Human-mouse cystic fibrosis transmembrane conductance regulator (CFTR) chimeras identify regions that partially rescue CFTR-ΔF508 processing and alter its gating defect.
The ΔF508 mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) gene is the most common cause of cystic fibrosis. The mutation disrupts biosynthetic processing, reduces channel opening rate, and decreases protein lifetime. In contrast to human CFTR (hCFTR)-ΔF508, mouse CFTR-ΔF508 is partially processed to the cell surface, although it exhibits a functional defect similar to hCFTR-ΔF508. To explore ΔF508 abnormalities, we generated human-mouse chimeric channels. Substituting mouse nucleotide-binding domain-1 (mNBD1) into hCFTR partially rescued the ΔF508-induced maturation defect, and substituting mouse membrane-spanning domain-2 or its intracellular loops (ICLs) into hCFTR prevented further ΔF508-induced gating defects. The protective effect of the mouse ICLs was reverted by inserting mouse NBDs. Our results indicate that the ΔF508 mutation affects maturation and gating via distinct regions of the protein; maturation of CFTR-ΔF508 depends on NBD1, and the ΔF508-induced gating defect depends on the interaction between the membrane-spanning domain-2 ICLs and the NBDs. These appear to be distinct processes, because none of the chimeras repaired both defects. This distinction was exemplified by the I539T mutation, which improved CFTR-ΔF508 processing but worsened the gating defect. Our results, together with previous studies, suggest that many different NBD1 modifications improve CFTR-ΔF508 maturation and that the effect of modifications can be additive. Thus, it might be possible to enhance processing by targeting several different regions of the domain or by targeting a network of CFTR-associated proteins. Because no one modification corrected both maturation and gating, perhaps more than a single agent will be required to correct all CFTR-ΔF508 defects.
Department of Internal Medicine, Roy J. and Lucille A. Carver College of Medicine, University of Iowa, Iowa City, IA 52242, USA.
Qian
Q
Dong
Lynda S
LS
Ostedgaard
Christopher
C
Rogers
Daniel W
DW
Vermeer
Yuping
Y
Zhang
Michael J
MJ
Welsh
D000595:Amino Acid Sequence; D000818:Animals; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000737:chemistry* / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D015640:Ion Channel Gating; D007700:Kinetics; D051379:Mice; D008969:Molecular Sequence Data; D011499:Protein Processing, Post-Translational; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000378:metabolism*
D011993:Recombinant Fusion Proteins; C093349:cystic fibrosis transmembrane conductance regulator delta F508; D019005:Cystic Fibrosis Transmembrane Conductance Regulator