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Structural characterization of mixed wt/mutant aquaporin‐2 tetramers using dual‐colored fluorescent subunit counting (1133.1)
Author(s) -
Lussier Yoann,
McGuire Hugo,
El Tarazi Abdulah,
Bissonnette Pierre,
Blunck Rikard,
Bichet Daniel
Publication year - 2014
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.28.1_supplement.1133.1
Subject(s) - tetramer , mutant , nephrogenic diabetes insipidus , protein subunit , xenopus , aquaporin 2 , reabsorption , chemistry , hek 293 cells , fluorescence , immunoprecipitation , wild type , microbiology and biotechnology , mutation , transfection , green fluorescent protein , biophysics , biology , gene , kidney , biochemistry , genetics , enzyme , physics , water channel , mechanical engineering , quantum mechanics , engineering , inlet
Mutations in Aquaporin‐2 (AQP2) induce nephrogenic diabetes insipidus, a water reabsorption defect of the kidney. Using co‐immunoprecipitation and functional assays, we have recently shown that (at least) some mild recessive (rec) AQP2 mutants can adequately associate to its wild‐type (wt) counterpart to generate fully functional wt/rec heterotetramers. To better characterize actual subunit distributions within wt/rec AQP2 populations, we have developed a dual‐colored fluorescent subunit counting approach in TIRF condition, in order to identify and quantify individual monomers through bleaching of linked fluorophores. Assays performed on both Xenopus oocyte and transfected cell line have confirmed that mild mutations (K228E, D150E and V24A), similar to wt‐AQP2, adequately oligomerize in tetramer, unlike the severe R187C mutant which only displays monomeric structures. In dual transfections (sfGFP‐wt‐AQP2 + RFP‐rec‐AQP2), we also confirm the presence of mixed wt/rec tetramers for mild mutations, but not with R187C, which remains monomeric. Ultimately, we aim at describing subunit distributions within wt/rec tetramers in order to evaluate the properties, limitations and functional outcome of such heteromeric associations.