Functional characterization of an AQP0 missense mutation, R33C, that causes dominant congenital lens cataract, reveals impaired cell-to-cell adhesion.

Aquaporin 0 (AQP0) performs dual functions in the lens fiber cells, as a water pore and as a cell-to-cell adhesion molecule. Mutations in AQP0 cause severe lens cataract in both humans and mice. An arginine to cysteine missense mutation at amino acid 33 (R33C) produced congenital autosomal dominant cataract in a Chinese family for five generations. We re-created this mutation in wild type human AQP0 (WT-AQP0) cDNA by site-directed mutagenesis, and cloned and expressed the mutant AQP0 (AQP0-R33C) in heterologous expression systems. Mutant AQP0-R33C showed proper trafficking and membrane localization like WT-AQP0. Functional studies conducted in Xenopus oocytes showed no significant difference (P > 0.05) in water permeability between AQP0-R33C and WT-AQP0. However, the cell-to-cell adhesion property of AQP0-R33C was significantly reduced (P < 0.001) compared to that of WT-AQP0, indicated by cell aggregation and cell-to-cell adhesion assays. Scrape-loading assay using Lucifer Yellow dye showed reduction in cell-to-cell adhesion affecting gap junction coupling (P < 0.001). The data provided suggest that this mutation might not have caused significant alterations in protein folding since there was no obstruction in protein trafficking or water permeation. Reduction in cell-to-cell adhesion and development of cataract suggest that the conserved positive charge of Extracellular Loop A may play an important role in bringing fiber cells closer. The proposed schematic models illustrate that cell-to-cell adhesion elicited by AQP0 is vital for lens transparency and homeostasis.

Functional expression of aquaporins in embryonic, postnatal, and adult mouse lenses.

Aquaporin 0 (AQP0) and AQP1 are expressed in the lens, each in a different cell type, and their functional roles are not thoroughly understood. Our previous study showed that these two AQPs function as water transporters. In order to further understand the functional significance of these two different aquaporins in the lens, we investigated their initiation and continued expression. AQP0 transcript and protein were first detected at embryonic stage (E) 11.25 in the differentiating primary fiber cells of the developing lens; its synthesis continued through the adult stage in the secondary fiber cells. Low levels of AQP1 expression were first seen in lens anterior epithelial cells at E17.5; following postnatal day (P) 6.5, the expression gradually progressed towards the equatorial epithelial cells. In the postnatal lens, the increase in membrane water permeability of epithelial cells and lens transparency coincides with the increase in AQP1 expression. AQP1 expression reaches its peak at P30 and continues through the adult stage both in the anterior and equatorial epithelial cells. The enhancement in AQP1 expression concomitant with the increase in the size of the lens suggests the progression in the establishment of the lens microcirculatory system. In vitro and in vivo studies show that both aquaporins share at least one important function, which is water transport in the lens microcirculatory system. However, the temporal expression of these two AQPs suggests an apparently unique role/s in lens development and transparency. To our knowledge, this is the first report on the expression patterns of AQP0 and AQP1 during lens development and differentiation and their relation to lens transparency.