1. ¹The Water and Salt Research Center, Institute of Anatomy, University of Aarhus, Aarhus, Denmark
2. ²Laboratory of Kidney and Electrolyte Metabolism, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland, USA

Correspondence: Robert A. Fenton, The Water and Salt Research Center, Institute of Anatomy (Building 1233), University of Aarhus, Aarhus DK-8000, Denmark. E-mail: rofe@ana.au.dk

Received 26 June 2008; Revised 6 August 2008; Accepted 19 August 2008; Published online 8 October 2008.

Abstract

Trafficking of the water channel aquaporin-2 to the apical plasma membrane of the collecting duct is mediated by arginine vasopressin, rendering the cell permeable to water. We recently identified a novel form of aquaporin-2 that is phosphorylated at serine-269 (pS269-AQP2). Using antibodies specific for this form of the water channel, we detected rat and mouse pS269-AQP2 in the connecting tubule and throughout the collecting duct system. Using confocal immunofluorescence microscopy with organelle-specific markers and immunogold electron microscopy, we found that pS269-AQP2 was found only on the apical plasma membrane of principal cells. In vasopressin-deficient Brattleboro rats, pS269-AQP2 was undetectable but dramatically increased in abundance after these rats were treated with [deamino-Cys-1, d-Arg-8]vasopressin (dDAVP). This increase occurred only at the apical plasma membrane, even after long-term dDAVP treatment. Following dDAVP there was a time-dependent redistribution of total aquaporin-2 from predominantly intracellular vesicles to the apical plasma membrane, clathrin-coated vesicles, early endosomal compartments, and lysosomes. However, pS269-AQP2 was found only on the apical plasma membrane at any time. Our results show that S269 phosphorylated aquaporin-2 is exclusively associated with the apical plasma membrane, where it escapes endocytosis to remain at the cell surface.