title: G551D-CFTR needs more bound actin than wild-type CFTR to maintain its presence in plasma membranes.
abstract: Cystic Fibrosis is due to mutations in the CFTR gene. The missense mutation G551D (approx. 5% of cases) encodes a CFTR chloride channel with normal cell surface expression but with an altered chloride channel activity, leading to a severe phenotype. Our aim was to identify specific interacting proteins of G551D-CFTR which could explain the channel defect. Wild-type CFTR (Wt-CFTR) was co-immunoprecipitated from stably transfected HeLa cells and resolved by 2D gel electrophoresis. Among the detected spots, one was expressed at a high level. Mass Spectrometry revealed that it corresponded to actin which is known to be involved in the CFTR's channel function. To assess whether actin could be involved in the altered G551D-CFTR function, its basal expression was studied. Because actin expression was the same in wt- and in G551D-CFTR expressing cells, its interaction with both wt- and G551D-CFTR was studied by co-immunoprecipitation, and we found that a higher amount of actin was bound onto G551D-CFTR than onto Wt-CFTR. The role of actin upon wt- and G551D-CFTR function was further studied by patch-clamp experiments after cytochalasin D treatment of the cells. We found a decrease of the very weak currents in G551D-CFTR expressing cells. Because a higher amount of actin is bound onto G551D-CFTR than onto Wt-CFTR, it is likely to be not involved in the mutated CFTR's defect. Nevertheless, because actin is necessary to maintain the very weak global currents observed in G551D-CFTR expressing HeLa cells, we conclude that more actin is necessary to maintain G551D-CFTR in the plasma membrane than for Wt-CFTR.
authors.affiliation: Inserm, UMR1078, Brest, F-29218, France.|Université de Bretagne Occidentale, Faculté de Médecine et des sciences de la santé, Brest, F-29200, France.
authors.forename: Pascal
authors.initials: P
authors.lastname: Trouvé
authors.affiliation: Inserm, UMR1078, Brest, F-29218, France.|Université de Bretagne Occidentale, Faculté de Médecine et des sciences de la santé, Brest, F-29200, France.
authors.forename: Mathieu
authors.initials: M
authors.lastname: Kerbiriou
authors.affiliation: Inserm, UMR1078, Brest, F-29218, France.|Université de Bretagne Occidentale, Faculté de Médecine et des sciences de la santé, Brest, F-29200, France.
authors.forename: Ling
authors.initials: L
authors.lastname: Teng
authors.affiliation: Inserm, UMR1078, Brest, F-29218, France.|Association de Biogénétique Gaëtan Salaün - Bretagne, Brest, F-29200, France.
authors.forename: Nathalie
authors.initials: N
authors.lastname: Benz
authors.affiliation: Service commun de spectrométrie de masse, Université de Bretagne Occidentale, Brest, F-29200, France.
authors.forename: Mehdi
authors.initials: M
authors.lastname: Taiya
authors.affiliation: Inserm, UMR1078, Brest, F-29218, France.|C.H.U. Brest, Hôpital Morvan, Laboratoire de Génétique Moléculaire, Brest, F-29200, France.
authors.forename: Sophie
authors.initials: S
authors.lastname: Le Hir
authors.affiliation: Inserm, UMR1078, Brest, F-29218, France.|Université de Bretagne Occidentale, Faculté de Médecine et des sciences de la santé, Brest, F-29200, France.|C.H.U. Brest, Hôpital Morvan, Laboratoire de Génétique Moléculaire, Brest, F-29200, France.|Etablissement Français du Sang - Bretagne, Brest, F-29200, France.
authors.forename: Claude
authors.initials: C
authors.lastname: Férec
mesh_terms: D000199:Actins / Q000378:metabolism*; D000595:Amino Acid Sequence; D002462:Cell Membrane / Q000378:metabolism; D018118:Chloride Channels / Q000235:genetics / Q000378:metabolism; D003550:Cystic Fibrosis / Q000235:genetics / Q000378:metabolism; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000235:genetics / Q000378:metabolism*; D015180:Electrophoresis, Gel, Two-Dimensional; D006367:HeLa Cells; D006801:Humans; D047468:Immunoprecipitation; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D018408:Patch-Clamp Techniques; D020641:Polymorphism, Single Nucleotide; D011485:Protein Binding
chemical_list: D000199:Actins; C505032:CFTR protein, human; D018118:Chloride Channels; D019005:Cystic Fibrosis Transmembrane Conductance Regulator
keywords: 2-DE; G551D-CFTR; actin; cystic fibrosis transmembrane conductance regulator