Influence of the Cytoplasmic Domains of Aquaporin-4 on Water Conduction and Array Formation |
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Authors: | Tadanori Mitsuma Kazutoshi Tani Yoko Hiroaki Hiroshi Suzuki Hiroshi Hibino Yoshinori Fujiyoshi |
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Institution: | 1 Department of Biophysics, Faculty of Science, Kyoto University, Oiwake, Kitashirakawa, Sakyo-ku, Kyoto 606-8502, Japan2 Japan Biological Informatics Consortium, Oiwake, Kitashirakawa, Sakyo-ku, Kyoto 606-8502, Japan3 Division of Molecular and Cellular Pharmacology, Department of Pharmacology, Graduate School of Medicine, Osaka University, 2-2 Yamada-oka, Suita, Osaka 565-0871, Japan4 Biological Information Research Center, National Institute of Advanced Industrial Science and Technology, 2-41-6, Aomi, Koto-ku, Tokyo 135-0064, Japan |
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Abstract: | Phosphorylation of Ser180 in cytoplasmic loop D has been shown to reduce the water permeability of aquaporin (AQP) 4, the predominant water channel in the brain. However, when the structure of the S180D mutant (AQP4M23S180D), which was generated to mimic phosphorylated Ser180, was determined to 2.8 Å resolution using electron diffraction patterns, it showed no significant differences from the structure of the wild-type channel. High-resolution density maps usually do not resolve protein regions that are only partially ordered, but these can sometimes be seen in lower-resolution density maps calculated from electron micrographs. We therefore used images of two-dimensional crystals and determined the structure of AQP4M23S180D at 10 Å resolution. The features of the 10-Å density map are consistent with those of the previously determined atomic model; in particular, there were no indications of any obstruction near the cytoplasmic pore entrance. In addition, water conductance measurements, both in vitro and in vivo, show the same water permeability for wild-type and mutant AQP4M23, suggesting that the S180D mutation neither reduces water conduction through a conformational change nor reduces water conduction by interacting with a protein that would obstruct the cytoplasmic channel entrance. Finally, the 10-Å map shows a cytoplasmic density in between four adjacent tetramers that most likely represents the association of four N termini. This finding supports the critical role of the N terminus of AQP4 in the stabilization of orthogonal arrays, as well as their interference through lipid modification of cysteine residues in the longer N-terminal isoform. |
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Keywords: | AQP aquaporin PKC protein kinase C 3D three-dimensional 2D two-dimensional LPR lipid-to-protein ratio HEK human embryonic kidney FFR freeze fracture replica hAQP1 human AQP1 |
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