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. 2018 Feb 22;175(7):1017–1038. doi: 10.1111/bph.14141

Figure 5.

Figure 5

Effects of CFFT‐004 on the stability of F508del‐CFTR‐mediated transepithelial Cl− currents. (A) Representative Ussing chamber recordings of F508del‐CFTR showing the effects of correction with C18 and CFFT‐004 on CFTR‐mediated transepithelial Cl− currents. F508del‐CFTR‐expressing FRT epithelia were incubated for 24 h at 37°C in the presence of either C18 (5 μM) or CFFT‐004 (10 μM) without reducing the FBS concentration of media. Fifteen minutes prior to mounting FRT epithelia in Ussing chambers (i.e. t = 0 h – 15 min), they were treated with cycloheximide (50 μg·mL−1), added to both the apical and basolateral solutions. At the indicated times, FRT epithelia were activated with forskolin (Fsk; 10 μM), potentiated with ivacaftor (VX; 1 μM) and inhibited by CFTRinh‐172 (I172; 10 μM); continuous lines indicate the presence of different compounds in the apical solution; cycloheximide (50 μg·mL−1) was present in the apical and basolateral solutions during Isc recordings. Data are normalized to baseline current so that ΔIsc represents the change in transepithelial current after CFTR activation by forskolin. The vertical lines denote the forskolin‐stimulated CFTR‐mediated Isc (IFsk) and the total CFTR‐mediated Isc (ITot). For representative CFTR‐mediated transepithelial Cl− currents of C18‐ and CFFT‐004‐rescued F508del‐CFTR recorded at different times after cycloheximide treatment, see Supporting Information Figure S2. (B and C) Rt and magnitude of forskolin‐stimulated CFTR‐mediated Isc (IFsk) expressed as a percentage of the total CFTR‐mediated Isc (ITot) (i.e. IFsk/ITot) for F508del‐CFTR‐expressing FRT epithelia pretreated with either C18 (5 μM) or CFFT‐004 (10 μM) for 24 h at 37°C prior to study. Fifteen minutes prior to t = 0 h, FRT epithelia were treated with cycloheximide (50 μg·mL−1) as described in (A). (D, E) Magnitude of forskolin‐stimulated CFTR‐mediated ΔIsc and the total CFTR‐mediated ΔIsc (i.e. the ivacaftor potentiated ΔIsc) for C18‐ and CFFT‐004‐rescued F508del‐CFTR at different times after cycloheximide treatment. (F) Magnitude of the absolute Isc prior to CFTR activation for C18‐ and CFFT‐004‐rescued F508del‐CFTR at different times after cycloheximide treatment. (G) Magnitude of the residual CFTR‐mediated Cl− current determined using CFTRinh‐172 (10 μM) for C18‐ and CFFT‐004‐rescued F508del‐CFTR at different times after cycloheximide treatment. For definition of residual CFTR‐mediated Cl− current (IRes), see Supporting Information Figure S2E. Positive values of IRes indicate that the CFTRinh‐172‐inhibited current did not fall below the value of basal current prior to F508del‐CFTR stimulation with forskolin. Negative values of IRes indicate that the CFTRinh‐172‐inhibited current has decreased below the value of basal current prior to F508del‐CFTR stimulation with forskolin. In (B–G), data are means ± SEM (C18, n = 6; CFFT‐004, n = 6, except t = 4 h, where n = 5); *, P < 0.05 versus C18 control, unpaired t‐test; †, P < 0.05 versus control at time 0 h, one‐way ANOVA with Dunnett's post test.