Clinical and molecular characterisation of the R751L-CFTR mutation

Figure 1: Short circuit current responses in F508del/R751L, F508del/F508del and non-CF paediatric human bronchial epithelial cultures.

A) Representative short circuit current (Isc) responses to amiloride, forskolin and CFTRinh-172 were measured using Ussing chamber experiments in F508del/R751L, non-CF and F508del/F508del air liquid interface paediatric human bronchial epithelial cultures (HBEs). Lines indicate reagent addition to the apical Ussing chamber. The dashed line indicates the baseline Isc prior to forskolin addition.

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 presented as mean (SD) responses for each donor, analysed using unpaired t test, * P < 0.05. n = 1 F508del/R751L donor; n = 5 non-CF donors; n = 3 F508del/F508del donors for each panel.

Figure 2. Transmembrane currents in wild-type and R751L-CFTR-expressing Xenopus oocytes.

A) Transmembrane current (I_M) responses to forskolin (5 mM), genistein (40 mM) and CFTR_inh-172 (10 mM) were measured in Xenopus oocytes expressing wild-type (WT) and R751L-CFTR with representative traces shown from n = 1 oocyte. Experiments were performed at room temperature. Downward deflections in I_M are indicative of anion efflux.

B) I_M measurement in oocytes expressing WT and R751L-CFTR demonstrated forskolin and genistein-induced I_M which were inhibited by CFTR_inh-172. Peak I_M values depicted after drug application from individual experiments with corresponding mean (SD). Data analysed using paired t test to demonstrate that added reagents affected the resultant I_M.

C) Comparative assessment of total CFTR I_M (the sum of forskolin and genistein-induced I_M) was the same in WT and R751L-CFTR-expressing oocytes. Peak I_M values depicted after drug application from individual experiments with corresponding mean (SD). Data analysed using unpaired t test.

D) The forskolin fraction of the CFTR current (percentage of total CFTR current) was the same in WT and R751L-CFTR-expressing oocytes. Data analysed 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 CFTR protein folding and transport, and cell-surface expression.

A and B) Human embryonic kidney 293 (HEK293) cells expressing wild-type (WT), R751L and F508del CFTR constructs were labelled with ³⁵S-methionine/cysteine for 15 min and chased for A) 0 (0’) and B) 2 h (120’) 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 ii) TMD1C (TMD1), iii) Mr. Pink (NBD1 and full-length CFTR), iv) TMD2C (TMD2), or v) 596 (NBD2) antibodies. * Indicates nonspecific bands, T1d-f: protease-resistant TMD1-specific fragments; N1a: protease-resistant NBD1-specific fragment; T2c: protease-resistant TMD2-specific fragment; N2a: protease-resistant NBD2-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 HEK293 cells expressing CFTR constructs in the presence of VX-809, VX-770 or DMSO control (ctrl) pre-treatment. Cells were lysed in 1% Triton X-100 and lysates were used for pull down of biotinylated proteins with Neutravidin beads. Proteins were analysed 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 four independent experiments is shown in Figure 4.

Figure 4. Quantification of total CFTR, percentage of Golgi-modified form and CFTR domain-specific fragments as shown in Figure 3.

Panels A-F show quantification data for four independent experiments together with mean and SD values for the following:

A) Fold increase in the amount of total CFTR (ER + G) relative to WT-CFTR. B) Percentage of Golgi-modified form (G/(ER+G). C-F) show the fold increase in the amount of folded CFTR domain specific fragments relative to WT-CFTR in DMSO as follows: C) TMD1 (T1d-f/(ER+G), D) NBD1 (N1a/(ER+G), E) TMD2 (T2c/(ER+G) and F) NBD2 (N2a/(ER+G).

Data in panels A) and D) have been normalised to WT-CFTR in DMSO (ctrl) at the 0-h (0’) chase for each experiment. Data in panels C), E), and F) have been normalised 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 normalised. * One data point per condition was removed from lanes where the signal/noise ratio was less than 1.5 x background.

Abbreviations: ER: Endoplasmic reticulum form of CFTR; G: complex-glycosylated Golgi form of CFTR which has left the ER and resides in or beyond the Golgi complex including the plasma membrane; WT: wild-type CFTR; T1d-f: protease-resistant TMD1-specific fragments; N1a: protease-resistant NBD1-specific fragment; T2c: protease-resistant TMD2-specific fragment; N2a: protease-resistant NBD2-specific fragment. T1, T2, and N2 fragments represent domain assembly which is a late folding stage of CFTR.