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Abstract: PO1402

Inhibition of Actin-Related Protein 2/3 Complex Blocks Vasopressin-Induced AQP2 Membrane Accumulation

Session Information

Category: Fluid, Electrolyte, and Acid-Base Disorders

  • 901 Fluid, Electrolyte, and Acid-Base Disorders: Basic


  • Liu, Chen-Chung, Massachusetts General Hospital, Boston, Massachusetts, United States
  • Cheung, Pui Susan Wen, Massachusetts General Hospital, Boston, Massachusetts, United States
  • Bouley, Richard, Massachusetts General Hospital, Boston, Massachusetts, United States
  • Brown, Dennis, Massachusetts General Hospital, Boston, Massachusetts, United States

Group or Team Name

  • MGH Program in Membrane Biology

Aquaporin 2 (AQP2) is a water channel protein located primarily on principal cells of the kidney collecting ducts and is crucial for regulating body water homeostasis. Regulation of AQP2 trafficking is subject to hormonal control, mainly via the canonical vasopressin (VP) signaling pathway which stimulates AQP2 membrane accumulation. Active actin cytoskeleton remodeling has been known to also play an important role in AQP2 trafficking, however, the mechanism is incompletely understood.


We applied CK-666, a pharmacological inhibitor of actin nucleator actin-related protein (Arp) 2/3 complex on our AQP2-transfected cells and animal (rat) models. Results were observed by using immunohistochemistry and confocal microscopy. VP signaling pathway and phosphorylation of AQP2 on various serine residues were studied with Western blotting.


Using CK-666, an Arp2/3 complex inhibitor, we found that VP induced AQP2 membrane accumulation was inhibited both in rat kidneys and LLC-AQP2 cells in vitro. Instead of distributing throughout the cytoplasm, AQP2 in cells treated with CK-666 was concentrated in vesicles forming a perinuclear patch, which was also positive for Rab-11 (a recycling endosome marker) and clathrin (a trans-Golgi Network (TGN) marker). Similar perinuclear AQP2 patches appear in cells incubated at 20°C (cold block), which allows endocytosis to continue, but prevents protein exit from the TGN. By rewarming the cells to 37°C, these perinuclear patches dissipate, and AQP2 quickly redistributes throughout the cytoplasm (cold block release). However, we found that in cells exposed to a 20°C cold block and treated with CK-666, AQP2 patches failed to dissipate upon rewarming, suggesting that CK-666 blocked the release of AQP2 from the TGN in the exocytotic pathway. This effect of CK-666 was independent of VP signaling and did not alter the VP-induced phosphorylation state of AQP2 at residues serine-256, S269, and S261.


Inhibition of the Arp2/3 complex blocks VP-induced AQP2 plasma membrane accumulation by blocking AQP2 exocytosis at the level of the TGN and the recycling endosome but did not affect VP signaling pathway. This result suggests that actin filament nucleation and growth via Arp2/3 activity is essential for AQP2 recycling and trafficking.