The Water Permeability of Lens Fibre Cells: The Relative Role of AQP0 and AQP5

Abstract

Transportation of water is essential for the maintenance of lens homeostasis, and members of the aquaporin (AQP) family have been found to mediate the water permeability of lens cells. In my thesis, I performed immunolabelling analysis of whole lens sections from three different species of mammalian lens to map the cellular and subcellular distribution of AQP5, a third water channel recently discovered in the lens. I found a common, yet species-dependent, subcellular redistribution of AQP5 from a cytoplasmic pool to the membranes of differentiating fibre cells that occurred in the outer cortex of mouse and bovine lenses, and in the transition zone between differentiating and mature fibre cells in the inner cortex of rat lenses. In addition to the programmed translocation of AQP5 that took place during fibre cell differentiation, I found that AQP5 translocation to the membrane of peripheral differentiating fibre cells can be induced by organ culturing of mouse lenses in solutions of varying extracellular osmolarity. In addition, I compared the subcellular distribution of AQP5 with the well-characterised distribution of AQP0 throughout adult, embryonic and postnatal lenses and found that each AQP contributes differently to membrane water permeability in different lens regions. I found that, during embryonic development, AQP5 is predominantly in the cytoplasm while AQP0 remained membranous, and that these spatial differences in the subcellular distribution of AQP0 and AQP5 were maintained into the early postnatal period. The most profound changes in the distribution of AQP5 and AQP0 took place during the first and second week of postnatal development. AQP0 underwent an abrupt differentiation-dependent truncation of its C-terminus that began in the core of the lens which was complemented by the translocation of AQP5 from the cytoplasm to the membranes of first the lens core, and then subsequently differentiating fibre cells in the outer cortex without undergoing C-terminal truncation. These changes in protein distribution were established at a postnatal stage when the hyaloid vascular system feeding the lens regresses, transforming the lens into an avascular tissue solely dependent on the lens microcirculation system to maintain its transparency and homeostasis. The existence of differences in the subcellular localisation of AQP5 in the outer cortex of mouse and rat lenses allowed me to propose the hypothesis that the relative functional contributions of AQP5 and AQP0 to cell water permeability in this peripheral region of the lens are different. To test this hypothesis, I developed and optimised a novel fluorescence-based functional assay using mouse and rat lens fibre cell membrane vesicles derived from the outer cortex. To further delineate between the relative functional contributions that AQP5 and AQP0 make to water permeability, I used mercury as a specific pharmacological blocker of AQP5 since AQP0 is insensitive to mercury. I found that mouse vesicles displayed higher water permeability than rat vesicles and, in addition, showed sensitivity to mercury. Taken together my data suggest that AQP5 is a regulated water channel that can be inserted into lens fibre cell membranes under osmotic stress conditions to restore lens water homeostasis. Furthermore, since AQP5 is not truncated in the lens core, it is likely to remain functional in this lens region to facilitate the circulating water fluxes that are proposed to maintain adequate water and refractive index gradients in the lens.