Importance of aquaporin-2 expression levels in genotype -phenotype studies in nephrogenic diabetes insipidus.
Aquaporin-2 (AQP2) water channel mutations cause autosomal recessive and dominant nephrogenic diabetes insipidus. Expressed in oocytes, a mutant in dominant (AQP2-E258K), but not in recessive (AQP2-R187C), NDI conferred a specific dominant-negative effect (DNE) on wild-type (WT) AQP2 water permeability (P(f)) but only at low expression levels. Here, we determined the cell biological basis for this requirement. Injection of different amounts of WT-AQP2 cRNAs revealed that a correlation between AQP2 protein levels and P(f) is only obtained with low expression levels. In coexpression studies of WT- and mutant AQP2 proteins, higher expression levels of AQP2-R187C also exerted a DNE on the P(f) of WT-AQP2. Immunoblot and immunoprecipitation analysis revealed that this DNE was caused by competitive inhibition of WT-AQP2 expression and escape of AQP2-R187C from the endoplasmic reticulum, resulting in oligomerization with WT-AQP2. Because many disease-related mutants of multimeric renal membrane transporters and channels are likely to be identified, our data provide important information for studying the effects of such mutants on the activity of WT transporters and channels in oocytes.
Kamsteeg
E J
EJ
Department of Cell Physiology, University of Nijmegen, 6500HB Nijmegen, The Netherlands.
Deen
P M
PM
D000818:Animals; D051399:Aquaporin 2; D051403:Aquaporin 6; D020346:Aquaporins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D018500:Diabetes Insipidus, Nephrogenic / Q000235:genetics* / Q000378:metabolism*; D005786:Gene Expression Regulation; D005799:Genes, Dominant; D005838:Genotype; D015151:Immunoblotting; D007150:Immunohistochemistry; D009154:Mutation; D009865:Oocytes / Q000378:metabolism; D010539:Permeability; D010641:Phenotype; D011233:Precipitin Tests; D012016:Reference Values; D014867:Water / Q000378:metabolism; D014982:Xenopus laevis
D051399:Aquaporin 2; D051403:Aquaporin 6; D020346:Aquaporins; D014867:Water