title: Characterization of D150E and G196D aquaporin-2 mutations responsible for nephrogenic diabetes insipidus: importance of a mild phenotype.
abstract: Aquaporin-2 (AQP2) is a water channel responsible for the final water reabsorption in renal collecting ducts. Alterations in AQP2 function induce nephrogenic diabetes insipidus (NDI), a condition characterized by severe polyuria and polydipsia. Three patients affected with severe NDI, who were compound heterozygous for the AQP2 mutations D150E and G196D, are presented here along with a mildly affected D150E homozygous patient from another family. Using Xenopus oocytes as an expression system, these two mutations (G196D and D150E) were compared with the wild-type protein (AQP2-wt) for functional activity (water flux analysis), protein maturation, and plasma membrane targeting. AQP2-wt induces a major increase in water permeability (P(f) = 47.4 +/- 12.2 x 10(-4) cm/s) whereas D150E displays intermediate P(f) values (P(f) = 12.5 +/- 3.0 x 10(-4) cm/s) and G196D presents no specific water flux, similar to controls (P(f) = 2.1 +/- 0.8 x 10(-4) cm/s and 2.2 +/- 0.7 x 10(-4) cm/s, respectively). Western blot and immunocytochemical evaluations show protein targeting that parallels activity levels with AQP2-wt adequately targeted to the plasma membrane, partial targeting for D150E, and complete sequestration of G196D within intracellular compartments. When coinjecting AQP2-wt with mutants, no (AQP2-wt + D150E) or partial (AQP2-wt + G196D) reduction of water flux were observed compared with AQP2-wt alone, whereas complete loss of function was found when both mutants were coinjected. These results essentially recapitulate the clinical profiles of the family members, showing a typical dominant negative effect when G196D is coinjected with either AQP2-wt or D150E but not between AQP2-wt and D150E mutant.
authors.affiliation: Groupe d'Etude des Protéines Membranaires, Département de Physiologie, Université de Montréal, Montréal, Québec, Canada.
authors.forename: Cécile
authors.initials: C
authors.lastname: Guyon
authors.forename: Yoann
authors.initials: Y
authors.lastname: Lussier
authors.forename: Pierre
authors.initials: P
authors.lastname: Bissonnette
authors.forename: Alexandre
authors.initials: A
authors.lastname: Leduc-Nadeau
authors.forename: Michèle
authors.initials: M
authors.lastname: Lonergan
authors.forename: Marie-Françoise
authors.initials: MF
authors.lastname: Arthus
authors.forename: Rafael Bedoya
authors.initials: RB
authors.lastname: Perez
authors.forename: Anatoly
authors.initials: A
authors.lastname: Tiulpakov
authors.forename: Jean-Yves
authors.initials: JY
authors.lastname: Lapointe
authors.forename: Daniel G
authors.initials: DG
authors.lastname: Bichet
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D051399:Aquaporin 2 / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002463:Cell Membrane Permeability; D048429:Cell Size; D018500:Diabetes Insipidus, Nephrogenic / Q000235:genetics* / Q000378:metabolism; D005260:Female; D020022:Genetic Predisposition to Disease; D006579:Heterozygote; D006720:Homozygote; D006801:Humans; D008297:Male; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D009865:Oocytes; D010375:Pedigree; D010641:Phenotype; D011487:Protein Conformation; D021381:Protein Transport; D012720:Severity of Illness Index; D013329:Structure-Activity Relationship; D014162:Transfection; D014867:Water / Q000378:metabolism; D014982:Xenopus laevis
chemical_list: C496023:AQP2 protein, human; D051399:Aquaporin 2; D014867:Water