Functional role of multiple phosphorylation sites in the carboxyl‐terminal tail of the water channel Aquaporin‐2

Abstract

Phosphorylation of Aquaporin‐2 (AQP2) at serine 256 (S256) is essential for its accumulation in the plasma membrane of collecting duct principal cells. We examined the role of additional phosphorylation sites in the C‐terminal tail on AQP2 function. Lack of AQP2 phosphorylation at S256 (S256A‐AQP2) reduced water permeability 3‐fold compared to wildtype (WT) AQP2‐injected Xenopus oocytes. Prevention of AQP2 phosphorylation at S261, S264, and S269 had no effect on water permeability. Oocytes expressing AQP2 mimicking phosphorylation at S264 and S269 had similar water permeabilites to WT‐AQP2‐expressing oocytes. Microscopy and biochemical analysis demonstrated that all AQP2 mutants, with the exception of S256A‐AQP2, had equal abundance in the oocyte plasma membrane. Water permeability relative to plasma membrane abundance demonstrated that lack of phosphorylation at S256, S261, S264 or S269 had no effect on AQP2 unit water transport. No effect on AQP2 unit water transort was observed for the 264D and 269D forms, indicating that phosphorylation of the C‐terminal tail of AQP2 is not involved in channel gating. Phosphospecific antibodies demonstrated that AQP2 S256 phosphorylation is not dependent on other phosphorylation sites, whereas S264 and S269 phosphorylation depend on prior S256 phosphorylation. In contrast, AQP2 S261 phosphorylation is independent of the phosphorylation status of S256.