title: Direct Measurement of Trafficking of the Cystic Fibrosis Transmembrane Conductance Regulator to the Cell Surface and Binding to a Chemical Chaperone.
abstract: Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) result in the disease cystic fibrosis. Deletion of Phe508, the most prevalent mutation associated with this disease, disrupts trafficking of the protein. Small molecule correctors yield moderate improvements in the trafficking of ΔF508-CFTR to the plasma membrane. It is currently not known if correctors increase the level of trafficking through improved cargo loading of transport vesicles or through direct binding to CFTR. Real-time measurements of trafficking were utilized to identify the mechanistic details of chemical, biochemical, and thermal factors that impact CFTR correction, using the corrector molecule VX-809, a secondary mutation (I539T), and low-temperature conditions. Each individually improved trafficking of ΔF508-CFTR to approximately 10% of wild-type levels. The combination of VX-809 with either low temperature or the I539T mutation increased the amount of CFTR on the plasma membrane to nearly 40%, indicating synergistic activity. The number of vesicles reaching the surface was significantly altered; however, the amount of channel in each vesicle remained the same. Direct binding measurements of VX-809 in native membranes using backscattering interferometry indicate tight binding to CFTR, which occurred in a manner independent of mutation. The similar values obtained for all forms of the channel indicate that the binding site is not compromised or enhanced by these mutations.
authors.affiliation: Department of Chemistry, University of Kentucky , Lexington, Kentucky 40506, United States.
authors.forename: Zhihui
authors.initials: Z
authors.lastname: Zhang
authors.affiliation: Department of Chemistry, Georgia Institute of Technology , Atlanta, Georgia 30332, United States.
authors.forename: Michael M
authors.initials: MM
authors.lastname: Baksh
authors.affiliation: Department of Chemistry, Georgia Institute of Technology , Atlanta, Georgia 30332, United States.
authors.forename: M G
authors.identifier: http://orcid.org/0000-0001-8247-3108
authors.initials: MG
authors.lastname: Finn
authors.affiliation: Department of Chemistry, University of Kentucky , Lexington, Kentucky 40506, United States.
authors.forename: David K
authors.initials: DK
authors.lastname: Heidary
authors.affiliation: Department of Chemistry, University of Kentucky , Lexington, Kentucky 40506, United States.
authors.forename: Christopher I
authors.identifier: http://orcid.org/0000-0003-0019-1989
authors.initials: CI
authors.lastname: Richards
mesh_terms: D000631:Aminopyridines / Q000378:metabolism*; D052117:Benzodioxoles / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism*; D003550:Cystic Fibrosis / Q000235:genetics / Q000378:metabolism; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000235:genetics / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007368:Interferometry / Q000379:methods; D008856:Microscopy, Fluorescence / Q000379:methods; D009154:Mutation; D011485:Protein Binding; D021381:Protein Transport / Q000235:genetics; D015203:Reproducibility of Results; D000072760:Single Molecule Imaging / Q000379:methods; D013696:Temperature; D022161:Transport Vesicles / Q000378:metabolism*
chemical_list: D000631:Aminopyridines; D052117:Benzodioxoles; C093349:cystic fibrosis transmembrane conductance regulator delta F508; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; C569105:lumacaftor