Three families with autosomal dominant nephrogenic diabetes insipidus caused by aquaporin-2 mutations in the C-terminus

The vasopressin-regulated water channel aquaporin-2 (AQP2) is known to tetr amerize in the apical membrane of the renal tubular cells and contributes t o urine concentration. We identified three novel mutations, each in a singl e allele of exon 4 of the AQP2 gene, in three families showing autosomal do minant nephrogenic diabetes insipidus (NDI). These mutations were found in the C-terminus of AQP2: a deletion of G at nucleotide 721 (721 delG), a del etion of 10 nucleotides starting at nucleotide 763 (763-772del), and a dele tion of 7 nucleotides starting at nucleotide 812 (812-818del). The wild-typ e AQP2 is predicted to be a 271-amino acid protein, whereas these mutant ge nes are predicted to encode proteins that are 330-333 amino acids in length , because of the frameshift mutations. Interestingly, these three mutant AQ P2s shared the same C-terminal tail of 61 amino acids. In Xenopus oocytes i njected with mutant AQP2 cRNAs, the osmotic water permeability (Pf) was muc h smaller than that of oocytes with the AQP2 wild-type (14%-17%). Immunoblo t analysis of the lysates of the oocytes expressing the mutant AQP2s detect ed a band at 34 kD, whereas the immunoblot of the plasma-membrane fractions of the oocytes and immunocytochemistry failed to show a significant surfac e expression, suggesting a defect in trafficking of these mutant proteins. Furthermore, coinjection of wild-type cRNAs with mutant cRNAs markedly decr eased the oocyte Pf in parallel with the surface expression of the wild-typ e AQP2. Immunoprecipitation with antibodies against wild-type and mutant AQ P2 indicated the formation of mixed oligomers composed of wild-type and mut ant AQP2 monomers. Our results suggest that the trafficking of mutant AQP2 is impaired because of elongation of the C-terminal tail, and the dominant- negative effect is attributed to oligomerization of the wild-type and mutan t AQP2s. Segregation of the mutations in the C-terminus of AQP2 with domina nt-type NDI underlies the importance of this domain in the intracellular tr afficking of AQP2.