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A. N. Van Hoek, D. Brown, S. Breton; Functional Expression of Human Aquaporin Water Channels AQP1, AQP4 and AQP9. Invest. Ophthalmol. Vis. Sci. 2010;51(13):692.
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© ARVO (1962-2015); The Authors (2016-present)
Aquaporin (AQP) water channels are present in cornea, ciliary body, lens and retina but their functional contributions to trans epithelial and endothelial water transport is not understood other then providing facilitated pathways for water movement. We have created CHO cell lines stably expressing human aquaporins with the goal to compare and contrast functional responses to anisosmotic solutions in intact CHO cells. Using intact cells it will permit measurement of direction-dependent osmotic water permeability coefficients of AQP1, AQP4 and AQP9. The dissection of properties specific to an aquaporin may provide novel concepts to how aquaporins contribute to water homeostasis in health and disease.
Large amount of cells were obtained by culturing CHO cells in suspension. The water transport function of CHO cells stably expressing human water channels AQP1, AQP4 and AQP9 was measured at 10 °C following rapid mixing in the stopped flow apparatus with an equal amount of a hyper- or hypo-osmotic solution. Cells in 275 mOsm HEPES buffered saline (HBS) were usually mixed with 3xHBS to produce a 2xHBS mixture, creating an outwardly directed 275 mOsm gradient across the cell plasma membrane, which shrink cells to 50% of their original size, as the result of volume flow of water. To produce an inwardly directed osmotic gradient of the same strength, cells in 1xHBS were premixed with 3xHBS, and the equilibrated cells in 2xHBS were then mixed in the stopped flow apparatus with an equal amount of plain water to produce the inwardly directed 275 mOsm gradient.
In shrinkage assays we found facilitated transport of water through AQP1 and AQP9 but not through AQP4. In re-swelling assays we found facilitated transport of water through all three water channels. In addition we observed overshoots (shrink-swell-shrink, respectively, swell-shrink-swell) in the osmotic responses and were able to fit traces with a damped sinusoidal function with a period of ~1 second superimposed on an exponential function. Revisiting our earlier stopped-flow experiments from almost 20 years ago multiple oscillations (period ~0.2 sec) of water transfer can be appreciated in kidney brush border membrane vesicles expressing AQP1.
This is the first demonstration of direction-dependent water transport through AQP4, signifying a rectifying property for this water channel. Further, rhythmic water permeation through aquaporins suggests inertial mass-movement (body of water), setting up a harmonic oscillation around the dissipating osmotic pressure, damped by a resisting force, possibly caused by the cytoskeleton.
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