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

1997 ◽  
Vol 273 (3) ◽  
pp. F451-F456 ◽  
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.

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

2009 ◽  
Vol 296 (3) ◽  
pp. F649-F657 ◽  
Author(s):  
Hanne B. Moeller ◽  
Nanna MacAulay ◽  
Mark A. Knepper ◽  
Robert A. Fenton
Keyword(s):  
Plasma Membrane ◽  
Water Transport ◽  
Water Permeability ◽  
Laser Scanning Microscopy ◽  
Apical Plasma Membrane ◽  
Phosphorylation Sites ◽  
Osmotic Water Permeability ◽  
Aquaporin 2 ◽  
Osmotic Water

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.

Sevenfold-reduced osmotic water permeability in primary astrocyte cultures from AQP-4-deficient mice, measured by a fluorescence quenching method

2004 ◽  
Vol 286 (2) ◽  
pp. C426-C432 ◽  
Author(s):  
Eugen Solenov ◽  
Hiroyuki Watanabe ◽  
Geoffrey T. Manley ◽  
A. S. Verkman
Keyword(s):  
Fluorescence Quenching ◽  
Water Transport ◽  
Water Permeability ◽  
Cell Swelling ◽  
Osmotic Water Permeability ◽  
Wild Type ◽  
Osmotic Water ◽  
Fluorescence Quenching Method ◽  
Quenching Method ◽  
Deficient Mice

A calcein fluorescence quenching method was applied to measure osmotic water permeability in highly differentiated primary cultures of brain astrocytes from wild-type and aquaporin-4 (AQP-4)-deficient mice. Cells grown on coverglasses were loaded with calcein for measurement of volume changes after osmotic challenge. Hypotonic shock producing twofold cell swelling resulted in a reversible ∼12% increase in calcein fluorescence, which was independent of cytosolic calcein concentration at levels well below where calcein self-quenching occurs. Calcein fluorescence was quenched in <200 ms in response to addition of cytosol in vitro, indicating that the fluorescence signal arises from changes in cytosol concentration. In astrocytes from wild-type CD1 mice, calcein fluorescence increased reversibly in response to hypotonic challenge with a half-time of 0.92 ± 0.05 s at 23°C, corresponding to an osmotic water permeability ( Pf) of ∼0.05 cm/s. Pf was reduced 7.1-fold in astrocytes from AQP-4-deficient mice. Temperature dependence studies indicated an increased Arrhenius activation energy for water transport in AQP-4-deficient astrocytes (11.3 ± 0.5 vs. 5.5 ± 0.4 kcal/mol). Our studies establish a calcein quenching method for measurement of cell membrane water permeability and indicate that AQP-4 provides the principal route for water transport in astrocytes.

Functional analysis of aquaporin-2 mutants associated with nephrogenic diabetes insipidus by yeast expression

1999 ◽  
Vol 277 (5) ◽  
pp. F734-F741 ◽  
Author(s):  
Itsuki Shinbo ◽  
Kiyohide Fushimi ◽  
Michihiro Kasahara ◽  
Kazushi Yamauchi ◽  
Sei Sasaki ◽  
...  
Keyword(s):  
Diabetes Insipidus ◽  
Water Permeability ◽  
Nephrogenic Diabetes Insipidus ◽  
Water Channel ◽  
Osmotic Water Permeability ◽  
Wild Type ◽  
The Third ◽  
Aquaporin 2 ◽  
Channel Water ◽  
Osmotic Water

Mutations of aquaporin-2 (AQP2) vasopressin water channel cause nephrogenic diabetes insipidus (NDI). It has been suggested that impaired routing of AQP2 mutants to the plasma membrane causes the disease; however, no determinations have been made of mutation-induced alterations of AQP2 channel water permeability. To address this issue, a series of AQP2 mutants were expressed in yeast, and the osmotic water permeability ( Pf) of the isolated vesicles was measured. Wild-type and mutant AQP2 were expressed equally well in vesicles. Pfof the vesicles containing wild-type AQP2 was 22 times greater than that of the control, which was sensitive to mercury and weakly dependent on the temperature. Pfmeasurements and mercury inhibition examinations suggested that mutants L22V and P262L are fully functional, whereas mutants N68S, R187C, and S216P are partially functional. In contrast, mutants N123D, T125M, T126M, A147T, and C181W had very low water permeability. Our results suggest that the structure between the third and fifth hydrophilic loops is critical for the functional integrity of the AQP2 water channel and that disruption of AQP2 water permeability by mutations may cause NDI.

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

2016 ◽  
Vol 310 (10) ◽  
pp. F1008-F1012 ◽  
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.

scholarly journals Dynamic Changes in the Osmotic Water Permeability of Protoplast Plasma Membrane

2004 ◽  
Vol 135 (4) ◽  
pp. 2301-2317 ◽  
Author(s):  
Menachem Moshelion ◽  
Nava Moran ◽  
François Chaumont
Keyword(s):  
Plasma Membrane ◽  
Water Permeability ◽  
Osmotic Water Permeability ◽  
Dynamic Changes ◽  
Osmotic Water

scholarly journals Relative CO2/NH3 selectivities of mammalian aquaporins 0–9

2013 ◽  
Vol 304 (10) ◽  
pp. C985-C994 ◽  
Author(s):  
R. Ryan Geyer ◽  
Raif Musa-Aziz ◽  
Xue Qin ◽  
Walter F. Boron
Keyword(s):  
Plasma Membrane ◽  
Water Permeability ◽  
Xenopus Oocytes ◽  
Video Microscopy ◽  
Osmotic Water Permeability ◽  
Permeability Ratio ◽  
Diverse Range ◽  
Surface Ph ◽  
Aquaporin 1 ◽  
Osmotic Water

Previous work showed that aquaporin 1 (AQP1), AQP4-M23, and AQP5 each has a characteristic CO2/NH3 and CO2/H2O permeability ratio. The goal of the present study is to characterize AQPs 0–9, which traffic to the plasma membrane when heterologously expressed in Xenopus oocytes. We use video microscopy to compute osmotic water permeability ( Pf) and microelectrodes to record transient changes in surface pH (ΔpHS) caused by CO2 or NH3 influx. Subtracting respective values for day-matched, H2O-injected control oocytes yields the channel-specific values Pf* and ΔpHS*. We find that Pf* is significantly >0 for all AQPs tested except AQP6. (ΔpHS*)CO2 is significantly >0 for AQP0, AQP1, AQP4-M23, AQP5, AQP6, and AQP9. (ΔpHS*)NH3 is >0 for AQP1, AQP3, AQP6, AQP7, AQP8, and AQP9. The ratio (ΔpHS*)CO2/ Pf* falls in the sequence AQP6 (∞) > AQP5 > AQP4-M23 > AQP0 ≅ AQP1 ≅ AQP9 > others (0). The ratio (ΔpHS*)NH3/ Pf* falls in the sequence AQP6 (∞) > AQP3 ≅ AQP7 ≅ AQP8 ≅ AQP9 > AQP1 > others (0). Finally, the ratio (ΔpHS*)CO2/(−ΔpHS*)NH3 falls in the sequence AQP0 (∞) ≅ AQP4-M23 ≅ AQP5 > AQP6 > AQP1 > AQP9 > AQP3 (0) ≅ AQP7 ≅ AQP8. The ratio (ΔpHS*)CO2/(−ΔpHS*)NH3 is indeterminate for both AQP2 and AQP4-M1. In summary, we find that mammalian AQPs exhibit a diverse range of selectivities for CO2 vs. NH3 vs. H2O. As a consequence, by expressing specific combinations of AQPs, cells could exert considerable control over the movements of each of these three substances.

scholarly journals Colon water transport in transgenic mice lacking aquaporin-4 water channels

2000 ◽  
Vol 279 (2) ◽  
pp. G463-G470 ◽  
Author(s):  
Kasper S. Wang ◽  
Tonghui Ma ◽  
Ferda Filiz ◽  
A. S. Verkman ◽  
J. Augusto Bastidas
Keyword(s):  
Water Content ◽  
Water Transport ◽  
Water Permeability ◽  
Basolateral Membrane ◽  
Distal Colon ◽  
Water Channels ◽  
Proximal Colon ◽  
Aquaporin 4 ◽  
Osmotic Water Permeability ◽  
Osmotic Water

Transgenic null mice were used to test the hypothesis that water channel aquaporin-4 (AQP4) is involved in colon water transport and fecal dehydration. AQP4 was immunolocalized to the basolateral membrane of colonic surface epithelium of wild-type (+/+) mice and was absent in AQP4 null (−/−) mice. The transepithelial osmotic water permeability coefficient ( P f) of in vivo perfused colon of +/+ mice, measured using the volume marker 14C-labeled polyethylene glycol, was 0.016 ± 0.002 cm/s. P f of proximal colon was greater than that of distal colon (0.020 ± 0.004 vs. 0.009 ± 0.003 cm/s, P < 0.01). P f was significantly lower in −/− mice when measured in full-length colon (0.009 ± 0.002 cm/s, P< 0.05) and proximal colon (0.013 ± 0.002 cm/s, P< 0.05) but not in distal colon. There was no difference in water content of cecal stool from +/+ vs. −/− mice (0.80 ± 0.01 vs. 0.81 ± 0.01), but there was a slightly higher water content in defecated stool from −/− mice (0.68 ± 0.01 vs. 0.65 ± 0.01, P < 0.05). Despite the differences in water permeability with AQP4 deletion, theophylline-induced secretion was not impaired (50 ± 9 vs. 51 ± 8 μl · min−1 · g−1). These results provide evidence that transcellular water transport through AQP4 water channels in colonic epithelium facilitates transepithelial osmotic water permeability but has little or no effect on colonic fluid secretion or fecal dehydration.

scholarly journals Droplet-based microfluidic platform for measurement of rapid erythrocyte water transport

Lab on a Chip ◽  
2015 ◽  
Vol 15 (16) ◽  
pp. 3380-3390 ◽  
Author(s):  
Byung-Ju Jin ◽  
Cristina Esteva-Font ◽  
A. S. Verkman
Keyword(s):  
Water Transport ◽  
Water Permeability ◽  
Droplet Microfluidics ◽  
Osmotic Water Permeability ◽  
Fluorescence Image ◽  
Microfluidic Platform ◽  
Single Time ◽  
Osmotic Water

Osmotic water permeability was measured from a single, time-integrated fluorescence image using droplet microfluidics.

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