The effect of the interaction between aquaporin 0 (AQP0) and the filensin tail region on AQP0 water permeability.

PURPOSE: To study the interaction between the lens-specific water channel protein, aquaporin 0 (AQP0) and the lens-specific intermediate filament protein, filensin, and the effect of this interaction on the water permeability of AQP0. The effect of other factors on the interaction was also investigated. METHODS: Expression plasmids were constructed in which glutathione-S-transferase (GST) was fused to the AQP0 COOH-terminal region (GST-AQP0-C), which contains the major phosphorylation sites of the protein. Plasmids for AQP0 COOH-terminal mutants were also constructed in which one, three or five sites were pseudophosphorylated, and the proteins expressed from these GST-fusion plasmids were assayed for their interaction with lens proteins. Expressed recombinant GST-fusion proteins were purified using glutathione beads and incubated with rat lens extract. Western blotting was used to identify the lens proteins that interacted with the GST-fusion proteins. Filensin tail and rod domains were also expressed as GST-fusion proteins and their interactions with AQPO were analyzed. Additionally, the water permeability of AQP0 was calculated by expressing AQP0 with or without the filensin peptide on the cell membrane of Xenopus oocytes by injecting cRNAs for AQP0 and filensin. RESULTS: The GST-AQP0-C construct interacted with the tail region of lens filensin and the GST-filensin-tail construct interacted with lens AQP0, but the GST-filensin-rod construct did not interact with AQP0. GST-AQP0-C also interacted with a purified recombinant filensin-tail peptide after cleavage from GST. The AQP0/filensin-tail interaction was not affected by pseudophosphorylation of the AQP0 COOH-terminal tail, nor was it affected by changes in pH. Xenopus oocytes expressing AQP0 on the plasma membrane showed increased water permeability, which was lowered when the filensin COOH-terminal peptide cRNA was coinjected with the cRNA for AQP0. CONCLUSIONS: The filensin COOH-terminal tail region interacted with the AQP0 COOH-terminal region and the results strongly suggested that the interaction was direct. It appears that interactions between AQP0 and filensin helps to regulate the water permeability of AQP0 and to organize the structure of lens fiber cells, and may also help to maintain the transparency of the lens.

Quantitative analysis of ascorbic acid permeability of aquaporin 0 in the lens.

Aquaporin 0 (AQP0) is a lens-specific protein comprising more than 30% of lens membrane protein content and is a member of the aquaporin family. Water permeates through AQP0 much more slowly than other aquaporin family members, and other compounds, such as glycerol, also permeate AQP0. In the lens, ascorbic acid (AA) is found at high concentrations, protecting the lens from photochemical events such as photo-oxidation. The aim of the present study was to clarify the function of AQP0. Mouse fibroblast L-cells stably expressing AQP0 were established and incubated in medium containing AA, and intracellular AA levels were measured by high-performance liquid chromatography (HPLC) and 2,6-dichlorophenol-indophenol (DCPIP) analysis. Intracellular AA levels in AQP0-expressing cells quickly rose and reached saturation 10 min after incubation in medium containing 1000 µM AA. In contrast, AA levels in cells slowly decreased when AA was washed out from the medium. Cells overexpressing AQP0 increased the cellular uptake of AA in a time- and concentration-dependent manner. These data suggest that AA as well as water permeates AQP0. AQP0 expression on Xenopus oocyte membranes was achieved by the injection of AQP0 cRNA into oocytes that were incubated in medium containing AA. Intracellular AA levels were then measured by HPLC. AA uptake was demonstrated in the AQP0-expressing oocytes and was shown to quickly reach saturation. Intracellular AA concentration in oocytes increased in a time- and concentration-dependent manner. The data in the present study show that AA permeates AQP0, reveal the role of AQP0 in AA permeability ex vivo, and also indicate that there is a difference between the import and export of AA via AQP0. These findings suggest that AQP0 plays an important role in controlling lens AA content.