Phosphorylation of Aquaporin-2 does not alter the Membrane Water Permeability of Rat Papillary Water Channel-containing Vesicles
| Title: | Phosphorylation of Aquaporin-2 does not alter the Membrane Water Permeability of Rat Papillary Water Channel-containing Vesicles |
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| Authors: | Lande, M.D., Marc; Jo, Inho; Zeidel, Mark L.; Somers, MD, Michael J.; Harris, H. William |
| Publisher: | Journal of Biological Chemistry |
| Date Published: | March 08, 1996 |
| Reference Number: | 434 |
Antidiuretic hormone modulates the water permeability (Pf) of epithelial cells in the rat kidney by vesicle-mediated insertion and removal of the aquaporin-2 (AQP-2) water channel. AQP-2 possesses a single consensus cAMP-dependent protein kinase A (PKA) phosphorylation site (Ser-256) hypothesized to regulate channel Pf (Kuwahara, M., Fushimi, K., Terada, Y., Bai, L., Sasaki, S., and Marumo, F. (1995) J. Biol. Chem. 270, 10384-10387). To test whether PKA phosphorylation of AQP-2 alters channel Pf, we compared the Pf values of purified AQP-2 endosomes after incubation with either PKA or alkaline phosphatase. Studies using [gamma-32P]ATP reveal that AQP-2 endosomes contain endogenous PKA and phosphatase activities that add and remove 32P label from AQP-2. However, the Pf (0.16 +/- 0.06 cm/s) of endosomes containing phosphorylated AQP-2 (0.7 +/- 0. 3 mol of PO4/mol of protein) is not significantly different from the same AQP-2 endosomes where 95 +/- 8% of the phosphate has been removed (Pf 0.14 +/- 0.06 cm/s). These data do not support a role for PKA phosphorylation in alteration of AQP-2's Pf. Instead, AQP-2 phosphorylation by PKA may modulate AQP-2's distribution between plasma membrane and intracellular vesicle compartments.
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This translation by the NDI Foundation is to assist the lay reader. To provide a clear, accessible interpretation of the original article, we eliminated or simplified some technical detail and complicated scientific language. We concentrated our translation on those aspects of the article dealing directly with NDI. The NDI Foundation thanks the researchers for their work toward understanding and more effectively treating this disorder.
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Researchers postulate that the AQP2 is a string of 271 amino acids. To picture how it looks, imagine a long, beaded string. (The beads are the amino acids.) The majority of the AQP2 lies within the cell membrane in six, folded clumps called transmembrane domains 1 - 6. Part of the AQP2 snakes outside the membrane to form extracellular loops A, and E; part of it snakes inside the cell to form intracellular loops B and D. One end of the AQP2 is called the amino-terminus; the other end is called the carboxy-terminus. (You can see a diagram of AQP2 here.)
At various points along the AQP2 there are sites where different metabolic processes can take place, depending on the amino acid located at the site. One such process is phosphorylation, the induction of a phosphate group into the AQP2. The carboxy-terminus is the location of several sites where phosphorylation may take place. The presence of these phosphorylation sites raises the question as to whether phosphorylation alters the water permeability of individual AQP2s.
To test this, Lande, et al., compared the water permeability values of purified AQP2 endosomes after incubating one group of endosomes with cAMP-dependent protein kinase A (PKA) and the other group with alkaline phosphate. PKA induces phosphorylation, and alkaline phosphate dephosphorylates AQP2s. If phosphorylation does increase AQP2 water permeability, there should be a measurable difference in AQP2 permeability values between the two groups of endosomes.
The authors found these endosomes (which are rich with AQP2s) naturally contain PKA and phosphatase activities that phosphorylate and dephosphorylate the AQP2s within them. However, when they measured the permeability values of both groups of endosomes, they found no differences between the endosmomes containing phosphorylated AQP2s and those that had been dephosphorylated by being incubated in alkaline phosphatase.
The authors conclude that their data does not support the idea that cAMP-mediated PKA phosphorylation plays a role in regulating the water permeability of the AQP2 water channel.