Water permeability of aquaporin-4 is decreased by protein kinase C and dopamine

Abstract

(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

Phosphorylation of Soybean Nodulin 26 on Serine 262 Enhances Water Permeability and Is Regulated Developmentally and by Osmotic Signals

“…Numerous MIP proteins are subject to phosphorylation, and the effects on activity are varied, ranging from the stimulation (e.g., the plant MIPs ␣ TIP [Maurel et al, 1995] and PM28A [Johansson et al, 1998]) to the inhibition of water transport (e.g., mammalian AQP4 [Han et al, 1998;Zelenina et al, 2002]) and alterations in water permeability by affecting membrane trafficking and subcellular localization (e.g., AQP4 [Madrid et al, 2001] and AQP2 [Nielsen et al, 1995;Fushimi et al, 1997]). The present study shows that phosphorylation of nodulin 26 enhances its water permeability both upon expression in Xenopus oocytes and in symbiosome membrane vesicles.…”

High-dose thiamine therapy counters dyslipidaemia in streptozotocin-induced diabetic rats

“…Decreased glucosuria may be linked to decreased washout of glucose by the concomitant decreased diuresis and effects on renal glucose transporters. Decreased diuresis may be linked to reversal of diabetes-induced activation of protein kinase C by highdose thiamine and benfotiamine [6] and consequent reversal of the inhibition of water re-uptake by aquaporins in renal collecting duct cells [21,22]. In any event, STZ diabetic rats with high-dose thiamine therapy consumed less food than control STZ diabetic rats whilst maintaining similar body weights.…”

Erythropoietin modulation of astrocyte water permeability as a component of neuroprotection

“…We used constructs encoding mouse AQP4.M23 tagged with green fluorescent protein (GFP) at the NH2 terminus (pGFP-AQP4.M23), described previously (35,36).…”

“…Water permeability (Pf) was measured using a method that we described in detail previously (35,37). By use of this method Pf in individual cells within cell monolayers can be determined and compared between cells that do or do not express GFP-labeled proteins.…”