title: New autosomal recessive mutations in aquaporin-2 causing nephrogenic diabetes insipidus through deficient targeting display normal expression in Xenopus oocytes.
abstract: Aquaporin-2 (AQP2), located at the luminal side of the collecting duct principal cells, is a water channel responsible for the final concentration of urine. Lack of function, often occurring through mistargeting of mutated proteins, induces nephrogenic diabetes insipidus (NDI), a condition characterized by large urinary volumes. In the present study, two new mutations (K228E and V24A) identified in NDI-affected individuals from distinct families along with the already reported R187C were analysed in comparison to the wild-type protein (AQP2-wt) using Xenopus laevis oocytes and a mouse collecting duct cell-line (mIMCD-3). Initial data in oocytes showed that all mutations were adequately expressed at reduced levels when compared to AQP2-wt. K228E and V24A were found to be properly targeted at the plasma membrane and exhibited adequate functionality similar to AQP2-wt, as opposed to R187C which was retained in internal stores and was thus inactive. In coexpression studies using oocytes, R187C impeded the functionality of all other AQP2 variants while combinations with K228E, V24A and AQP2-wt only showed additive functionalities. When expressed in mIMCD-3 cells, forskolin treatment efficiently promoted the targeting of AQP2-wt at the plasma membrane (>90%) while K228E only weakly responded to the same treatment (approximately 20%) and both V24A and R187C remained completely insensitive to the treatment. We concluded that both V24A and K228E are intrinsically functional water channels that lack a proper response to vasopressin, which leads to NDI as found in both compound mutations studied (K228E + R187C and V24A + R187C). The discrepancies in plasma membrane targeting response found in both expression systems stress the need to evaluate such data using mammalian cell systems.
authors.affiliation: Groupe d' Etude des Protéines Membranaires (GEPROM), département de Physiologie, Université de Montréal, Montréal, Québec, Canada.
authors.forename: Alexandre
authors.initials: A
authors.lastname: Leduc-Nadeau
authors.forename: Yoann
authors.initials: Y
authors.lastname: Lussier
authors.forename: Marie-Françoise
authors.initials: MF
authors.lastname: Arthus
authors.forename: Michèle
authors.initials: M
authors.lastname: Lonergan
authors.forename: Alejandro
authors.initials: A
authors.lastname: Martinez-Aguayo
authors.forename: Eva
authors.initials: E
authors.lastname: Riveira-Munoz
authors.forename: Olivier
authors.initials: O
authors.lastname: Devuyst
authors.forename: Pierre
authors.initials: P
authors.lastname: Bissonnette
authors.forename: Daniel G
authors.initials: DG
authors.lastname: Bichet
mesh_terms: D000328:Adult; D000595:Amino Acid Sequence; D000818:Animals; D051399:Aquaporin 2 / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism*; D005576:Colforsin / Q000494:pharmacology; D018500:Diabetes Insipidus, Nephrogenic / Q000235:genetics / Q000378:metabolism* / Q000503:physiopathology; D005260:Female; D005808:Genes, Recessive; D020022:Genetic Predisposition to Disease; D006801:Humans; D007685:Kidney Tubules, Collecting / Q000187:drug effects / Q000378:metabolism* / Q000503:physiopathology; D008297:Male; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation; D009865:Oocytes / Q000378:metabolism*; D010375:Pedigree; D021381:Protein Transport; D014162:Transfection; D014667:Vasopressins / Q000378:metabolism; D014867:Water / Q000378:metabolism; D014982:Xenopus laevis / Q000235:genetics / Q000378:metabolism*
chemical_list: C496023:AQP2 protein, human; D051399:Aquaporin 2; D014867:Water; D014667:Vasopressins; D005576:Colforsin
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