls. Each bar represents mean ± S.E. (error bars) of three independent experiments. *, significant increase comparing CBZ + MI, CBZ + MI + Diaz, or CBZ + MI + VU with Vehicle + MI (p < 0.05 by one-way ANOVA and Dunnett's post hoc test).