Recycling of AQP2 occurs through a temperature- and bafilomycin-sensitivetrans-Golgi-associated compartment

Abstract

The exo- and endocytotic pathway in which aquaporin-2 (AQP2) travels between the plasma membrane and intracellular vesicles is only partially characterized. It is known that the antidiuretic hormone vasopressin induces a translocation of AQP2 from an intracellular to a plasma membrane location, both in kidney collecting duct principal cells and in transfected epithelial cells. Here we provide evidence suggesting that while AQP2 shifts from an intracellular location to the cell surface in response to vasopressin, AQP2 also constitutively recycles through a similar pathway in transfected LLC-PK(1) cells even in the absence of hormonal stimulation. Incubating cells at 20 degrees C blocks AQP2 recycling in a perinuclear compartment, regardless of whether vasopressin is present. The H(+)-ATPase inhibitor bafilomycin A1 also blocks the recycling pathway of AQP2 in a perinuclear compartment adjacent to the Golgi in the presence and absence of vasopressin stimulation, indicating a role of vesicle acidification in both the constitutive and regulated recycling of AQP2. Colocalization of AQP2 with clathrin, but not with giantin, after both bafilomycin treatment and a 20 degrees C block suggests that the compartment in which recycling AQP2 is blocked may be the trans-Golgi, and not cis- and medial-Golgi cisternae.

Acute Hypertonicity Alters Aquaporin-2 Trafficking and Induces a MAPK-dependent Accumulation at the Plasma Membrane of Renal Epithelial Cells

“…Compartments-AQP2 perinuclear accumulation following hypertonic challenge observed in the present study was previously observed in LLC-AQP2 cells exposed to low temperature or treated with bafilomycin (34). In that study, the perinuclear compartment was identified as the trans-Golgi network (TGN).…”

“…Importantly, bafilomycin additionally abolished hypertonicity-induced AQP2 accumulation at the cell surface, suggesting that AQP2 constitutively recycles between the plasma membrane and the trans-Golgi region under hypertonic conditions. Bafilomycin-induced accumulation of AQP2 at the trans-Golgi region was previously shown to be unaffected by VP stimulation (34). Interestingly, 10 nM VP greatly reduced the hypertonicityinduced (500 mosmol/kg) accumulation of AQP2 at the trans-Golgi region (Fig.…”

“…Morphological studies have detected AQP2 in distinct intracellular pools, including early and recycling endosomes, TGN, and exocytic vesicles (34,61). AQP2 was previously shown to constitutively recycle between recycling vesicles and the transGolgi region both under base-line conditions and following VP stimulation (34).…”

“…Constitutive AQP2 recycling between the plasma membrane and the trans-Golgi region was previously revealed in LLC-AQP2 cells by treating cells with bafilomycin, an H ϩ -ATPase inhibitor reported to perturb intracellular trafficking (34). We investigated the effect of bafilomycin on hypertonicity-induced AQP2 cell surface accumulation by subjecting cells to isotonic or hypertonic (500 mosmol/kg) medium for 30 min in the presence of 125 nM bafilomycin (Fig.…”

“…4A, right). Clathrin is concentrated in transport vesicles derived from the plasma membrane and the TGN (35,36) and can be used as a TGN marker (34,35), although a subpopulation of recycling endosomes also consists of clathrin-coated vesicles (37). Confocal imaging of the basal side of cells revealed that, under hypertonic conditions, whereas AQP2 (red) was concentrated at the proximity of the plasma membrane, golgin-97 (green) was predominantly perinuclear.…”