title: Revertant mutants G550E and 4RK rescue cystic fibrosis mutants in the first nucleotide-binding domain of CFTR by different mechanisms.
abstract: The revertant mutations G550E and 4RK [the simultaneous mutation of four arginine-framed tripeptides (AFTs): R29K, R516K, R555K, and R766K] rescue the cell surface expression and function of F508del-cystic fibrosis (CF) transmembrane conductance regulator (-CFTR), the most common CF mutation. Here, we investigate their mechanism of action by using biochemical and functional assays to examine their effects on F508del and three CF mutations (R560T, A561E, and V562I) located within a conserved region of the first nucleotide-binding domain (NBD1) of CFTR. Like F508del, R560T and A561E disrupt CFTR trafficking. G550E rescued the trafficking defect of A561E but not that of R560T. Of note, the processing and function of V562I were equivalent to that of wild-type (wt)-CFTR, suggesting that V562I is not a disease-causing mutation. Biochemical studies revealed that 4RK generates higher steady-state levels of mature CFTR (band C) for wt- and V562I-CFTR than does G550E. Moreover, functional studies showed that the revertants rescue the gating defect of F508del-CFTR with different efficacies. 4RK modestly increased F508del-CFTR activity by prolonging channel openings, whereas G550E restored F508del-CFTR activity to wt levels by altering the duration of channel openings and closings. Thus, our data suggest that the revertants G550E and 4RK might rescue F508del-CFTR by distinct mechanisms. G550E likely alters the conformation of NBD1, whereas 4RK allows F508del-CFTR to escape endoplasmic reticulum retention/retrieval mediated by AFTs. We propose that AFTs might constitute a checkpoint for endoplasmic reticulum quality control.
authors.affiliation: Department of Chemistry and Biochemistry, Faculty of Sciences, University of Lisbon, Campo Grande, 1749-016 Lisbon, Portugal.
authors.forename: Mónica
authors.initials: M
authors.lastname: Roxo-Rosa
authors.forename: Zhe
authors.initials: Z
authors.lastname: Xu
authors.forename: André
authors.initials: A
authors.lastname: Schmidt
authors.forename: Mário
authors.initials: M
authors.lastname: Neto
authors.forename: Zhiwei
authors.initials: Z
authors.lastname: Cai
authors.forename: Cláudio M
authors.initials: CM
authors.lastname: Soares
authors.forename: David N
authors.initials: DN
authors.lastname: Sheppard
authors.forename: Margarida D
authors.initials: MD
authors.lastname: Amaral
mesh_terms: D000818:Animals; D002460:Cell Line; D002712:Chlorides / Q000378:metabolism; D006224:Cricetinae; D003550:Cystic Fibrosis / Q000235:genetics*; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D006026:Glycoside Hydrolases / Q000378:metabolism; D006801:Humans; D007454:Iodides / Q000378:metabolism; D015640:Ion Channel Gating; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D018408:Patch-Clamp Techniques; D017434:Protein Structure, Tertiary
chemical_list: C505032:CFTR protein, human; D002712:Chlorides; D007454:Iodides; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D006026:Glycoside Hydrolases