The subcellular localization of an aquaporin-2 tetramer depends on the stoichiometry of phosphorylated and nonphosphorylated monomers

In renal principal cells, vasopressin regulates the shuttling of the aquapo rin (AQP)2 water channel between intracellular vesicles and the apical plas ma membrane. Vasopressin-induced phosphorylation of AQP2 at serine 256 (S25 6) by protein kinase A (PKA) is essential for its localization in the membr ane. However, phosphorylated AQP2 (p-AQP2) has also been detected in intrac ellular vesicles of noninduced principal cells. As AQP2 is expressed as hom otetramers, we hypothesized that the number of p-AQP2 monomers in a tetrame r might be critical for the its steady state distribution. Expressed in ooc ytes, AQP2-S256D and AQP2-S256A mimicked p-AQP2 and non-p-AQP2, respectivel y, as routing and function of AQP2-S256D and wild-type AQP2 (wt-AQP2) were identical, whereas AQP2-S256A was retained intracellularly. In coinjection experiments, AQP2-S256A and AQP2-S256D formed heterotetramers. Coinjection of different ratios of AQP2-S256A and AQP2-S256D cRNAs revealed that minima lly three AQP2-S256D monomers in an AQP2 tetramer were essential for its pl asma membrane localization. Therefore, our results suggest that in principa l cells, minimally three monomers per AQP2 tetramer have to be phosphorylat ed for its steady state localization in the apical membrane. As other multi subunit channels are also regulated by phosphorylation, it is anticipated t hat the stoichiometry of their phosphorylated and nonphosphorylated subunit s may fine-tune the activity or subcellular localization of these complexes .