Dataset Information

Opposing Effects of cAMP and T259 Phosphorylation on Plasma Membrane Diffusion of the Water Channel Aquaporin-5 in Madin-Darby Canine Kidney Cells.

ABSTRACT: Aquaporin-5 (AQP5) facilitates passive water transport in glandular epithelia in response to secretory stimuli via intracellular pathways involving calcium release, cAMP and protein kinase A (PKA). In epithelial plasma membranes, AQP5 may be acutely regulated to facilitate water transport in response to physiological stimuli by changes in protein modifications, interactions with proteins and lipids, nanoscale membrane domain organization, and turnover rates. Such regulatory mechanisms could potentially be associated with alteration of diffusion behavior, possibly resulting in a change in the plasma membrane diffusion coefficient of AQP5. We aimed to test the short-term regulatory effects of the above pathways, by measuring lateral diffusion of AQP5 and an AQP5 phospho-mutant, T259A, using k-space Image Correlation Spectroscopy of quantum dot- and EGFP-labeled AQP5. Elevated cAMP and PKA inhibition significantly decreased lateral diffusion of AQP5, whereas T259A mutation showed opposing effects; slowing diffusion without stimulation and increasing diffusion to basal levels after cAMP elevation. Thus, lateral diffusion of AQP5 is significantly regulated by cAMP, PKA, and T259 phosphorylation, which could be important for regulating water flow in glandular secretions.

SUBMITTER: Koffman JS

PROVIDER: S-EPMC4517861 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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Opposing Effects of cAMP and T259 Phosphorylation on Plasma Membrane Diffusion of the Water Channel Aquaporin-5 in Madin-Darby Canine Kidney Cells.

Koffman Jennifer S JS Arnspang Eva C EC Marlar Saw S Nejsum Lene N LN

PloS one 20150728 7

Aquaporin-5 (AQP5) facilitates passive water transport in glandular epithelia in response to secretory stimuli via intracellular pathways involving calcium release, cAMP and protein kinase A (PKA). In epithelial plasma membranes, AQP5 may be acutely regulated to facilitate water transport in response to physiological stimuli by changes in protein modifications, interactions with proteins and lipids, nanoscale membrane domain organization, and turnover rates. Such regulatory mechanisms could pote ...[more]

PMID: 26218429

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Project description:Madin-Darby canine kidney (MDCK) cells were cultured on polycarbonate filters to study the synthesis and sorting of proteoglycans in polarized epithelial cells. Two strains of MDCK cells were used. MDCK I cells resemble distal tubule epithelial cells, and MDCK II cells share some characteristics with proximal tubule cells. Both strains were grown to confluency and labelled with [35S]sulphate for 24 h. The apical and basolateral media and the cell fractions were harvested and analysed by DEAE ion-exchange chromatography. A large portion of the [35S]sulphate-labelled macromolecules bound strongly to the ion-exchange columns, and could be eluted in three distinct peaks. The latest eluting peak was demonstrated to contain almost exclusively chondroitin sulphate, whereas peak 2 contained mostly heparan sulphate, demonstrated by using chondroitinase ABC and nitrous acid (pH 1.5) respectively to depolymerize the [35S]glycosaminoglycan chains. Peak 1 contained negligible amounts of proteoglycans. Large differences could be observed in proteoglycan sorting in MDCK I and II cells. Strain I secreted approx. 67% of the proteoglycans to the apical side and 17% to the basolateral side. The cell fraction contained 17% of the proteoglycans after 24 h of labelling. In contrast, 19% of the proteoglycans were sorted to the apical side of MDCK II cells and 61% to the basolateral side, whereas the cell fraction contained 20%. Furthermore, the level of [35S]proteoglycan biosynthesis (apical and basolateral media and cell fraction total) was higher in MDCK I cells than in strain II. Based on the amount of material degraded by chondroitinase ABC and nitrous acid respectively, and the total amounts of [35S]proteoglycans recovered from the cells, it was calculated that the MDCK I strain synthesized approx. 56% chondroitin sulphate and 44% heparan sulphate. In contrast, the MDCK II strain synthesized 69% heparan sulphate and 31% chondroitin sulphate. To further identify the [35S]proteoglycans synthesized by MDCK I and II cells, antibodies against perlecan, versican and syndecan were used. The antibody against mouse syndecan did not cross-react with any of the proteoglycans produced in MDCK I or II cells. Both MDCK I and II cells expressed perlecan; 57-61% could be recovered from the basolateral fractions and 18-34% from the apical medium. Versican was also found in both MDCK I and II cells. Compared with perlecan, a larger percentage of versican (43-53%) was found in the cell fractions.

Dataset Information

Opposing Effects of cAMP and T259 Phosphorylation on Plasma Membrane Diffusion of the Water Channel Aquaporin-5 in Madin-Darby Canine Kidney Cells.

Publications

Opposing Effects of cAMP and T259 Phosphorylation on Plasma Membrane Diffusion of the Water Channel Aquaporin-5 in Madin-Darby Canine Kidney Cells.

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