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The role of Cysteine 227 in subcellular localization, water permeability, and multimerization of aquaporin-11.

Takahashi S, Muta K, Sonoda H, Kato A, Abdeen A, Ikeda M - FEBS Open Bio (2014)

Bottom Line: Interestingly, cells expressing the mutants had significantly higher osmotic water permeability.In contrast, the mutation lowered the cell surface expression and multimerization levels.Our observations suggest that Cys(227) is crucial for the proper molecular function of AQP11.

View Article: PubMed Central - PubMed

Affiliation: Department of Veterinary Pharmacology, University of Miyazaki, Miyazaki 889-2192, Japan.

ABSTRACT
Aquaporin-11 (AQP11) is the latest member of the mammalian water channel protein family to be described. Recent in vivo studies have shown that mutation at Cys(227) causes renal failure. However the importance of Cys(227) for the molecular function of AQP11 is largely unknown. In this study, we examined the subcellular localization, water permeability, and multimerization of AQP11 with a mutation at Cys(227). Interestingly, cells expressing the mutants had significantly higher osmotic water permeability. In contrast, the mutation lowered the cell surface expression and multimerization levels. Our observations suggest that Cys(227) is crucial for the proper molecular function of AQP11.

No MeSH data available.


Related in: MedlinePlus

Osmotic water permeability of cells expressing AQP11 mutant at Cys227. (A) CHO cells were transfected with DsRM-AQP11, DsRM-hAQP11-C227S, or DsRM-hAQP11-C227A expression plasmid. After 24 h, the cell diameter was measured under a microscope. The cells were exposed to hypotonic solution (300–150 m Osm/kg solution) at time point of 0. An image of the cell was recorded every 1.12 s with the microscope equipped with a time-lapse image-capture system. Values are presented as mean ± SE. The numbers of cells tested are given in parentheses. (B) Pf values were calculated from osmotic swelling data from A using the formula as indicated in the Materials and Methods. Values are shown as mean ± SE. ∗P < 0.05 vs. DsRM-hAQP11 (Dunnett’s test).
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f0015: Osmotic water permeability of cells expressing AQP11 mutant at Cys227. (A) CHO cells were transfected with DsRM-AQP11, DsRM-hAQP11-C227S, or DsRM-hAQP11-C227A expression plasmid. After 24 h, the cell diameter was measured under a microscope. The cells were exposed to hypotonic solution (300–150 m Osm/kg solution) at time point of 0. An image of the cell was recorded every 1.12 s with the microscope equipped with a time-lapse image-capture system. Values are presented as mean ± SE. The numbers of cells tested are given in parentheses. (B) Pf values were calculated from osmotic swelling data from A using the formula as indicated in the Materials and Methods. Values are shown as mean ± SE. ∗P < 0.05 vs. DsRM-hAQP11 (Dunnett’s test).

Mentions: Cell surface biotinylation experiments showed that significant amounts of AQP11 and its mutants were also expressed at the plasma membrane (Fig. 2), and we had previously reported that the level of AQP11 cell surface expression allowed us to measure water permeability by an osmotic swelling assay [8]. Therefore, we employed this assay system and examined the osmotic water permeability of cells expressing DsRM-hAQP11, DsRM-hAQP11-C227S, or DsRM-hAQP11-C227A. As shown in Fig. 3A, hypotonic stimulation unexpectedly caused more rapid swelling of cells expressing DsRM-hAQP11-C227S and DsRM-hAQP11-C227A than that of cells expressing the wild type. The Pf values (Fig. 3B) of DsRM-hAQP11-C227S and -C227A were significantly higher than that of wild-type AQP11. It was noted that the Pf value shown as mean ± SE was 4.7 ± 0.9 cm/s × 10−4 (n = 6) for the mock cells. As the plasma membrane expression levels of mutants were significantly lower than that of the wild type (Fig. 2), these data suggest that the mutation of AQP11 at Cys227 conferred a gain of function for water permeability.


The role of Cysteine 227 in subcellular localization, water permeability, and multimerization of aquaporin-11.

Takahashi S, Muta K, Sonoda H, Kato A, Abdeen A, Ikeda M - FEBS Open Bio (2014)

Osmotic water permeability of cells expressing AQP11 mutant at Cys227. (A) CHO cells were transfected with DsRM-AQP11, DsRM-hAQP11-C227S, or DsRM-hAQP11-C227A expression plasmid. After 24 h, the cell diameter was measured under a microscope. The cells were exposed to hypotonic solution (300–150 m Osm/kg solution) at time point of 0. An image of the cell was recorded every 1.12 s with the microscope equipped with a time-lapse image-capture system. Values are presented as mean ± SE. The numbers of cells tested are given in parentheses. (B) Pf values were calculated from osmotic swelling data from A using the formula as indicated in the Materials and Methods. Values are shown as mean ± SE. ∗P < 0.05 vs. DsRM-hAQP11 (Dunnett’s test).
© Copyright Policy - CC BY-NC-ND
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4048847&req=5

f0015: Osmotic water permeability of cells expressing AQP11 mutant at Cys227. (A) CHO cells were transfected with DsRM-AQP11, DsRM-hAQP11-C227S, or DsRM-hAQP11-C227A expression plasmid. After 24 h, the cell diameter was measured under a microscope. The cells were exposed to hypotonic solution (300–150 m Osm/kg solution) at time point of 0. An image of the cell was recorded every 1.12 s with the microscope equipped with a time-lapse image-capture system. Values are presented as mean ± SE. The numbers of cells tested are given in parentheses. (B) Pf values were calculated from osmotic swelling data from A using the formula as indicated in the Materials and Methods. Values are shown as mean ± SE. ∗P < 0.05 vs. DsRM-hAQP11 (Dunnett’s test).
Mentions: Cell surface biotinylation experiments showed that significant amounts of AQP11 and its mutants were also expressed at the plasma membrane (Fig. 2), and we had previously reported that the level of AQP11 cell surface expression allowed us to measure water permeability by an osmotic swelling assay [8]. Therefore, we employed this assay system and examined the osmotic water permeability of cells expressing DsRM-hAQP11, DsRM-hAQP11-C227S, or DsRM-hAQP11-C227A. As shown in Fig. 3A, hypotonic stimulation unexpectedly caused more rapid swelling of cells expressing DsRM-hAQP11-C227S and DsRM-hAQP11-C227A than that of cells expressing the wild type. The Pf values (Fig. 3B) of DsRM-hAQP11-C227S and -C227A were significantly higher than that of wild-type AQP11. It was noted that the Pf value shown as mean ± SE was 4.7 ± 0.9 cm/s × 10−4 (n = 6) for the mock cells. As the plasma membrane expression levels of mutants were significantly lower than that of the wild type (Fig. 2), these data suggest that the mutation of AQP11 at Cys227 conferred a gain of function for water permeability.

Bottom Line: Interestingly, cells expressing the mutants had significantly higher osmotic water permeability.In contrast, the mutation lowered the cell surface expression and multimerization levels.Our observations suggest that Cys(227) is crucial for the proper molecular function of AQP11.

View Article: PubMed Central - PubMed

Affiliation: Department of Veterinary Pharmacology, University of Miyazaki, Miyazaki 889-2192, Japan.

ABSTRACT
Aquaporin-11 (AQP11) is the latest member of the mammalian water channel protein family to be described. Recent in vivo studies have shown that mutation at Cys(227) causes renal failure. However the importance of Cys(227) for the molecular function of AQP11 is largely unknown. In this study, we examined the subcellular localization, water permeability, and multimerization of AQP11 with a mutation at Cys(227). Interestingly, cells expressing the mutants had significantly higher osmotic water permeability. In contrast, the mutation lowered the cell surface expression and multimerization levels. Our observations suggest that Cys(227) is crucial for the proper molecular function of AQP11.

No MeSH data available.


Related in: MedlinePlus