Fig. 1.

(A) Raw fluorescence signal traces recorded on fluorometric imaging plate reader (FLIPR) with HEK 293 cells stably expressing human Ether-à-go-go related gene (hERG) channels. Recording started 10 s before addition of stimulation solution and continued for another 90 s. Cisapride was shown to reduce signals in a dose-dependent manner (error bars were not shown). Initial slope of curves was plotted against concentrations of cisapride (triplicate set) and fitted to a four-parameter logistic function (B) in the Prism 5.0. (C) Voltage protocol (upper) and corresponding hERG tail current recording (lower) for the automated planar patch clamp (APPC) assay. (D) The captured screen in the DataXpress program of a representative whole-cell patch clamp recordings of hERG tail currents in the absence and presence of cisapride. Red vertical line indicated addition of buffer control and green vertical triple lines indicated triple additions of drugs. Cisapride concentrations (μM) are listed above the green lines. Red cross indicates the time point when hERG tail current was measured by the system. After washout (blue vertical line), hERG channel was shown to partially recover from complete inhibition. The hERG tail currents were extracted from the DataXpress, plotted against cisapride concentrations, and fitted to a four-parameter logistic function (E) in the Prism 5.0. (F) Side-by-side comparison of dose-dependent relationships of cisapride in (•) [³H]dofetilide competition binding assay, (▪) Tl⁺ flux assay, and (▴) APPC assay. Results from multiple assays (n ≥ 2) were normalized to percentage inhibition and pooled for curve-fitting in Prism. The potency or affinity value and corresponding Hill slope from the best fitting model were reported in Table 2.