Haq IJ , Althaus M , Gardner AI , Yeoh HY , Joshi U , Saint-Criq V , Verdon B , Townshend J , O'Brien C , Ben-Hamida M , Thomas M , Bourke S , van der Sluijs P , Braakman I , Ward C , Gray MA , Brodlie M .

203520/Z/16/Z Wellcome Trust (Wellcome), MR/M008797/1 Medical Research Council (MRC), SRC003 and SRC013 Cystic Fibrosis Trust, ZonMw TOP 40-00812-98-14103 Netherlands Organisation for Health Research and Development (ZonMw), LIFT 731.017.404 Dutch Research Council, BRAAKM14XX0 Cystic Fibrosis Foundation (CFF), MR/M008797/1 Medical Research Council , Wellcome Trust

	Figure 1. Short-circuit current responses in F508del/R751L, non-cystic fibrosis (CF), and F508del/F508del human bronchial epithelial (HBE) cultures. A: short-circuit current (Isc) responses to amiloride (100 µM), forskolin (10 µM), and CF transmembrane conductance regulator (CFTR)inh-172 (20 µM) were measured using Ussing chamber experiments in F508del/R751L, non-CF, and F508del/F508del HBEs. Representative traces shown from n = 1 donor in each graph. Lines indicate reagent addition to the apical Ussing chamber, which remained present in solution throughout the recordings. Functional CFTR is represented by a positive Isc deflection arising from net anion basolateral-apical secretion following forskolin addition. Dashed line indicates the baseline Isc prior to forskolin addition. CFTR is inhibited by the addition of CFTRinh-172. Amiloride addition inhibits epithelial sodium channel (ENaC)-mediated sodium absorption, resulting in a downward Isc deflection. B: comparative assessment of Isc showed greater responses to forskolin and CFTRinh-172 in non-CF compared with F508del/R751L HBEs. F508del/R751L HBEs demonstrated the smallest response to amiloride. Data are presented as mean (SD) responses for each donor and analyzed using unpaired t test. F508del/R751L HBEs were not included in statistical comparisons; n = 1 F508del/R751L donor, n = 5 non-CF donors, and n = 3 F508del/F508del donors for each graph. Minimum 3 culture inserts used from each donor. *P < 0.05.
	Figure 2. Transmembrane currents in wild-type (WT) and R751L-CFTR-expressing Xenopus oocytes. A: transmembrane current (IM) responses to forskolin (5 µM), genistein (40 µM), and cystic fibrosis transmembrane conductance regulator (CFTR)inh-172 (10 µM) were measured in Xenopus oocytes expressing WT and R751L-CFTR, with representative traces shown from n = 1 oocyte. Experiments were performed at room temperature. Arrows indicate reagent addition to the bathing solutions, with evidence of a time lag between reagent addition and response. Downward deflections in IM are indicative of anion efflux. Dashed lines indicate zero current for representative recordings. B: IM measurement in oocytes expressing WT and R751L-CFTR demonstrated forskolin and genistein-induced IM, which were inhibited by CFTRinh-172. Peak IM values depicted after drug application from individual experiments with corresponding mean (SD). Data were analyzed using paired t test to demonstrate that added reagents affected the resultant IM. C: comparative assessment of total CFTR IM (the sum of forskolin and genistein-induced IM) was the same in WT and R751L-CFTR-expressing oocytes. Peak IM values depicted after drug application from individual experiments with corresponding mean (SD). Data analyzed using unpaired t test. D: the forskolin fraction of the CFTR current (%total CFTR current) was the same in WT and R751L-CFTR-expressing oocytes. Data were analyzed using unpaired t test; n = 6 WT-CFTR and n = 8 R751L-CFTR-expressing oocytes. *P < 0.05; **P < 0.01; ***P < 0.001. ns, not significant.
	Figure 3. Impact of R751L on cystic fibrosis transmembrane conductance regulator (CFTR) protein folding and transport and cell surface expression. A and B: i) human embryonic kidney 293 (HEK-293) cells expressing wild-type (WT), R751L, and F508del CFTR constructs were labeled with 35S-methionine/cysteine for 15 min and chased for 0 (0′; A) and 2 h (120′; B) in the presence of VX-770 (3 µM), VX-809 (3 µM), or DMSO control (ctrl). Cells were lysed in 1% Triton X-100, and lysates were treated or not with proteinase K (25 µg/mL) for 15 min. CFTR and fragments were immunoprecipitated using TMD1C (TMD1; ii), Mr. Pink (NBD1 and full-length CFTR; iii), TMD2C (TMD2; iv), or 596 (NBD2) antibodies (v). *Nonspecific bands. N1a, protease-resistant NBD1-specific fragment; N2a, protease-resistant NBD2-specific fragment; T1d-f, protease-resistant TMD1-specific fragments; T2c, protease-resistant TMD2-specific fragment. T1, T2, and N2 fragments represent domain assembly, which is a late-folding stage of CFTR. C: cell surface biotinylation was performed in HEK-293 cells expressing CFTR constructs in the presence of VX-809, VX-770, or DMSO ctrl pretreatment. Cells were lysed in 1% Triton X-100, and lysates were used for pulldown of biotinylated proteins with Neutravidin beads. Proteins were analyzed on 7.5% SDS-PAA gels and transferred to PVDF membrane and blotted for CFTR (596) or actin. R751L was similar to WT-CFTR in transport to the Golgi (120′ chase), protein folding (protease resistance) of all 4 domains at both time points, and presence at the cell surface. Quantification for this data from 4 independent experiments is shown in Fig. 4.
	Figure 4. Quantification of total cystic fibrosis transmembrane conductance regulator (CFTR), %Golgi-modified form, and CFTR domain-specific fragments as shown in Fig. 3. A–F: quantification data for 4 independent experiments together with mean and SD values for the following. A: fold increase in the amount of total CFTR [endoplasmic reticulum form of CFTR (ER) + complex-glycosylated Golgi form of CFTR which has left the ER and resides in or beyond the Golgi complex including the plasma membrane (G)] relative to wild-type (WT)-CFTR. B: %Golgi-modified form [G/(ER + G). C–F shows the fold increase in the amount of folded CFTR domain-specific fragments relative to WT-CFTR in DMSO as follows: TMD1 (T1d-f/(ER + G) (C), NBD1 (N1a/(ER + G) (D), TMD2 (T2c/(ER + G) (E), and NBD2 (N2a/(ER + G) (F). Data in A and D have been normalized to WT-CFTR in DMSO (ctrl) at the 0-h (0′) chase for each experiment. Data in C, E, and F have been normalized to WT-CFTR in DMSO (ctrl) at the 2-h (120′) chase for each experiment. No samples at the 0-h chase contained N2a, and this time point has not been normalized. *One data point/condition was removed from lanes where the signal/noise ratio was less than 1.5× background. N1a, protease-resistant NBD1-specific fragment; N2a, protease-resistant NBD2-specific fragment; T1d-f, protease-resistant TMD1-specific fragments; T2c, protease-resistant TMD2-specific fragment. T1, T2, and N2 fragments represent domain assembly, which is a late-folding stage of CFTR.

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