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K. Kalman, K. Németh–Cahalan, A. Froger, J.E. Hall; Role of the AQP0 C–Terminus in Calcium–Mediated Regulation of Water Permeability . Invest. Ophthalmol. Vis. Sci. 2006;47(13):5421.
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We previously reported that water permeability of AQP0 expressed in oocytes increases when calcium concentration is lowered from two to zero mM external calcium and involves calmodulin, probably bound to the C–terminus. Here we investigated the role of phosphorylated and positively charged amino–acids of the C–terminus in calcium regulation of AQP0 water permeability.
Serines (known to be phosphorylated in vivo) were mutated to either negatively charged aspartates (to mimic phosphorylation) or to neutral or positive amino acids as controls. We also replaced charged residues with a small neutral amino acid or by an opposite charge. We expressed the mutants in Xenopus oocytes and measured the water permeability in response to an osmotic challenge at different calcium concentration (0, 2 or 5 mM).
Mutations resulted in three distinct phenotypes: wild–type calcium sensitivity (S229N, S235A, S235K, R233A, K238A, R241E); elimination of calcium sensitivity (S235N/I236S, S229D, S231N, S231D); and reversal of calcium sensitivity such that increasing calcium concentration from two to five mM increased water permeability and lowering calcium concentration to zero had no effect (S235D, R241A). Furthermore, co–expression of "crippled" calmodulin (unable to bind calcium) with AQP0, S235D or R241A eliminated any calcium regulation of water permeability.
Phosphorylation of serines in the C–terminus plays a critical role in determining the response of AQP0 water permeability to changes in calcium concentration. Calmodulin is involved in both low and high calcium regulation. Also, the charges at position 233 and 238 are not necessary for calcium regulation whereas the position 241 is required for calcium regulation.
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