We have previously demonstrated that renal cortical collecting duct cells (RCCD 1 ), responded to hypotonic stress with a rapid activation of regulatory volume decrease (RVD) mechanisms. This process requires the presence of the water channel AQP2 and calcium influx, opening the question about the molecular identity of this calcium entry path. Since the calcium permeable nonselective cation channel TRPV4 plays a crucial role in the response to mechanical and osmotic perturbations in a wide range of cell types, the aim of this work was to test the hypothesis that the increase in intracellular calcium concentration and the subsequent rapid RVD, only observed in the presence of AQP2, could be due to a specific activation of TRPV4. We evaluated the expression and function of TRPV4 channels and their contribution to RVD in WT-RCCD 1 (not expressing aquaporins) and in AQP2-RCCD 1 (transfected with AQP2) cells. Our results demonstrated that both cell lines endogenously express functional TRPV4, however, a large activation of the channel by hypotonicity only occurs in cells that express AQP2. Blocking of TRPV4 by ruthenium red abolished calcium influx as well as RVD, identifying TRPV4 as a necessary component in volume regulation. Even more, this process is dependent on the translocation of TRPV4 to the plasma membrane. Our data provide evidence of a novel association between TRPV4 and AQP2 that is involved in the activation of TRPV4 by hypotonicity and regulation of cellular response to the osmotic stress, suggesting that both proteins are assembled in a signaling complex that responds to anisosmotic conditions. J. Cell. Biochem. 113: 580-589, 2012. ß 2011 Wiley Periodicals, Inc.KEY WORDS: AQUAPORIN 2; TRANSIENT RECEPTOR POTENTIAL VANILLOID 4; CELL VOLUME REGULATION; INTRACELLULAR CALCIUM M any cell types have evolved specialized mechanisms of volume regulation to counteract damage induced by either cell swelling or shrinking. Under hypotonic conditions, cells first respond by swelling and second by initiating a mechanism that allows them to recover their original volume, this mechanism is called regulatory volume decrease (RVD) [O'Neill, 1999;Okada et al., 2001;Lang, 2007]. Essentially all cells respond to hypotonic swelling with this complex mechanism but the response is usually incomplete probably due to the fact that a new steady-state volume is achieved [O'Neill, 1999]. RVD depends on the release of inorganic and organic osmolytes that reverse the osmotic gradient and direction of water flow [Okada et al., 2001;Strange, 2004;Lang, 2007]. A number of ion channels and transporters were identified as the pathways for volume-regulatory ionic flux [Lang, 2007], however, the underlying mechanism that senses the change in osmolarity and/or cell volume to initiate volume regulation is not yet completely understood.We have previously demonstrated, in a rat cortical collecting duct cell line (RCCD 1 ) which exhibits many major functional properties of the cortical collecting duct (CCD) [Blot-Chabaud et al., 1996;Dje...

Aquaporins (AQPs) function as tetrameric structures in which each monomer has its own permeable pathway. The combination of structural biology, molecular dynamics simulations, and experimental approaches has contributed to improve our knowledge of how protein conformational changes can challenge its transport capacity, rapidly altering the membrane permeability. This review is focused on evidence that highlights the functional relationship between the monomers and the tetramer. In this sense, we address AQP permeation capacity as well as regulatory mechanisms that affect the monomer, the tetramer, or tetramers combined in complex structures. We therefore explore: (i) water permeation and recent evidence on ion permeation, including the permeation pathway controversy—each monomer versus the central pore of the tetramer—and (ii) regulatory mechanisms that cannot be attributed to independent monomers. In particular, we discuss channel gating and AQPs that sense membrane tension. For the latter we propose a possible mechanism that includes the monomer (slight changes of pore shape, the number of possible H-bonds between water molecules and pore-lining residues) and the tetramer (interactions among monomers and a positive cooperative effect).

Galizia L, Flamenco MP, Rivarola V, Capurro C, Ford P. Role of AQP2 in activation of calcium entry by hypotonicity: implications in cell volume regulation. Am J Physiol Renal Physiol 294: F582-F590, 2008. First published December 19, 2007 doi:10.1152/ajprenal.00427.2007.-We previously reported in a rat cortical collecting duct cell line (RCCD1) that the presence of aquaporin 2 (AQP2) in the cell membrane is critical for the rapid activation of regulatory volume decrease mechanisms (RVD) (Ford et al. Biol Cell 97: 687-697, 2005). The aim of our present work was to investigate the signaling pathway that links AQP2 to this rapid RVD activation. Since it has been previously described that hypotonic conditions induce intracellular calcium ([Ca 2ϩ ]i) increases in different cell types, we tested the hypothesis that AQP2 could have a role in activation of calcium entry by hypotonicity and its implication in cell volume regulation. Using a fluorescent probe technique, we studied [Ca 2ϩ ]i and cell volume changes in response to a hypotonic shock in WT-RCCD1 (not expressing aquaporins) and in AQP2-RCCD1 (transfected with AQP2) cells. We found that after a hypotonic shock only AQP2-RCCD1 cells exhibit a substantial increase in [Ca 2ϩ ]i. This [Ca 2ϩ ]i increase is strongly dependent on extracellular Ca 2ϩ and is partially inhibited by thapsigargin (1 M) indicating that the rise in [Ca 2ϩ ]i reflects both influx from the extracellular medium and release from intracellular stores. Exposure of AQP2-RCCD1 cells to 100 M gadolinium reduced the increase in [Ca 2ϩ ]i suggesting the involvement of a mechanosensitive calcium channel. Furthermore, exposure of cells to all of the above described conditions impaired rapid RVD. We conclude that the expression of AQP2 in the cell membrane is critical to produce the increase in [Ca 2ϩ ]i which is necessary to activate RVD in RCCD1 cells. aquaporin 2; intracellular calcium; renal cells THE KIDNEY COLLECTING DUCT plays an important role in the process of urine concentration through a mechanism regulated by arginine vasopressin. This hormone induces an increase in osmotic water permeability (P f ) by triggering translocation and fusion of intracellular vesicles containing aquaporin 2 (AQP2) to the apical membrane of principal cells (32,33,48). In this condition, two-thirds of the hyposmotic luminal fluid entering the cortical collecting duct (CCD) is reabsorbed. Therefore, CCD cells are faced with both, changes in apical osmolarity and important volume flows which could result in cell volume increases. For this reason, potent volume regulatory mechanisms are needed to maintain cellular homeostasis and epithelial transport (29).Most cells respond to decrease in tonicity first by swelling and second by initiating mechanisms that allow them to recover their original volume (21,35). This complex mechanism, called regulatory volume decrease (RVD), depends on the activation of different ion permeabilities (usually K ϩ , Cl Ϫ , and/or HCO 3 Ϫ ) that reverse the osmotic gradient an...