Direct demonstration of aquaporin-2 water channel recycling in stably transfected LLC-PK1 epithelial cells.
Vasopressin-dependent membrane insertion of aquaporin-2 (AQP-2) in collecting duct principal cells has been demonstrated in vivo and in vitro. However, the hypothesis that the AQP-2 molecule recycles between intracellular vesicles and the plasma membrane in response to hormonal stimulation and withdrawal remains to be demonstrated directly. In the present study, we examined AQP-2 recycling between intracellular vesicles and the plasma membrane in the absence of de novo protein synthesis using LLC-PK1 cells transfected with an AQP-2-c-myc construct. Cells were treated with cycloheximide for 30 min prior to vasopressin stimulation, and all subsequent treatments were performed in the continued presence of cycloheximide. Complete inhibition of AQP-2 biosynthesis by cycloheximide was verified by immuno-precipitation. Immunofluorescence revealed that AQP-2 was located on intracellular vesicles in nonstimulated cells but was relocated to the plasma membrane after vasopressin treatment, even in the presence of cycloheximide. After vasopressin washout, AQP-2 was retrieved to intracellular vesicles and was relocated to the plasma membrane after restimulation with forskolin. Subsequent forskolin washout resulted in AQP-2 endocytosis, and a second stimulation with forskolin resulted in relocation to the plasma membrane. These data, obtained in the absence of de novo protein synthesis, clearly indicate that AQP-2 can be recycled multiple times between intracellular vesicles and the plasma membrane.
Katsura
T
T
Renal Unit, Massachusetts General Hospital, USA.
Ausiello
D A
DA
Brown
D
D
D000818:Animals; D051399:Aquaporin 2; D051403:Aquaporin 6; D020346:Aquaporins; D001692:Biological Transport; D004848:Epithelium / Q000378:metabolism; D007473:Ion Channels / Q000235:genetics / Q000378:metabolism*; D007684:Kidney Tubules / Q000378:metabolism*; D018374:LLC-PK1 Cells; D013552:Swine; D014162:Transfection; D014867:Water / Q000378:metabolism*
D051399:Aquaporin 2; D051403:Aquaporin 6; D020346:Aquaporins; D007473:Ion Channels; D014867:Water