Full Rescue of F508del-CFTR Processing and Function by CFTR Modulators Can Be Achieved by Removal of Two Regulatory Regions
Abstract
:1. Introduction
2. Results
2.1. Removal of Short Regulatory Extension (RES) Alone or with Regulatory Insertion (RI) has No Impact on 508del-CFTR Processing
2.2. Simultaneous Removal of Long Regulatory Extension (REL) and Helix H9 Significantly Reduces wt-CFTR Processing but Increases Levels of F508del-CFTR Immature Form
2.3. ΔRIS, but Not ΔRES, Abolishes the Plasma Membrane Rescue of F508del-CFTR by Revertants
2.4. ΔRIL Synergises with VX-809, but Not with Revertants to Rescue ΔRES-F508del-CFTR Processing
2.5. ΔRIL-ΔRES-F508del-CFTR Levels at the Plasma Membrane are Equivalent to Those of wt-CFTR
2.6. VX-809 Jointly with RE and RI Removal Completely Restored F508del-CFTR Function as Chloride Channel
2.7. VX-770-Stimulated Currents of CFTR Variants are Dramatically Decreased by ΔRIL but not by ΔRIL-ΔRES
3. Discussion
3.1. Impact of RE and RI on CFTR Processing and Function
3.2. Effect of VX-809 on F508del-CFTR Variants Lacking RE and RI
3.3. Impact of F508del-Revertants on CFTR Variants Lacking RE and RI
4. Materials and Methods
4.1. CFTR Variants, Cells, and Culture Conditions
4.2. Western Blot
4.3. Pulse-Chase and Immunoprecipitation
4.4. Iodide Efflux
4.5. Biochemical Determination of the Plasma Membrane Levels of CFTR
4.6. Multiple Sequence Alignment
4.7. Data and Statistical Analyses
4.8. Reagents
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
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wt-Background | F508del-Background | ||||
---|---|---|---|---|---|
Control | VX-809 | Control | VX-809 | ||
CFTR Variant | % to wt | % to wt | % to wt | % to wt | |
- | 100 ± 1 | 103 ± 1 | 8 ± 1 | 17 ± 2 | |
ΔRES | - | 98 ± 2 | 100 ± 1 | 3 ± 2 | 9 ± 4 |
ΔRIS | 54 ± 9 | 93 ± 3 | 7 ± 2 | 20 ± 4 | |
ΔRIL | 100 ± 1 | 97 ± 2 | 71 ± 3 | 96 ± 2 | |
R1070W | 71 ± 6 | 90 ± 3 | 33 ± 7 | 26 ± 6 | |
G550E | 101 ± 1 | 102 ± 1 | 37 ± 5 | 39 ± 4 | |
ΔREL | - | 88 ± 5 | 87 ± 3 | 9 ± 2 | 8 ± 2 |
ΔH9 | 55 ± 3 | 69 ± 3 | 0 ± 0 | 0 ± 0 | |
ΔH9 | 72 ± 4 | 69 ± 3 | 1 ± 0 | 0 ± 0 | |
ΔRIS | - | 44 ± 2 | 90 ± 3 | 3 ± 2 | 4 ± 3 |
R1070W | 11 ± 3 | 56 ± 8 | 2 ± 0 | 4 ± 2 | |
G550E | 82 ± 3 | 98 ± 1 | 6 ± 4 | 5 ± 3 | |
ΔRIL | - | 101 ± 1 | 101 ± 0 | 78 ± 5 | 92 ± 4 |
R1070W | 92 ± 2 | 94 ± 3 | 96 ± 2 | 96 ± 3 | |
G550E | 97 ± 2 | 92 ± 2 | 92 ± 4 | 97 ± 2 | |
R1070W | 69 ± 7 | 93 ± 4 | 34 ± 3 | 46 ± 4 | |
G550E | 101 ± 1 | 101 ± 1 | 42 ± 4 | 73 ± 6 |
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Uliyakina, I.; Botelho, H.M.; da Paula, A.C.; Afonso, S.; Lobo, M.J.; Felício, V.; Farinha, C.M.; Amaral, M.D. Full Rescue of F508del-CFTR Processing and Function by CFTR Modulators Can Be Achieved by Removal of Two Regulatory Regions. Int. J. Mol. Sci. 2020, 21, 4524. https://doi.org/10.3390/ijms21124524
Uliyakina I, Botelho HM, da Paula AC, Afonso S, Lobo MJ, Felício V, Farinha CM, Amaral MD. Full Rescue of F508del-CFTR Processing and Function by CFTR Modulators Can Be Achieved by Removal of Two Regulatory Regions. International Journal of Molecular Sciences. 2020; 21(12):4524. https://doi.org/10.3390/ijms21124524
Chicago/Turabian StyleUliyakina, Inna, Hugo M. Botelho, Ana C. da Paula, Sara Afonso, Miguel J. Lobo, Verónica Felício, Carlos M. Farinha, and Margarida D. Amaral. 2020. "Full Rescue of F508del-CFTR Processing and Function by CFTR Modulators Can Be Achieved by Removal of Two Regulatory Regions" International Journal of Molecular Sciences 21, no. 12: 4524. https://doi.org/10.3390/ijms21124524