Aldosterone classically modulates Na transport in tight epithelia such as the renal collecting duct (CD) through the transcellular route, but it is not known whether the hormone could also affect paracellular permeability. Such permeability is controlled by tight junctions (TJ) that form a size- and charge-selective barrier. Among TJ proteins, claudin-4 has been highlighted as a key element to control paracellular charge selectivity. In RCCD2 CD cells grown on filters, we have identified novel early aldosterone effects on TJ. Endogenous claudin-4 abundance and cellular localization were unaltered by aldosterone. However, the hormone promoted rapid (within 15-20 min) and transient phosphorylation of endogenous claudin-4 on threonine residues, without affecting tyrosine or serine; this event was fully developed at 10 nM aldosterone and appeared specific for aldosterone (because it is not observed after dexamethasone treatment and it depends on mineralocorticoid receptor occupancy). Within the same delay, aldosterone also promoted an increased apical-to-basal passage of 125I (a substitute for 36Cl), whereas 22Na passage was unaffected; paracellular permeability to [3H]mannitol was also reduced. Later on (45 min), a fall in transepithelial resistance was observed. These data indicate that aldosterone modulates TJ properties in renal epithelial cells.

Long-term effects of arginine vasopressin (AVP) in the kidney involve the transcription of unidentified genes. By subtractive hybridization experiments performed on the RCCD 1 cortical collecting duct cell line, we identified calcyclin as an early AVP-induced gene (1 h). Calcyclin is a calcium-binding protein involved in the transduction of intracellular signals. In the kidney, calcyclin was localized at the mRNA level in the glomerulus, all along the collecting duct, and in the epithelium lining the papilla. In RCCD 1 cells and in m-IMCD 3 inner medullary collecting duct cells, calcyclin was evidenced in the cytoplasm. Calcyclin mRNA levels were progressively increased by AVP treatment in RCCD 1 (1.7-fold at 4 h) and m-IMCD 3 (2-fold at 7.5 h) cells. In RCCD 1 cells, calcyclin protein levels were increased by 4 h of AVP treatment. In vivo, treatment of genetically vasopressindeficient Brattleboro rats with AVP for 4 days induced an increase in both calcyclin and aquaporin-2 mRNA expression. Finally, introduction of anti-calcyclin antibodies into RCCD 1 cells by permeabilizing the plasma membrane prevented the long-term (but not short-term) increase in short-circuit current induced by AVP. Taken together, these results suggest that calcyclin is an early vasopressin-induced gene that participates in the late phase of the hormone response in transepithelial ion transport. Arginine vasopressin (AVP)1 is a polypeptide hormone involved in the regulation of renal water and ion transport. In the collecting duct, AVP coordinately increases water and NaCl reabsorption by a two-step mechanism. The first mechanism is responsible for the short-term effects of AVP. These effects consist of the translocation of aquaporin-2 (AQP2) water channels and amiloride-sensitive sodium channels from intracellular pools to the apical membrane, promoting an increase in both sodium and water entry (1-4). This increase induces, in turn, a coordinate rise in transepithelial water and sodium reabsorption (5-10). These effects are rapid and transient because the effect is down-regulated after ϳ1 h in the rat collecting duct (11). In addition to this short-term effect, AVP induces a long-term effect. This effect consists of a late increase in transepithelial water, sodium, and chloride transport after several hours. This late effect is mediated through a transcriptional/translational mechanism and involves an increase in the mRNA and de novo synthesis of different proteins such as AQP2; the ␤ and ␥ (but not ␣) subunits of ENaC (epithelial Na ϩ channel); the ␣ 1 (but not ␤ 1 ) subunit of Na ϩ /K ϩ -ATPase; and CFTR (cystic fibrosis transmembrane conductance regulator) (12-15). This long-term effect may involve the genomic pathway by activation of cAMP-responsive elements in the promoter region of these genes and may ensure a sustained increase in sodium, chloride, and water transport in this segment of the nephron. A recent study has focused on the effects of 4 h of treatment with vasopressin on the transcriptome of a mouse kidney cortical colle...

BackgroundThe large conductance calcium-activated potassium channel alpha-subunit (Slo) is widely distributed throughout the body and plays an important role in a number of diseases. Prior work has shown that Slo, through its S10 region, interacts with β-catenin, a key component of the cytoskeleton framework and the Wnt signaling pathway. However, the physiological significance of this interaction was not clear.Methodology/Principal FindingsUsing a combination of proteomic and cell biology tools we show the existence of additional multiple binding sites in Slo, and explore in detail β-catenin interactions with the S10 region. We demonstrate that deletion of this region reduces Slo surface expression in HEK cells, which indicates that interaction with beta-catenin is important for Slo surface expression. This is confirmed by reduced expression of Slo in HEK cells and chicken (Gallus gallus domesticus leghorn white) hair cells treated with siRNA to β-catenin. HSlo reciprocally co-immunoprecipitates with β-catenin, indicating a stable binding between these two proteins, with the S10 deletion mutant having reduced binding with β-catenin. We also observed that mutations of the two putative GSK phosphorylation sites within the S10 region affect both the surface expression of Slo and the channel's voltage and calcium sensitivities. Interestingly, expression of exogenous Slo in HEK cells inhibits β-catenin-dependent canonical Wnt signaling.Conclusions and SignificanceThese studies identify for the first time a central role for β-catenin in mediating Slo surface expression. Additionally we show that Slo overexpression can lead to downregulation of Wnt signaling.