Constitutive and regulated membrane expression of aquaporin 1 and aquaporin 2 water channels in stably transfected LLC-PK1 epithelial cells.
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.
Katsura
T
T
Renal Unit, Massachusetts General Hospital, Boston 02114, USA.
Verbavatz
J M
JM
Farinas
J
J
Ma
T
T
Ausiello
D A
DA
Verkman
A S
AS
Brown
D
D
D000818:Animals; D051398:Aquaporin 1; D051399:Aquaporin 2; D051403:Aquaporin 6; D020346:Aquaporins; D002462:Cell Membrane / Q000378:metabolism; D002463:Cell Membrane Permeability / Q000187:drug effects; D018076:DNA, Complementary / Q000235:genetics; D004705:Endocytosis / Q000187:drug effects; D005786:Gene Expression Regulation; D007473:Ion Channels / Q000235:genetics* / Q000378:metabolism; D018374:LLC-PK1 Cells; D013552:Swine; D014162:Transfection; D014667:Vasopressins / Q000494:pharmacology; D014867:Water / Q000378:metabolism
D051399:Aquaporin 2; D051403:Aquaporin 6; D020346:Aquaporins; D018076:DNA, Complementary; D007473:Ion Channels; D014867:Water; D014667:Vasopressins; D051398:Aquaporin 1