Fate of Antidiuretic Hormone Water Channel Proteins after Retrieval from Apical Membrane

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Title: Fate of Antidiuretic Hormone Water Channel Proteins after Retrieval from Apical Membrane
Authors: Zeidel, Mark L.; Hammond, Timothy G.; Wade, James B.; Tucker, Julia; Harris, H. William
Publisher: American Journal of Physiology
Date Published: September 01, 1993
Reference Number: 268
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In toad bladder granular cells, antidiuretic hormone (ADH) stimulates insertion of vesicles containing water channels (WCV), markedly increasing apical membrane osmotic water permeability (Pf). After withdrawal of ADH stimulation, WCV are removed from the apical membrane and fluid-phase markers endocytosed from the apical solution appear predominantly in endosomes at 10-15 min and multivesicular bodies at 30-60 min. Although the luminal contents of this endocytic pathway have been well characterized, the fate of membrane proteins, including functional ADH water channels in these vesicles remains unclear. Using electron microscopic, flow cytometric, and stopped-flow fluorescence measurements and characterization of labeled vesicle proteins, we examined the fate of membrane proteins contained within WCV. The protein complements of endosomes harvested after 10, 30, and 60 min of ADH withdrawal were similar. Selective covalent labeling of apical proteins during ADH stimulation followed by ADH reversal for 30 or 60 min showed that apical proteins colocalize with fluid-phase marker-labeled endosomes at all times, and most apically labeled protein bands present in the 10-min fraction were also present in the 30- and 60-min endosome fractions. Endosomes at 10 and 30 min but not at 60 min contained functional water channels revealed by high Pf and proton permeability, low activation energy of Pf, and sensitivity of Pf to mercurial reagents. We conclude that a portion of apically exposed membrane proteins, including candidate water channel proteins, travel together with fluid-phase markers from 10-min endosomes into later endosomal compartments. Functional water channels may be inactivated or some essential protein component selectively sorted away between 30 and 60 min after ADH withdrawal.

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.
© Copyright NDI Foundation 2007 (JC)

The principal cells of the toad urinary bladder and the mammalian duct must, when necessary, increase the amount of body water that can flow through them. This increase in the water permeability of these cells enables toads and mammals to concentrate urine and properly balance body water. This increased water permeability is achieved by the cells when the antidiuretic hormone (ADH) binds with receptors on these cells and stimulates little sacs called water channel vesicles (WCVs) to travel from inside the cells to the cells' apical membranes. The WCVs contain water-transporting proteins. When they reach the apical membranes, they fuse with them and insert the water-transporting proteins (called water channels) into the membranes. The water channels (WCs) act as channels through which water can flow. It is this insertion of the WCs that increases the cells' water permeability.

When the ADH removes itself from the cells, the WCVs take themselves out of the apical membranes. They move into pits on the cell surface that are coated with a protein called clathrin. These clathrin-coated pits, with the WCVs inside them, descend back inside the cell. When the pits, still containing the WCVs, move inside the cells, they lose their clathrin coating. At this point they are called endosomes.

Zeidel, et al., wanted to know the fate of the membrane proteins, including the WCs, as they sat inside their WCVs inside the endosomes. Using toad urinary bladder cells, the authors were able to track and harvest the WCVs and examine the membrane proteins contained within them. They harvested the endosomes from the bladder cells at different times after ADH was withdrawn from the cell: 10 minutes, 30 minutes and 60 minutes.

The authors found that the protein components within the WCVs were similar in the endosomes harvested at all three time periods. The authors were also able to determine that the fluid phase markers they used to track the membrane protein fragments always stayed with the membrane proteins during the entire time span of their testing period.

Next, the authors tested the membrane proteins contained within the WCVs to see if they could still increase apical cell water permeability. If they could, it would indicate that the WCs remain functional after they reenter the cell and perhaps they can be used by the cell again when ADH once again binds with a receptor on the cell's surface. In other words, it could indicate that the WCs stay intact and functional and therefore could be used by the cell repeatedly to increase its water permeability.

Zeidel, et al., found that the WCVs in the endosomes harvested at 10 and 30 minutes still contained functional WCs. This was determined because they still increased the cell water permeability, they required little energy to do so, and they remained sensitive to mercury compounds. The endosomes harvested at 60 minutes did not contain functional water channels.

There are two possible ways to explain why the WCs are inactive 60 minutes after they recycle back inside the cell. It may be that the endosomes somehow can separate the WC from the fluid phase marker that researchers used to track the WC, but the authors test results determined that this was not the case. The other explanation is that between 30 and 60 minutes after the WCs recycle from the apical membrane back into the cell they are inactivated. The authors feel this is a much more likely scenario.