title: Regulated recycling of mutant CFTR is partially restored by pharmacological treatment.
abstract: Efficient trafficking of the cystic fibrosis transmembrane conductance regulator (CFTR) to and from the cell surface is essential for maintaining channel density at the plasma membrane (PM) and ensuring proper physiological activity. The most common mutation, F508del, exhibits reduced surface expression and impaired function despite treatment with currently available pharmacological small molecules, called correctors. To gain more detailed insight into whether CFTR enters compartments that allow corrector stabilization in the cell periphery, we investigated the peripheral trafficking itineraries and kinetics of wild type (WT) and F508del in living cells using high-speed fluorescence microscopy together with fluorogen activating protein detection. We directly visualized internalization and accumulation of CFTR WT from the PM to a perinuclear compartment that colocalized with the endosomal recycling compartment (ERC) markers Rab11 and EHD1, reaching steady-state distribution by 25 minutes. Stimulation by protein kinase A (PKA) depleted this intracellular pool and redistributed CFTR channels to the cell surface, elicited by reduced endocytosis and active translocation to the PM. Corrector or temperature rescue of F508del also resulted in targeting to the ERC and exhibited subsequent PKA-stimulated trafficking to the PM. Corrector treatment (24 hours) led to persistent residence of F508del in the ERC, while thermally destabilized F508del was targeted to lysosomal compartments by 3 hours. Acute addition of individual correctors, C4 or C18, acted on peripheral trafficking steps to partially block lysosomal targeting of thermally destabilized F508del. Taken together, corrector treatment redirects F508del trafficking from a degradative pathway to a regulated recycling route, and proteins that mediate this process become potential targets for improving the efficacy of current and future correctors.
authors.affiliation: University of Pittsburgh School of Medicine, Department of Cell Biology, BSTS 225, 3500 Terrace St, Pittsburgh, PA 15261, USA.
authors.forename: John P
authors.initials: JP
authors.lastname: Holleran
authors.forename: Jianxin
authors.initials: J
authors.lastname: Zeng
authors.forename: Raymond A
authors.initials: RA
authors.lastname: Frizzell
authors.forename: Simon C
authors.initials: SC
authors.lastname: Watkins
mesh_terms: D002460:Cell Line; D002462:Cell Membrane / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000235:genetics / Q000378:metabolism; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000235:genetics* / Q000378:metabolism*; D004705:Endocytosis / Q000235:genetics / Q000502:physiology; D011992:Endosomes / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D057809:HEK293 Cells; D006801:Humans; D008247:Lysosomes / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D008856:Microscopy, Fluorescence / Q000379:methods; D009154:Mutation; D021381:Protein Transport; D059748:Proteolysis; D033921:Vesicular Transport Proteins / Q000235:genetics / Q000378:metabolism; D020691:rab GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism; D000099193:rab11 GTP-Binding Proteins
chemical_list: D017868:Cyclic AMP-Dependent Protein Kinases; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D033921:Vesicular Transport Proteins; D020691:rab GTP-Binding Proteins; D000099193:rab11 GTP-Binding Proteins; C505032:CFTR protein, human; C120700:EHD1 protein, human
keywords: CFTR; Correctors; Endosomal recycling compartment; F508del; Fluorescence microscopy; Mutant; PKA; Recycling; Trafficking