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W.E. C. Harries, D. Akhavan, L.J. W. Miercke, S. Khademi, R.M. Stroud; The Channel Architecture and Functional Characterization of Bovine AQP0 . Invest. Ophthalmol. Vis. Sci. 2005;46(13):1126.
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Purpose: The purpose of our work was to determine the three–dimensional structure of bovine AQP0 (bAQP0) to atomic resolution and elucidate the relationship between the molecular structure and the function of AQP0 as a water channel and a putative cell adhesion molecule. Methods: Bovine AQP0 was isolated from fresh lenses and purified by HPLC ion exchange and gel filtration chromatography. Three dimensional crystals were grown and X–ray diffraction patterns were obtained at the Advanced Light Source, Beamline 8.3.1, Berkeley National Laboratory. The structure was determined by molecular replacement using AQP1 as a search model. We measured the water transport rate across AQP0 proteoliposome membranes formed using E.coli total polar lipids. An osmotic challenge assay measuring proteoliposome volume changes used an Applied Photophysics stopped–flow apparatus. Results: The structure of bAQP0 suggests that the selectivity of bAQP0 for water transport is based on the identity and location of signature residues in the channel that are the hallmarks of the aquaporin family. The channel lumen is narrowed only by two, quasi two–fold related tyrosine side chains that might account for reduced water conductance relative to other AQPs. Our high–resolution structure shows the presence of eight discreet water molecules within the channel, located at well–defined hydrogen bond acceptor/donor locations that line the channel. Measured water transport rates were 2–4 X above the background membrane permeation rate. Conclusions: Our structural and functional data provide both static and dynamic evidence that the bAQP0 channel is indeed open to the passage of water. Comparisons of rates and channel dimensions with the rest of the AQP family show AQP0 to have the smallest channel diameter and consequently the lowest water conductivity. These measurements do not however, rule out AQP0 as an integral part of the osmo–regulatory mechanism of the lens interior. Comparison of this structure with the recent electron diffraction structure of sheep AQP0 provides an opportunity to compare almost identical structures at pHs (6.0 for sheep AQP0 and 10 for bAQP0) that span a range that has been reported to alter the water transport rates through a pH gating mechanism. We saw no significant structural changes between the two structures that could be interpreted to be a gating mechanism. Our structure also aids analysis of the interaction of the extracellular domains and the possibility of a cell–cell adhesion role for AQP0, as well as, the basis for the formation of certain types of cataracts that are the result of mutations.
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