Vilas G et al. (2015), Increased water flux induced by an aquaporin-1/...

XB-ART-50397

Mol Biol Cell 2015 Mar 15;266:1106-18. doi: 10.1091/mbc.E14-03-0812.

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Increased water flux induced by an aquaporin-1/carbonic anhydrase II interaction.

Vilas G , Krishnan D , Loganathan SK , Malhotra D , Liu L , Beggs MR , Gena P , Calamita G , Jung M , Zimmermann R , Tamma G , Casey JR , Alexander RT .

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Aquaporin-1 (AQP1) enables greatly enhanced water flux across plasma membranes. The cytosolic carboxy terminus of AQP1 has two acidic motifs homologous to known carbonic anhydrase II (CAII) binding sequences. CAII colocalizes with AQP1 in the renal proximal tubule. Expression of AQP1 with CAII in Xenopus oocytes or mammalian cells increased water flux relative to AQP1 expression alone. This required the amino-terminal sequence of CAII, a region that binds other transport proteins. Expression of catalytically inactive CAII failed to increase water flux through AQP1. Proximity ligation assays revealed close association of CAII and AQP1, an effect requiring the second acidic cluster of AQP1. This motif was also necessary for CAII to increase AQP1-mediated water flux. Red blood cell ghosts resealed with CAII demonstrated increased osmotic water permeability compared with ghosts resealed with albumin. Water flux across renal cortical membrane vesicles, measured by stopped-flow light scattering, was reduced in CAII-deficient mice compared with wild-type mice. These data are consistent with CAII increasing water conductance through AQP1 by a physical interaction between the two proteins.

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Species referenced: Xenopus laevis
Genes referenced: actl6a alb aqp1 aqp2 ca2 npdc1.2 prss1 slc4a1 utp25

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	FIGURE 1:. The cytosolic C- terminus of AQP1 has two CAII binding motifs. Examination of the human AQP1 amino acid sequence identified two consensus carbonic anhydrase binding (CAB) sites in the cytosolic C-terminus. (A) Sequence alignment of the CABs found in AQP1 genes in the indicated species. CAB1 is depicted in blue and CABII in red throughout. (B) Comparison of the same region of the other aquaporins does not reveal this motif.
	FIGURE 2:. CAII and AQP1 colocalize in mouse proximal tubular brush border membrane. (A, B) A kidney section immunostained for CAII (green) and with the nuclear stain 4′,6-diamidino-2-phenylindole. (A) A low-power cortical image, with arrows pointing to proximal tubular staining. (B) A high-power image with an arrow pointing to the brush border. Individual channels are displayed at both high and low power. (C, D) Serial sections immunostained for (C) CAII) and (D) AQP1. (E–E′′) Image of the renal cortex, immunostained for CAII E and with a proximal tubular marker, L. tetragonolobus lectin (E′). An overlay of the two channels is presented in E′′.
	FIGURE 3:. CAII expression increases water flux through AQP in Xenopus oocytes. (A) Oocytes injected with the indicated cRNAs were perfused alternately with isotonic (dark blue bar) and hypotonic (light blue bar) buffers. Oocyte volume was calculated from images captured digitally every 10 s and is plotted as fraction of initial volume (V/V0). Representative traces are displayed. (B) Oocyte swelling rate determined by linear regression of data from A over the first 60 s after switching to hypotonic perfusion buffer. (C) After cell-swelling assays, membrane lysates were prepared, and 50 μg of protein along with 4.8 ng of HA-tagged AE1 or 20 ng of CAII standard was probed on immunoblots with anti-HA or anti-CAII antibodies. Densitometry of the immunoblots was used to calculate the number of AQP1 and CAII molecules present per oocyte. (D) Swelling rates and the number of HA-tagged AQP1 molecules per oocyte were used to calculate AQP1 transport activity (number of water molecules permeated by a single AQP1 monomer per second). Transport activity values for each condition are expressed as percentage of AQP1 activity alone. Bars represent mean ± SEM from three separate experiments with 10 oocytes/assay. Asterisk represents a statistically significant difference (p < 0.05) compared with AQP1, and number sign (#) represents a statistically significant difference from AQP1 + CAII.
	FIGURE 4:. CAII expression increases water flux through AQP1 expressed in HEK293 cells. (A) AQP1 water transport activity was measured by confocal fluorescence microscopy on transfected HEK293 cells. Cells were perfused alternately with isotonic (dark blue bar) and hypotonic (light blue bar) medium, and GFP fluorescence was quantified digitally. GFP concentration as assessed by fluorescence in a representative region of interest (ROI) normalized to initial fluorescence in that ROI (F/F0) is plotted vs. time. (B) Rate of fluorescence change corrected for activity of vector-transfected cells and normalized to AQP1 cell surface expression. Cell surface expression was determined by biotinylation and is presented in Supplemental Figure S7. Asterisk represents a statistically significant difference (p < 0.05) compared with AQP1, and number sign (#) represents a statistically significant difference from AQP1 + CAII.
	FIGURE 5:. CAII expression does not increase water flux through AQP2 expressed in HEK293 cells. Aquaporin-mediated (AQP1 and AQP2) water transport activity was measured by confocal fluorescence microscopy of transfected HEK293 cells as per Figure 4. (A) Rate of fluorescence change corrected for activity of vector-transfected cells and normalized to AQP1 or AQP2 cell surface expression. Cell surface expression was determined by biotinylation and is presented in Supplemental Figure S7. Number sign (#) represents a statistically significant difference (p < 0.05) compared with AQP1; n > 3 per condition. (B) Immunoblots demonstrating total cellular expression of AQP1, AQP2, CAII, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH).
	FIGURE 6:. CAII interacts with the acidic clusters in the C-terminus of AQP1 by peptide overlay assay. Fifteen–amino acid peptides corresponding to overlapping progressively more C-terminal sequences in AQP1 were spotted onto cellulose membranes and visualized with ultraviolet fluorescence (tryptophan fluorescence). The exact peptide sequence is detailed above the blot. Recombinant CAII or albumin was incubated with the membrane before a primary anti-CAII antibody, followed by the appropriate secondary antibody and then visualization. Representative overlays from three separate experiments.
	FIGURE 7:. AQP1 and CAII are in close proximity. (A) The CAB mutants generated. The CAB1 motif is in blue, and the CABII motif is in red. (B) Immunoblots of cell lysates from transfections used in the proximity ligation assays. The primary antibody used is depicted under the blot, and the cDNA transfected is displayed in bold above the blot. GAPDH was probed as a loading control. (C) Cells transfected with the indicated cDNAs were processed in a proximity ligation assay, using an anti-CAII antibody in each case, along with anti-AE1 (AE1), anti-AQP1 (AQP1, CAB1AQP1, CABIIAQP1, CABI/IIAQP1), or anti-hCNT3 (hCNT3).
	FIGURE 8:. CABII is necessary to mediate increased water flux through AQP1 by CAII. AQP1 Water transport activity was measured by confocal fluorescence microscopy of HEK293 cells transfected with AQP1 CAB mutants in the presence and absence of CAII. (A) Cells were perfused initially with isotonic (dark blue bar) and then hypotonic (light blue bar) medium, and eGFP fluorescence was recorded. eGFP concentration as assessed by fluorescence in a representative ROI normalized to initial fluorescence in that ROI (F/F0) is plotted vs. time. The constructs transfected are indicated on the left side of the curves. (B) Rate of fluorescence change corrected for activity of vector-transfected cells and normalized to AQP1 amount at the cell surface. Cell surface expression was determined by biotinylation and is presented in Supplemental Figure S7. Asterisk represents a statistically significant difference (p < 0.05) compared with AQP1, and number sign (#) represents a statistically significant difference from AQP1 + CAII.
	FIGURE 9:. CAII increases the water permeability of red blood cell ghosts. (A) Immunoblot of ghost lysate resealed in the presence of BSA or CAII and probed with anti-CAII. (B) Immunoblot of ghost lysate resealed in the presence of CAII and treated with trypsin, Triton X-100, or both. (C) Representative light scattering traces from red blood cell ghosts supplemented with BSA or CAII. (D) Coefficient of osmotic water permeability (Pf) of red blood cell ghosts supplemented with bovine serum albumin or CAII. Asterisk represents a statistically significant difference (p < 0.05) compared with BSA-supplemented sample.
	FIGURE 10:. CAII-deficient cortical kidney membrane vesicles have reduced water permeability. (A) Representative traces of the light scattering induced by renal cortical membranes from WT and CAII-deficient mice. (B) Osmotic water permeability of kidney cortical membrane vesicles prepared from WT and CAII-deficient mice. (C) Representative immunoblots of cortical kidney membranes isolated from six WT and six CAII-deficient (CAII-def) mice probed for AQP1 and β-actin. (D) Quantification of AQP1 membrane expression normalized to β-actin. Asterisk represents a statistically significant difference (p < 0.05) compared with WT.

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