title: The CFTR trafficking mutation F508del inhibits the constitutive activity of SLC26A9.
abstract: Several members of the SLC26A family of anion transporters associate with CFTR, forming complexes in which CFTR and SLC26A functions are reciprocally regulated. These associations are thought to be facilitated by PDZ scaffolding interactions. CFTR has been shown to be positively regulated by NHERF-1, and negatively regulated by CAL in airway epithelia. However, it is unclear which PDZ-domain protein(s) interact with SLC26A9, a SLC26A family member found in airway epithelia. We have previously shown that primary, human bronchial epithelia (HBE) from non-CF donors exhibit constitutive anion secretion attributable to SLC26A9. However, constitutive anion secretion is absent in HBE from CF donors. We examined whether changes in SLC26A9 constitutive activity could be attributed to a loss of CFTR trafficking, and what role PDZ interactions played. HEK293 coexpressing SLC26A9 with the trafficking mutant F508del CFTR exhibited a significant reduction in constitutive current compared with cells coexpressing SLC26A9 and wt CFTR. We found that SLC26A9 exhibits complex glycosylation when coexpressed with F508del CFTR, but its expression at the plasma membrane is decreased. SLC26A9 interacted with both NHERF-1 and CAL, and its interaction with both significantly increased with coexpression of wt CFTR. However, coexpression with F508del CFTR only increased SLC26A9's interaction with CAL. Mutation of SLC26A9's PDZ motif decreased this association with CAL, and restored its constitutive activity. Correcting aberrant F508del CFTR trafficking in CF HBE with corrector VX-809 also restored SLC26A9 activity. We conclude that when SLC26A9 is coexpressed with F508del CFTR, its trafficking defect leads to a PDZ motif-sensitive intracellular retention of SLC26A9.
authors.affiliation: Departments of Pediatrics and Cell Biology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania; cbertra@pitt.edu.
authors.forename: Carol A
authors.initials: CA
authors.lastname: Bertrand
authors.affiliation: Departments of Pediatrics and Cell Biology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania.
authors.forename: Shalini
authors.initials: S
authors.lastname: Mitra
authors.affiliation: Departments of Pediatrics and Cell Biology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania.
authors.forename: Sanjay K
authors.initials: SK
authors.lastname: Mishra
authors.affiliation: Departments of Pediatrics and Cell Biology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania.
authors.forename: Xiaohui
authors.initials: X
authors.lastname: Wang
authors.affiliation: Department of Biochemistry and Cell Biology, Geisel School of Medicine at Dartmouth, Hanover, New Hampshire.
authors.forename: Yu
authors.initials: Y
authors.lastname: Zhao
authors.affiliation: Department of Pulmonary, Allergy, and Critical Care Medicine, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania; and.
authors.forename: Joseph M
authors.initials: JM
authors.lastname: Pilewski
authors.affiliation: Department of Biochemistry and Cell Biology, Geisel School of Medicine at Dartmouth, Hanover, New Hampshire.
authors.forename: Dean R
authors.initials: DR
authors.lastname: Madden
authors.affiliation: Departments of Pediatrics and Cell Biology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania.
authors.forename: Raymond A
authors.initials: RA
authors.lastname: Frizzell
mesh_terms: D048868:Adaptor Proteins, Signal Transducing; D020816:Amino Acid Motifs; D017920:Antiporters / Q000737:chemistry / Q000378:metabolism*; D002352:Carrier Proteins; D003550:Cystic Fibrosis / Q000378:metabolism / Q000473:pathology; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000378:metabolism*; D004847:Epithelial Cells / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D000077614:Golgi Matrix Proteins; D057809:HEK293 Cells; D006801:Humans; D047468:Immunoprecipitation; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D008954:Models, Biological; D009154:Mutation / Q000235:genetics*; D054731:PDZ Domains; D010455:Peptides / Q000378:metabolism; D010750:Phosphoproteins / Q000378:metabolism; D017923:Sodium-Hydrogen Exchangers / Q000378:metabolism; D000076988:Sulfate Transporters
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D017920:Antiporters; D002352:Carrier Proteins; C434484:GOPC protein, human; D000077614:Golgi Matrix Proteins; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D010455:Peptides; D010750:Phosphoproteins; C455628:SLC26A9 protein, human; D017923:Sodium-Hydrogen Exchangers; D000076988:Sulfate Transporters; C093349:cystic fibrosis transmembrane conductance regulator delta F508; C093376:sodium-hydrogen exchanger regulatory factor; D019005:Cystic Fibrosis Transmembrane Conductance Regulator
keywords: CAL; NHERF-1; PDZ domain; chloride channel; intracellular trafficking