Phosphorylation of Ser(256), in a PKA consensus site, in AQP2 (p-AQP2) appears to be critically involved in the vasopressin-induced trafficking of AQP2. In the present study, affinity-purified antibodies that selectively recognize AQP2 phosphorylated at Ser(256) were developed. These antibodies were used to determine 1) the subcellular localization of p-AQP2 in rat kidney and 2) changes in distribution and/or levels of p-AQP2 in response to [desamino-Cys(1),D-Arg(8)]vasopressin (DDAVP) treatment or V(2)-receptor blockade. Immunoelectron microscopy revealed that p-AQP2 was localized in both the apical plasma membrane and in intracellular vesicles of collecting duct principal cells. Treatment of rats with V(2)-receptor antagonist for 30 min resulted in almost complete disappearance of p-AQP2 labeling of the apical plasma membrane with only marginal labeling of intracellular vesicles remaining. Immunoblotting confirmed a marked decrease in p-AQP2 levels. In control Brattleboro rats (BB), lacking vasopressin secretion, p-AQP2 labeling was almost exclusively present in intracellular vesicles. Treatment of BB rats with DDAVP for 2 h induced a 10-fold increase in p-AQP2 labeling of the apical plasma membrane. The overall abundance of p-AQP2, however, was not increased, as determined both by immunoelectron microscopy and immunoblotting. Consistent with this, 2 h of DDAVP treatment of normal rats also resulted in unchanged p-AQP2 levels. Thus the results demonstrate that AQP2 phosphorylated in Ser(256) is present in the apical plasma membrane and in intracellular vesicles and that both the intracellular distribution/trafficking, as well as the abundance of p-AQP2, are regulated via V(2) receptors by altering phosphorylation and/or dephosphorylation of Ser(256) in AQP2.

Astrocytes play a key role for maintenance of brain water homeostasis, but little is known about mechanisms of short-term regulation of astrocyte water permeability. Here, we report that glutamate increases astrocyte water permeability and that the molecular target for this effect is the aquaporin-4 (AQP4) serine 111 residue, which is in a strategic position for control of the water channel gating. The glutamate effect involves activation of group I metabotropic glutamate receptors (mGluR), intracellular calcium release, and activation of calcium/calmodulin-dependent protein kinase II (CaMKII) and nitric oxide synthase (NOS). The physiological impact of our results is underlined by the finding that mGluR activation increases the rate of hypoosmotic tissue swelling in acute rat hippocampal slices. Cerebral ischemia is associated with an excessive release of glutamate, and in postischemic cerebral edema ablation of AQP4 attenuates the degree of damage. Thus, we have identified AQP4 as the molecular target for drugs that may attenuate the development of brain edema.

(AQP4) plays an important role in the basolateral movement of water in the collecting duct. Here we show that this water channel can be dynamically regulated. Water permeability (P f) was measured in individual LLC-PK 1 cells that were transiently transfected with AQP4. To identify which cells were transfected, AQP4 was tagged at the NH 2 terminus with green fluorescent protein.Transfected cells showed a strong fluorescent signal in basolateral membrane and a low-to-negligible signal in the cytosol and apical membrane. Activation of protein kinase C (PKC) with phorbol 12,13-dibutyrate (PDBu) significantly decreased P f of cells expressing AQP4 but had no effect on neighboring untransfected cells. No redistribution of AQP4 in response to PDBu was detected. Dopamine also decreased the P f in transfected cells. The effect was abolished by the PKC inhibitor Ro 31-8220. Reduction of AQP4 water permeability by PDBu and dopamine was abolished by point mutation of Ser 180 , a consensus site for PKC phosphorylation. We conclude that PKC and dopamine decrease AQP4 water permeability via phosphorylation at Ser 180 and that the effect is likely mediated by gating of the channel. water channels; protein kinase C phosphorylation; LLC-PK 1 cells; green fluorescent protein; water transport CELLS THAT REQUIRE A HIGH water permeability, such as kidney epithelial cells, express specific water channels, aquaporins (AQPs; recently reviewed in Refs. 3 and 23). Because regulation of cell and total body water content is an essential homeostatic function, the question has been raised of whether the activity of AQPs is dynamically regulated by G protein-coupled receptors and intracellular messengers. Short-term regulation of the activity of AQPs by G protein-coupled receptors has until now mostly been studied for aquaporin-2 (AQP2) (18), the water channel that is expressed in kidney collecting duct and that is regulated by vasopressin (AVP) (6,8,9,17,27,28,40,45,48,50).Aquaporin-4 (AQP4) (22, 25) is expressed in collecting duct principal cells (13,14,55) and is important for concentration of urine (5). It has been shown that AQP4 can be phosphorylated by protein kinase C (PKC) in vitro, and, when expressed in Xenopus laevis oocytes, the water permeability of AQP4 is decreased by PKC activation (21). The physiological significance of these findings has not yet been revealed. Studies of the dynamic regulation of water permeability in welldifferentiated mammalian cells have so far been associated with a number of methodological problems. Here we employ a method that allows simultaneous studies of water permeability of renal epithelial cells that do or do not express AQP4 tagged with green fluorescent protein (GFP). By using this method, we show that the water permeability of AQP4 is downregulated by PKC activation and by dopamine. To examine whether the effect of PKC was direct, or mediated via an intermediary protein, studies were also performed with AQP4, where the consensus site for PKC phosphorylation, Ser 180

Aquaporin-2 (AQP2), the protein that mediates arginine vasopressin (AVP)-regulated apical water transport in the renal collecting duct, possesses a single consensus phosphorylation site for cAMP-dependent protein kinase A (PKA) at Ser256. The aim of this study was to examine whether AVP, and other agents that increase cAMP levels, could stimulate the phosphorylation of AQP2 in intact rat renal tissue. Rat renal papillae were prelabeled with32P and incubated with vehicle or drugs, and then AQP2 was immunoprecipitated. Two polypeptides corresponding to nonglycosylated (29 kDa) and glycosylated (35–48 kDa) AQP2 were identified by SDS-PAGE. AVP caused a time- and dose-dependent increase in phosphorylation of both glycosylated and nonglycosylated AQP2. The threshold dose for a significant increase in phosphorylation was 10 pM, which corresponds to a physiological serum concentration of AVP. Maximal phosphorylation was reached within 1 min of AVP incubation. This effect on AQP2 phosphorylation was mimicked by the vasopressin (V2) agonist, 1-desamino-[8-d-arginine]vasopressin (DDAVP), or forskolin. Two-dimensional phosphopeptide mapping indicated that AVP and forskolin stimulated the phosphorylation of the same site in AQP2. Immunoblot analysis using a phosphorylation state-specific antiserum revealed an increase in phosphorylation of Ser256 after incubation of papillae with AVP. The results indicate that AVP stimulates phosphorylation of AQP2 at Ser256via activation of PKA, supporting the idea that this is one of the first steps leading to increased water permeability in collecting duct cells.