Constitutive and Regulated Membrane Expression of Aquaporin 1 and Aquaporin 2 Water Channels in Stably Transfected LLC-PK1 Epithelial Cells

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Title: Constitutive and Regulated Membrane Expression of Aquaporin 1 and Aquaporin 2 Water Channels in Stably Transfected LLC-PK1 Epithelial Cells
Authors: Verkman, Alan S.; Farinas, Javier; Katsura, Toshiya; Verbavatz, Jean-Marc; Ma, Tonghui; Brown, Dennis; Ausiello, M.D., Dennis A.
Publisher: Proceedings of the National Academy of Sciences of the U.S.A.
Date Published: August 01, 1995
Reference Number: 208
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The aquaporins (AQPs) are a family of homologous water-channel proteins that can be inserted into epithelial cell plasma membranes either constitutively (AQP1) or by regulated exocytosis following vasopressin stimulation (AQP2). LLC-PK1 porcine renal epithelial cells were stably transfected with cDNA encoding AQP2 (tagged with a C-terminal c-Myc epitope) or rat kidney AQP1 cDNA in an expression vector containing a cytomegalovirus promoter. Immunofluorescence staining revealed that AQP1 was mainly localized to the plasma membrane, whereas AQP2 was predominantly located on intracellular vesicles. After treatment with vasopressin or forskolin for 10 min, AQP2 was relocated to the plasma membrane, indicating that this relocation was induced by cAMP. The location of AQP1 did not change. The basal water permeability of AQP1-transfected cells was 2-fold greater than that of nontransfected cells, whereas the permeability of AQP2-transfected cells increased significantly only after vasopressin treatment. Endocytotic uptake of fluorescein isothiocyanate-coupled dextran was stimulated 6-fold by vasopressin in AQP2-transfected cells but was only slightly increased in wild-type or AQP1-transfected cells. This vasopressin-induced endocytosis was inhibited in low-K+ medium, which selectively affects clathrin-mediated endocytosis. These water channel-transfected cells represent an in vitro system that will allow the detailed dissection of mechanisms involved in the processing, targeting, and trafficking of proteins via constitutive versus regulated intracellular transport pathways.
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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.
© Copyright NDI Foundation 2007 (JC)

Proteins are delivered to the cell membrane in two different ways:

  1. the constitutive (non-regulated) way,
  2. the stimulated (regulated) way.

The constitutive way delivers proteins to the cell membrane constantly or in fixed amounts, regardless of environmental demands. The regulated way delivers proteins to the cell membrane upon stimulation by something such as a hormone.

Proteins that are constitutively inserted into the cell membrane generally do not accumulate in significant amounts within the cell before they are inserted into the cell membrane. Proteins that are inserted in the cell in the regulated way do build up within the cell before insertion. They are contained in little sacs called vesicles and they wait inside the cell until the appropriate stimulus occurs. The stimulus signals the protein-carrying vesicles to fuse with the cell membrane so the proteins within them can be inserted into the membrane.

Aquaporins (AQPs) are a family of proteins that, when inserted in cell membranes, make those membranes more water permeable. They do this by acting as channels through which water can flow in greater amounts than it can by simply diffusing through the membrane itself. One such AQP, AQP1, is located in cell membranes found in those areas of the kidney tubule called the proximal tubule and the thin descending limbs of Henle. AQP1 is delivered constitutively.

Another AQP, AQP2, is located in the principal cells of the kidney collecting duct. It is delivered to the cell membrane in a regulated way. It awaits within the cell until stimulated by the antidiuretic hormone, vasopressin (VP). Then the vesicles that carry it travel to and fuse with the cell membrane. This allows AQP2 to insert itself into the membrane. Thus, AQP2 is regulated by VP; it is delivered to the membrane when stimulated to do so by the presence of VP.

Detailed investigation as to how AQP1 and AQP2 are packaged, sorted and delivered to the cell membrane have been hampered due to the lack of a laboratory cell culture in which the cellular pathways (constitutive or regulated) could be maintained. Katsura, et al., were able to find a cell culture (derived from pig kidney) wherein AQP1 and AQP2 could be delivered to the cell membranes by their respective pathways, constitutive and regulated, respectively.

The authors were able to track the pathways of the two AQPs by staining them with an immunoflourescent dye. The staining revealed that most of the AQP1 was located in the cell membrane, and that most of the AQP2 was located in intracellular vesicles beneath the apical membrane (the apex of the cell membrane).

When the cell culture was infused with VP (or forskolin), AQP1 remained where it was while AQP2 was shuttled to the apical membrane. When VP was removed from the cell culture, AQP2 relocated within its vesicles in subapical areas of the cell.

To ensure that AQP1 and AQP2 were functional with the cell culture, the authors tested the cell culture's water permeability in response to the presence of the AQPs. The water permeability of cells with just AQP1 was twice as great as the cell culture without any AQPs. The permeability of cells with just AQP2 dramatically increased after VP was added to the culture. (As AQP1 is not regulated by VP, adding VP to the cell culture with AQP1 did not additionally increase its water permeability.) Thus, both AQPs functioned normally in the cell cultures.

In kidney collecting duct cells in mammalian bodies a protein called clathrin helps the AQP2-bearing vesicles recycle from the cell's apical membrane back to the inside of the cell. The authors tested their cell culture to see if clathrin was present to perform this function. They did this by depleting the culture of potassium. Clathrin cannot operate without it. When they infused the cell culture with VP after depleting it of potassium and then removed the VP, the AQP2 could not cycle back inside the cell. Since they had no problem doing this when the cell culture was not potassium deficient, it indicated that clathrin was present in the cell culture.

Thus, the cell culture the authors used was found to hold the conditions required for both regulated and constitutive delivery of AQPs to the cell membrane. Since the cell culture mimics the conditions occurring within living mammalian cell systems with regards to AQP delivery, now researchers can examine in detail the mechanisms involved in the processing, targeting and trafficking of AQP1 and AQP2 (and other, similar, proteins) to cellular membranes.

For example, the authors' research indicated that both AQP1 and AQP2 contain within them information that is sufficient to direct their packaging into constitutive versus regulated transporting vesicles. The VP-regulated recycling pathway is absent from the cell culture when it is not infused with AQP2. This shows that AQP2 is not simply a passenger in vesicles that already travel this cyclic path from inside the cell to the membrane and back again. Somehow AQP2 induces the appearance of its transport vesicles.