Abstract

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.

Highlights

  • IntroductionShort-term urine concentration is regulated in the renal collecting duct principal cells inShort-term urine concentration regulated in the renal collecting principal (AVP), cells in response response to circulating levels of theis antidiuretic hormone arginine duct vasopressin which to circulating levels offrom the antidiuretic hormone arginine vasopressin (AVP), which water mediates water uptake the renal ultra-filtrate.Water crosses the epithelial plasmamediates membranes uptakeaquaporin from the renal ultra-filtrate.Waterdriven crossesby theanepithelial membranes aquaporin through (AQP)water channels, osmotic plasma gradient.AQPs are through homotetrameric (AQP) water channels, by an osmotic gradient.homotetrameric transmembrane proteins, driven expressed in multiple epithelial AQPs tissuesare including lung [1], transmembrane sweat glands in multiple epithelial tissues including lung [1], sweatselective glands [2], and [2],proteins, skin [3],expressed and kidney (for review [4])

  • AQP2-enhanced green fluorescent protein (EGFP) has been stably expressed in LLC-PK1 cells, where it localized correctly to the plasma membrane and intracellular vesicles [44]

  • AQP2 aggregation due to EGFP was rescued by co-transfection of AQP2-EGFP and untagged AQP2

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Summary

Introduction

Short-term urine concentration is regulated in the renal collecting duct principal cells inShort-term urine concentration regulated in the renal collecting principal (AVP), cells in response response to circulating levels of theis antidiuretic hormone arginine duct vasopressin which to circulating levels offrom the antidiuretic hormone arginine vasopressin (AVP), which water mediates water uptake the renal ultra-filtrate.Water crosses the epithelial plasmamediates membranes uptakeaquaporin from the renal ultra-filtrate.Waterdriven crossesby theanepithelial membranes aquaporin through (AQP)water channels, osmotic plasma gradient.AQPs are through homotetrameric (AQP) water channels, by an osmotic gradient.homotetrameric transmembrane proteins, driven expressed in multiple epithelial AQPs tissuesare including lung [1], transmembrane sweat glands in multiple epithelial tissues including lung [1], sweatselective glands [2], and [2],proteins, skin [3],expressed and kidney (for review [4]). Short-term urine concentration is regulated in the renal collecting duct principal cells in. Short-term urine concentration regulated in the renal collecting principal (AVP), cells in response response to circulating levels of theis antidiuretic hormone arginine duct vasopressin which to circulating levels offrom the antidiuretic hormone arginine vasopressin (AVP), which water mediates water uptake the renal ultra-filtrate. AQPs are through homotetrameric (AQP) water channels, by an osmotic gradient. Homotetrameric transmembrane proteins, driven expressed in multiple epithelial AQPs tissuesare including lung [1], transmembrane sweat glands in multiple epithelial tissues including lung [1], sweatselective glands [2], and [2],proteins, skin [3],expressed and kidney (for review [4]). Each monomer contains a pore selective for water (for review [4])

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