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Int. J. Mol. Sci. 2016, 17(11), 1804;

AQP2 Plasma Membrane Diffusion Is Altered by the Degree of AQP2-S256 Phosphorylation

Department of Clinical Medicine, Aarhus University, DK-8000 Aarhus C, Denmark
The Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892, USA
Department of Interdisciplinary Nanoscience Center, Aarhus University, DK-8000 Aarhus C, Denmark
Current address: Department of Chemical Engineering, Biotechnology and Environmental Technology, University of Southern Denmark, Campusvej 55, 5230 Odense M, Denmark.
Current address: Janelia Research Campus, Ashburn, VA 20147, USA.
Author to whom correspondence should be addressed.
Academic Editor: Kenichi Ishibashi
Received: 19 July 2016 / Revised: 15 September 2016 / Accepted: 22 September 2016 / Published: 28 October 2016
(This article belongs to the Special Issue Aquaporin)
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Fine tuning of urine concentration occurs in the renal collecting duct in response to circulating levels of arginine vasopressin (AVP). AVP stimulates intracellular cAMP production, which mediates exocytosis of sub-apical vesicles containing the water channel aquaporin-2 (AQP2). Protein Kinase A (PKA) phosphorylates AQP2 on serine-256 (S256), which triggers plasma membrane accumulation of AQP2. This mediates insertion of AQP2 into the apical plasma membrane, increasing water permeability of the collecting duct. AQP2 is a homo-tetramer. When S256 on all four monomers is changed to the phosphomimic aspartic acid (S256D), AQP2-S256D localizes to the plasma membrane and internalization is decreased. In contrast, when S256 is mutated to alanine (S256A) to mimic non-phosphorylated AQP2, AQP2-S256A localizes to intracellular vesicles as well as the plasma membrane, with increased internalization from the plasma membrane. S256 phosphorylation is not necessary for exocytosis and dephosphorylation is not necessary for endocytosis, however, the degree of S256 phosphorylation is hypothesized to regulate the kinetics of AQP2 endocytosis and thus, retention time in the plasma membrane. Using k-space Image Correlation Spectroscopy (kICS), we determined how the number of phosphorylated to non-phosphorylated S256 monomers in the AQP2 tetramer affects diffusion speed of AQP2 in the plasma membrane. When all four monomers mimicked constitutive phosphorylation (AQP2-S256D), diffusion was faster than when all four were non-phosphorylated (AQP2-S256A). AQP2-WT diffused at a speed similar to that of AQP2-S256D. When an average of two or three monomers in the tetramer were constitutively phosphorylated, the average diffusion coefficients were not significantly different to that of AQP2-S256D. However, when only one monomer was phosphorylated, diffusion was slower and similar to AQP2-S256A. Thus, AQP2 with two to four phosphorylated monomers has faster plasma membrane kinetics, than the tetramer which contains just one or no phosphorylated monomers. This difference in diffusion rate may reflect behavior of AQP2 tetramers destined for either plasma membrane retention or endocytosis. View Full-Text
Keywords: diffusion coefficients; kICS: k-space Image Correlation Spectroscopy; MDCK; AQP2; kidney; urine; phosphorylation diffusion coefficients; kICS: k-space Image Correlation Spectroscopy; MDCK; AQP2; kidney; urine; phosphorylation

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This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited (CC BY 4.0).

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Arnspang, E.C.; Login, F.H.; Koffman, J.S.; Sengupta, P.; Nejsum, L.N. AQP2 Plasma Membrane Diffusion Is Altered by the Degree of AQP2-S256 Phosphorylation. Int. J. Mol. Sci. 2016, 17, 1804.

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