Importance of aquaporin-2 expression levels in genotype-phenotype studies in nephrogenic diabetes insipidus

Aquaporin-2 (AQP2) water channel mutations cause autosomal recessive and do minant nephrogenic diabetes insipidus. Expressed in oocytes, a mutant in do minant (AQP2-E258K), but not in recessive (AQP2-R187C), NDI conferred a spe cific dominant-negative effect (DNE) on wild-type (WT) AQP2 water permeabil ity (P-f) but only at low expression levels. Here, we determined the cell b iological 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 exer ted a DNE on the P-f of WT- AQP2. Immunoblot and immunoprecipitation analys is revealed that this DNE was caused by competitive inhibition of WT- AQP2 expression and escape of AQP2-R187C from the endoplasmic reticulum, resulti ng in oligomerization with WT- AQP2. Because many disease-related mutants o f multimeric renal membrane transporters and channels are likely to be iden tified, our data provide important information for studying the effects of such mutants on the activity of WT transporters and channels in oocytes.