Figure 4. Functional characterization of S955P-CFTR in the CFBE cell line.

CFBE cells stably expressing (A) wt-CFTR or (B–D) S955P-CFTR, were pre-incubated for 24h with DMSO (0.1% v/v) (A,B) as vehicle control, (C) 3μM VX-809, or (D) 5 μM VX-661 and analyzed under CFTR potentiation with genistein. (E) Graph summarizing equivalent short-circuit currents (I_eq-sc) after apical stimulation with forskolin+IBMX (I/F 2 μM and 100 μM, respectively) obtained in A–D. A low Cl⁻ Ringer solution was used at apical side to stablish a Cl⁻ gradient. Negative transepithelial voltage (Vte) deflections were observed following the addition of apical I/F which were fully reverted by addition of CFTR inhibit Inh172 (30 μM). (F) Representative single-channel CFTR current traces at different membrane potentials in the presence of 2 mM ATP. (G) Single-channel I-V relationships of S955P-CFTR (blue points) vs WT-CFTR (fitting from WT (Yeh et al, 2014)). (H) GLPG1837 further increases macroscopic S955P-CFTR currents in the presence of P-dATP (a high affinity ATP analogue). (I) Comparison of residual currents after incubation with GLPG1837 (G7) shows an increase in 955P-CFTR gating of ~4-fold and ~6-fold in the absence or presence of P-dATP, respectively. (J) Single-channel behavior of GLPG1837-potentiated S955P-CFTR. Comparing these two 20-s traces, longer open events and shorter closed events after potentiation with GLPG1837 were noted. (K) Summary of the additive effect of GLPG1837 on single-channel kinetics of S955P-CFTR. Po, open time (τo), and closed time (τc) were as follows: 0.19±0.03; 368±46ms, and 1841±490 ms for ATP alone (n=4); and 0.75±0.02, 689±56 ms, and 212±35 ms for ATP+GLPG1837 (n=3). Error bars represent the SEM of the mean. Asterisks indicate degree of significant difference calculated by unpaired t-test. ** - p value <0.01, *** - p value <0.001.