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Graphical Abstract
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Fig. 1. Schematic representation of mouse AQP0. The monomer structure shows folds, helix assignment, and the location at the membrane. The membrane-spanning α-helices are shown as H1–H6, the three extracellular loops as ELA, ELC and ELE and the two intracellular loops as LB, LD. The two water pore-lining helices are labeled as HB and HE. Two highly conserved asparagine–proline–alanine (NPA) motifs in HB and HE that line the water pore of aquaporin and considered responsible for water-selectivity are shaded in yellow. N, amino terminus; C, carboxyl terminus. ‘+’ and ‘−’ represent amino acid charges in the extracellular and cytoplasmic domains. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
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Fig. 2. Schematics of AQP0 showing WT and the sites targeted for site-directed mutagenesis and extracellular loop (EL) domain substitutions. (a) WT; (b) AQP0-AQP1ELA chimera (ELA of AQP0 is replaced with AQP1ELA (indicated in red)); (c) AQP0-AQP1ELC chimera (ELC of AQP0 is replaced with AQP1ELC (indicated in red)); (d) Site-directed mutants of ELA (R33Q, H40Q; indicated with asterisks on the loop) or ELC (R113Q, H122Q; shown by asterisks on the loop) had a charged residue (arginine, R or histidine, H) replaced with a neutral amino acid residue, glutamine (Q). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
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Fig. 3. Expression of wild type and mutant AQP0, and characterization of water channel function. (A). Expression of WT and mutants of AQP0 in the Xenopus oocytes. Cryosections of oocytes injected with distilled water or complementary RNAs (cRNAs) of WT or mutant AQP0 were immunostained using a polyclonal AQP0 antibody; the secondary antibody was conjugated to FITC: (a) Water-injected oocyte; (b–h) cRNA-injected: (b) WT AQP0; (c) AQP0-AQP1ELA; (d) AQP0-AQP1ELC; (e) R33Q; (f) H40Q; (g) R113Q; (h) H122Q. White solid arrows pointing to the plasma membrane indicate the absence or expression of AQP0. (B). Functional expression of WT AQP0, extracellular loop domain chimeras (AQP0-AQP1ELA and AQP0-AQP1ELC) and missense mutants (R33Q, H40Q, R113Q, and H122Q) in Xenopus laevis oocytes. For the study, the oocytes were injected with either distilled water or cRNA of WT or mutant AQP0 (25 ng/oocyte). Average Pf of twelve oocytes (mean ± SD) of WT or mutant AQP0 is shown. Star represents lack of Pf in AQP0-AQP1ELC, which is similar to dH2O-injected control. ‘+’ denotes the significant increase in Pf compared to WT AQP0.
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Fig. 4. Verification of WT and mutant AQP0 expression and localization in MDCK cells. (A). Plasma membrane localization of WT and mutant AQP0 by FRET analysis. Colocalization of EGFP tagged (a–c) WT AQP0, (d–f) AQP0-AQP1ELA, (g–i) AQP0-AQP1ELC, (j–l) R33Q, (m–o) H40Q, (p–r) R113Q, (s–u) H122Q coexpressed with plasma membrane (PM-RFP (CellLight® Red Fluorescent Protein, PM-RFP). Coexpressing cells were viewed under an EGFP fluorescent filter (column 1) and the same cells were viewed under Texas Red fluorescent filter to see protein localization at the plasma membrane (column 2). Plasma membrane localization was assessed using Förster Resonance Energy Transfer (FRET) signal intensity, shown in red (column 3). FRET studies were conducted using MDCK cells expressing WT AQP0-EGFP (a) or mutant AQP0-EGFP (d, g, j, m, p, s) and PM-RFP (b, e, h, k, n, q, t). Column 1 (a, d, g, j, m, p, s), cells excited at 488 nm and emission recorded at 507 nm; Column 2 (b, e, h, k, n, q, t), cells excited at 587 nm and emission recorded at 610 nm; Column 3 (c, f, i, l, o, r, u), cells excited at 470 nm and emission recorded at 640 nm; fluorescence due to FRET indicates colocalization of WT AQP0-EGFP or mutant AQP0-EGFP with PM-RFP within 100 Å. Nuclei were stained with DAPI (blue). Compared to other AQP0 mutants and WT AQP0, AQP0-AQP1ELC (g–i) did not exhibit considerable FRET signal. (B). WT and mutant AQP0 protein expression levels at the plasma membranes of adhesion-deficient mouse fibroblast L-cells. Expression levels of the proteins were tested by Western blotting using a C-terminal-specific AQP0 antibody. Top panel: Western blotting. Immunoreactivity of EGFP tagged WT AQP0 (lane 2), AQP0-AQP1ELC (lane 3), AQP0-AQP1ELA (lane 4), mutants (R33Q lane 5), H40Q (lane 6), R113Q (lane 7) and H122Q (lane 8). Lane 1 was left empty. A ∼55 kDa band reacted intensely in all samples except in AQP0-AQP1ELC. Expression level of Na/K-ATPase α1 housekeeping protein (~100 kDa) served as a loading control (bottom panel of the Western blot; anti-Na/K-ATPase α1; Research Diagnostics Inc., Pleasant Hill Road, Flanders, NJ). Immunoreactive band intensities were quantified and represented in the bottom panel. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
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Fig. 6. CTCA assay. (A). Over a monolayer of L-cells expressing empty vector, AQP1, E-cadherin, WT AQP0 or mutant AQP0 (AQP0-AQP1ELA, AQP0-AQP1ELC, R33Q, H40Q, R113Q or H122Q) corresponding cells loaded with CellTracker Red were plated. At the end of the procedure, the cells were imaged under an epifluorescent microscope. Cells/aggregates were counted and plotted. (B). Bar graph represents the number of dye-loaded L-cells expressing empty vector, AQP1, E-cadherin, WT AQP0 or mutant AQP0 (AQP0-AQP1ELA, AQP0-AQP1ELC, R33Q, H40Q, R113Q or H122Q) that remained attached to the matching cDNA construct-transfected L-cells (without dye) due to CTCA. Samples were tested using the fluorescence assay and incubated for 1 h for CTCA. Compared to WT-AQP0, mutant AQP0 exhibited significantly low (P < 0.05) CTCA, denoted with a star for each sample. E-cadherin - positive control. (C). CTCA assay testing the possible mechanism of CTCA. The number of dye-loaded cells expressing empty vector, AQP1, E-cadherin, WT AQP0 or mutant AQP0 (AQP0-AQP1ELA, AQP0-AQP1ELC, R33Q, H40Q, R113Q or H122Q) that remained attached to untransfected L-cells, due to CTCA in the samples tested using the fluorescence assay with 1 h of incubation are represented. Star on the mutants denotes a reduction in CTCA in comparison with WT AQP0. Note: Number of E-cadherin transfected cells that remained attached was much less than that of WT AQP0 transfected cells and represented with two stars. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
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Fig. 7. Cell-to-Lipid vesicle adhesion assay. (A). Bar graph representing the number of dye-loaded unilamellar phosphatidylserine (PS) lipid vesicles (negatively charged) attached to L-cells expressing empty vector, AQP1, E-cadherin, WT AQP0 or mutant AQP0 (AQP0-AQP1ELA, AQP0-AQP1ELC, R33Q, H40Q, R113Q, H122Q) without dye. Samples were incubated for 1 h. PS lipid vesicles exhibited increased adhesion to WT-AQP0 and most of the mutants compared to vector-transfected cells (P < 0.05), denoted with a star for each sample. (B). Histogram representing the number of dye-loaded unilamellar phosphatidylcholine (PC) neutral lipid vesicles attached to L-cells expressing empty vector, AQP1, E-cadherin, WT AQP0 or mutant AQP0 (AQP0-AQP1ELA, AQP0-AQP1ELC, R33Q, H40Q, R113Q, H122Q) without dye. Samples were incubated for 1 h. In contrast to PS vesicles, PC lipid vesicles exhibited no significant adhesion to WT-AQP0 or mutant AQP0 (P > 0.05).
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