Role of multiple phosphorylation sites in the COOH-terminal tail of aquaporin-2 for water transport: evidence against channel gating

Arginine vasopressin (AVP)-regulated phosphorylation of the water channel aquaporin-2 (AQP2) at serine 256 (S256) is essential for its accumulation in the apical plasma membrane of collecting duct principal cells. In this study, we examined the role of additional AVP-regulated phosphorylation sites in the COOH-terminal tail of AQP2 on protein function. When expressed in Xenopus laevis oocytes, prevention of AQP2 phosphorylation at S256A (S256A-AQP2) reduced osmotic water permeability threefold compared with wild-type (WT) AQP2-injected oocytes. In contrast, prevention of AQP2 single phosphorylation at S261 (S261A), S264 (S264A), and S269 (S269A), or all three sites in combination had no significant effect on water permeability. Similarly, oocytes expressing S264D-AQP2 and S269D-AQP2, mimicking AQP2 phosphorylated at these residues, had similar water permeabilities to WT-AQP2-expressing oocytes. The use of high-resolution confocal laser-scanning microscopy, as well as biochemical analysis demonstrated that all AQP2 mutants, with the exception of S256A-AQP2, had equal abundance in the oocyte plasma membrane. Correlation of osmotic 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. Similarly, no effect on AQP2 unit water transport was observed for the 264D and 269D forms, indicating that phosphorylation of the COOH-terminal tail of AQP2 is not involved in gating of the channel. The use of phosphospecific antibodies demonstrated that AQP2 S256 phosphorylation is not dependent on any of the other phosphorylation sites, whereas S264 and S269 phosphorylation depend on prior phosphorylation of S256. In contrast, AQP2 S261 phosphorylation is independent of the phosphorylation status of S256.

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Metformin, an AMPK activator, stimulates the phosphorylation of aquaporin 2 and urea transporter A1 in inner medullary collecting ducts

AJP Renal Physiology ◽

10.1152/ajprenal.00102.2016 ◽

2016 ◽

Vol 310 (10) ◽

pp. F1008-F1012 ◽

Cited By ~ 25

Author(s):

Janet D. Klein ◽

Yanhua Wang ◽

Mitsi A. Blount ◽

Patrick A. Molina ◽

Lauren M. LaRocque ◽

...

Keyword(s):

Plasma Membrane ◽

Water Permeability ◽

Therapeutic Option ◽

Apical Plasma Membrane ◽

Osmotic Water Permeability ◽

Urea Transporter ◽

Aquaporin 2 ◽

Urea Permeability ◽

Osmotic Water ◽

Collecting Ducts

Nephrogenic diabetes insipidus (NDI) is characterized by production of very large quantities of dilute urine due to an inability of the kidney to respond to vasopressin. Congenital NDI results from mutations in the type 2 vasopressin receptor (V2R) in ∼90% of families. These patients do not have mutations in aquaporin-2 (AQP2) or urea transporter UT-A1 (UT-A1). We tested adenosine monophosphate kinase (AMPK) since it is known to phosphorylate another vasopressin-sensitive transporter, NKCC2 (Na-K-2Cl cotransporter). We found AMPK expressed in rat inner medulla (IM). AMPK directly phosphorylated AQP2 and UT-A1 in vitro. Metformin, an AMPK activator, increased phosphorylation of both AQP2 and UT-A1 in rat inner medullary collecting ducts (IMCDs). Metformin increased the apical plasma membrane accumulation of AQP2, but not UT-A1, in rat IM. Metformin increased both osmotic water permeability and urea permeability in perfused rat terminal IMCDs. These findings suggest that metformin increases osmotic water permeability by increasing AQP2 accumulation in the apical plasma membrane but increases urea permeability by activating UT-A1 already present in the membrane. Lastly, metformin increased urine osmolality in mice lacking a V2R, a mouse model of congenital NDI. We conclude that AMPK activation by metformin mimics many of the mechanisms by which vasopressin increases urine-concentrating ability. These findings suggest that metformin may be a novel therapeutic option for congenital NDI due to V2R mutations.

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Importance of the mercury-sensitive cysteine on function and routing of AQP1 and AQP2 in oocytes

AJP Renal Physiology ◽

10.1152/ajprenal.1997.273.3.f451 ◽

1997 ◽

Vol 273 (3) ◽

pp. F451-F456 ◽

Cited By ~ 3

Author(s):

S. M. Mulders ◽

J. P. Rijss ◽

A. Hartog ◽

R. J. Bindels ◽

C. H. van Os ◽

...

Keyword(s):

Endoplasmic Reticulum ◽

Plasma Membrane ◽

Water Transport ◽

Water Permeability ◽

Nephrogenic Diabetes Insipidus ◽

Osmotic Water Permeability ◽

Wild Type ◽

Mutant Proteins ◽

Osmotic Water ◽

Structural Differences

To discriminate between water transport of of aquaporin-2 (AQP2) mutants in nephrogenic diabetes insipidus and that of an AQP2 molecule used to drag them to the oolemma, we investigated the mercury sensitivity of wild-type and AQP2 C181S proteins in oocytes. Incubation with HgCl2 inhibited the osmotic water permeability (Pf) of human (h) AQP2 by 40%, whereas inhibition of hAQP1 was 75%. Oocytes expressing hAQP1 C189S revealed a Pf comparable to wild-type hAQP1, but mercury sensitivity was lost. In contrast, no increase in Pf was obtained when hAQP2 C181S was expressed. Also, expression of rat AQP2 C181A and C181S mutants did not increase the Pf, which contrasts with published observations. Immunocytochemistry and immunoblotting revealed that only AQP1, AQP1 C189S, and AQP2 were targeted to the plasma membrane and that AQP2 mutant proteins are retarded in the endoplasmic reticulum. In conclusion, water transport through AQP2 is less sensitive to mercury inhibition than through AQP1. Furthermore, substitution of the mercury-sensitive cysteine for a serine results in an impaired routing of human and rat AQP2. Similar mutations have no effect on AQP1 function, which is indicative of structural differences between AQP1 and AQP2.

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Faculty Opinions recommendation of The role of ABA and the transpiration stream in the regulation of the osmotic water permeability of leaf cells.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1004186.27054 ◽

2001 ◽

Author(s):

Walter Kockenberger

Keyword(s):

Water Permeability ◽

Osmotic Water Permeability ◽

Transpiration Stream ◽

Osmotic Water

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Role of PKC and AMPK in hypertonicity-stimulated water reabsorption in rat inner medullary collecting ducts

AJP Renal Physiology ◽

10.1152/ajprenal.00491.2017 ◽

2019 ◽

Vol 316 (2) ◽

pp. F253-F262 ◽

Cited By ~ 4

Author(s):

Josephine K. Liwang ◽

Joseph A. Ruiz ◽

Lauren M. LaRocque ◽

Fitra Rianto ◽

Fuying Ma ◽

...

Keyword(s):

Water Permeability ◽

Collecting Duct ◽

Adenosine Monophosphate ◽

Osmotic Water Permeability ◽

Water Reabsorption ◽

Compound C ◽

Ampk Phosphorylation ◽

Osmotic Water ◽

Medullary Collecting Duct

Hypertonicity increases water permeability, independently of vasopressin, in the inner medullary collecting duct (IMCD) by increasing aquaporin-2 (AQP2) membrane accumulation. We investigated whether protein kinase C (PKC) and adenosine monophosphate kinase (AMPK) are involved in hypertonicity-regulated water permeability. Increasing perfusate osmolality from 150 to 290 mosmol/kgH2O and bath osmolality from 290 to 430 mosmol/kgH2O significantly stimulated osmotic water permeability. The PKC inhibitors chelerythrine (10 µM) and rottlerin (50 µM) significantly reversed the increase in osmotic water permeability stimulated by hypertonicity in perfused rat terminal IMCDs. Chelerythrine significantly increased phosphorylation of AQP2 at S261 but not at S256. Previous studies show that AMPK is stimulated by osmotic stress. We tested AMPK phosphorylation under hypertonic conditions. Hypertonicity significantly increased AMPK phosphorylation in inner medullary tissues. Blockade of AMPK with Compound C decreased hypertonicity-stimulated water permeability but did not alter phosphorylation of AQP2 at S256 and S261. AICAR, an AMPK stimulator, caused a transient increase in osmotic water permeability and increased phosphorylation of AQP2 at S256. When inner medullary tissue was treated with the PKC activator phorbol dibutyrate (PDBu), the AMPK activator metformin, or both, AQP2 phosphorylation at S261 was decreased with PDBu or metformin alone, but there was no additive effect on phosphorylation with PDBu and metformin together. In conclusion, hypertonicity regulates water reabsorption by activating PKC. Hypertonicity-stimulated water reabsorption by PKC may be related to the decrease in endocytosis of AQP2. AMPK activation promotes water reabsorption, but the mechanism remains to be determined. PKC and AMPK do not appear to act synergistically to regulate water reabsorption.

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