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The Ca2+-permeable transient receptor potential vanilloid type 4 (TRPV4) channel serves as the sensor of tubular flow, thus being well suited to govern mechanosensitive K+ transport in the distal renal tubule. Here, we directly tested whether the TRPV4 function is significant in affecting K+ balance. We used balance metabolic cage experiments and systemic measurements with different K+ feeding regimens [high (5% K+), regular (0.9% K+), and low (<0.01% K+)] in newly created transgenic mice with selective TRPV4 deletion in the renal tubule (TRPV4fl/fl-Pax8Cre) and their littermate controls (TRPV4fl/fl). Deletion was verified by the absence of TRPV4 protein expression and lack of TRPV4-dependent Ca2+ influx. There were no differences in plasma electrolytes, urinary volume, and K+ levels at baseline. In contrast, plasma K+ levels were significantly elevated in TRPV4fl/fl-Pax8Cre mice on high K+ intake. K+-loaded knockout mice exhibited lower urinary K+ levels than TRPV4fl/fl mice, which was accompanied by higher aldosterone levels by day 7. Moreover, TRPV4fl/fl-Pax8Cre mice had more efficient renal K+ conservation and higher plasma K+ levels in the state of dietary K+ deficiency. H+-K+-ATPase levels were significantly increased in TRPV4fl/fl-Pax8Cre mice on a regular diet and especially on a low-K+ diet, pointing to augmented K+ reabsorption in the collecting duct. Consistently, we found a significantly faster intracellular pH recovery after intracellular acidification, as an index of H+-K+-ATPase activity, in split-opened collecting ducts from TRPV4fl/fl-Pax8Cre mice. In summary, our results demonstrate an indispensable prokaliuretic role of TRPV4 in the renal tubule in controlling K+ balance and urinary K+ excretion during variations in dietary K+ intake. NEW & NOTEWORTHY The mechanoactivated transient receptor potential vanilloid type 4 (TRPV4) channel is expressed in distal tubule segments, where it controls flow-dependent K+ transport. Global TRPV4 deficiency causes impaired adaptation to variations in dietary K+ intake. Here, we demonstrate that renal tubule-specific TRPV4 deletion is sufficient to recapitulate the phenotype by causing antikaliuresis and higher plasma K+ levels in both states of K+ load and deficiency. [ABSTRACT FROM AUTHOR]

The renal collecting duct is known to play a critical role in many physiological processes, including systemic water–electrolyte homeostasis, acid–base balance, and the salt sensitivity of blood pressure. ClC-K2 (ClC-Kb in humans) is a Cl−-permeable channel expressed on the basolateral membrane of several segments of the renal tubule, including the collecting duct intercalated cells. ClC-Kb mutations are causative for Bartters' syndrome type 3 manifested as hypotension, urinary salt wasting, and metabolic alkalosis. However, little is known about the significance of the channel in the collecting duct with respect to the normal physiology and pathology of Bartters' syndrome. In this review, we summarize the available experimental evidence about the signaling determinants of ClC-K2 function and the regulation by systemic and local factors as well as critically discuss the recent advances in understanding the collecting-duct-specific roles of ClC-K2 in adaptations to changes in dietary Cl− intake and maintaining systemic acid–base homeostasis. [ABSTRACT FROM AUTHOR]

We used patchclamp electrophysiology on isolated, split-open murine collecting ducts (CD) to test the hypothesis that regulation of epithelial sodium channel (ENaC) activity is a physiologically important effect of vasopressin. Surprisingly, this has not been tested directly before. We ask whether vasopressin affects ENaC activity distinguishing between acute and chronic effects, as well as, parsing the cellular signaling pathway and molecular mechanism of regulation. In addition, we quantified possible synergistic regulation of ENaC by vasopressin and aldosterone associating this with a requirement for distal nephron [Na.sup.+] reabsorption during water conservation vs. maintenance of Na+ balance. We find that vasopressin significantly increases ENaC activity within 2-3 min by increasing open probability ([P.sub.o]). This activation was dependent on adenylyl cyclase (AC) and PKA. Water restriction (18-24 h) and pretreatment of isolated CD with vasopressin (~30 min) resulted in a similar increase in [P.sub.o]. In addition, this also increased the number (N) of active ENaC in the apical membrane. Similar to [P.sub.o], increases in N were sensitive to inhibitors of AC. Stressing animals with water and salt restriction separately and jointly revealed an important effect of vasopressin: conservation of water and [Na.sup.+] each independently increased ENaC activity and jointly had a synergistic effect on channel activity. These results demonstrate a quantitatively important action of vasopressin on ENaC suggesting that distal nephron [Na.sub.+] reabsorption mediated by this channel contributes to maintenance of water reabsorption. In addition, our results support that the combined actions of vasopressin and aldosterone are required to achieve maximally activated ENaC. hypertension; [Na.sup.+] reabsorption; aldosterone; cAMP; PKA doi: 10.1152/ajprenal.00371.2009.

Bile acids, originally known to emulsify dietary lipids, are now established signalling molecules that regulate physiological processes. Signalling targets several proteins that include the ion channels involved in regulating intestinal motility and bile viscosity. Studies show that bile acids regulate the epithelial sodium channel (ENaC) in cultured cell models and heterologous expression systems. ENaC plays both local and systemic roles in regulating extracellular fluids. Here we investigated whether bile acids regulate ENaC expressed in native tissues. We found that taurocholic acid and taurohyodeoxycholic acid regulated ENaC in both the distal nephron and distal colon. We also tested the hypothesis that regulation occurs through direct binding. Using photoaffinity labelling, we found evidence for specific binding to both the β and γ subunits of the channel. In functional experiments, we found that the α subunit was sufficient for regulation. We also found that regulation by at least one bile acid was voltage‐sensitive, suggesting that one binding site may be closely associated with the pore‐forming helices of the channel. Our data provide evidence that bile acids regulate ENaC by binding to multiple sites to influence the open probability of the channel. Key points: Recent studies have shown that bile acids regulate the epithelial sodium channel (ENaC) in vitro. Here we investigated whether bile acids regulate ENaC in native tissues and whether bile acids directly bind the channel.We found that bile acids regulate ENaC expressed in the mouse cortical collecting duct and mouse colon by modulating open probability.Photoaffinity labelling experiments showed specific binding to the β and γ subunits of the channel, while channels comprising only α subunits were sensitive to taurocholic acid in functional experiments using Xenopus oocytes.Taurocholic acid regulation of ENaC was voltage‐dependent, providing evidence for binding to pore‐forming helices.Our data indicate that bile acids are ENaC regulatory effectors that may have a role in the physiology and pathophysiology of several systems. [ABSTRACT FROM AUTHOR]

We used patch-clamp electrophysiology to investigate regulation of the epithelial [Na.sup.+] channel (ENaC) by endothelin-1 (ET-1) in isolated, split-open rat collecting ducts. ET-1 significantly decreases ENaC open probability by about threefold within 5 min. ET-1 decreases ENaC activity through basolateral membrane E[T.sub.B] but not E[T.sub.A] receptors. In rat collecting duct, we find no role for phospholipase C or protein kinase C in the rapid response of ENaC to ET-1. ET-1, although, does activate src family tyrosine kinases and their downstream MAPK1/2 effector cascade in renal principal cells. Both src kinases and MAPK1/2 signaling are necessary for ET-l-dependent decreases in ENaC open probability in the split-open collecting duct. We conclude that ET-1 in a physiologically relevant manner rapidly suppresses ENaC activity in native, mammalian principal cells. These findings may provide a potential mechanism for the natriuresis observed in vivo in response to ET-1, as well as a potential cause for the salt-sensitive hypertension found in animals with impaired endothelin signaling. salt-sensitive hypertension; systemic blood pressure

Activity of the epithelial sodium channel (ENaC) is limiting for [Na.sup.+] reabsorption at the distal nephron. Phosphoinositides, such as phosphatidylinositol 4,5-biphosphate [PI(4,5)[P.sub.2]] modulate the activity of this channel. Activation of purinergic receptors triggers multiple events, including activation of PKC and PLC, with the latter depleting plasma membrane PI(4,5)[P.sub.2]. Here, we investigate regulation of ENaC in renal principal cells by purinergic receptors via PLC and PI(4,5)[P.sub.2]. Purinergic signaling rapidly decreases ENaC open probability and apical membrane PI(4,5)[P.sub.2] levels with similar time courses. Moreover, inhibiting purinergic signaling with suramin rescues ENaC activity. The PLC inhibitor U73122, but not U73343, its inactive analog, recapitulates the action of suramin. In contrast, modulating PKC signaling failed to affect purinergic regulation of ENaC. Unexpectedly, inhibiting either purinergic receptors or PLC in resting cells dramatically increased ENaC activity above basal levels, indicating tonic activation of purinergic signaling in these polarized renal epithelial cells. Increased ENaC activity was associated with elevation of apical membrane PI(4,5)[P.sub.2] levels. Subsequent treatment with ATP in the presence of inhibited purinergic signaling failed to decrease ENaC activity and apical membrane PI(4,5)[P.sub.2] levels. Dwell-time analysis reveals that depletion of PI(4,5)[P.sub.2] forces ENaC toward a closed state. In contrast, increasing PI(4,5)[P.sub.2] levels above basal values locks the channel in an open state interrupted by brief closings. Thus our results suggest that purinergic control of apical membrane PI(4,5)[P.sub.2] levels is a major regulator of ENaC activity in renal epithelial cells. phosphoinositides; PLC signaling; P2Y receptors; sodium reabsorption; hypertension

Phosphatidylinositol 4,5-bisphosphate (PI(4,5)[P.sub.2]) and phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)[P.sub.3]) are physiologically important second messengers. These molecules bind effector proteins to modulate activity. Several types of ion channels, including the epithelial [Na.sup.+] channel (ENaC), are phosphoinositide effectors capable of directly interacting with these signaling molecules. Little, however, is known of the regions within ENaC and other ion channels important to phosphoinositide binding and modulation. Moreover, the molecular mechanism of this regulation, in many instances, remains obscure. Here, we investigate modulation of ENaC by PI(3,4,5)[P.sub.3] and PI (4,5)[P.sub.2] to begin identifying the molecular determinants of this regulation. We identify intracellular regions near the inner membrane interface just following the second transmembrane domains in [beta]- and [gamma]- but not [alpha]-ENaC as necessary for PI (3,4,5) [P.sub.2] but not PI (4,5) [P.sub.2] modulation. Charge neutralization of conserved basic amino acids within these regions demonstrated that these polar residues are critical to phosphoinositide regulation. Single channel analysis, moreover, reveals that the regions just following the second transmembrane domains in [beta]- and [gamma]-ENaC are critical to PI (3,4 5) [P.sub.3] augmentation of ENaC open probability, thus, defining mechanism. Unexpectedly, intracellular domains within the extreme N terminus of [beta- and [gamma]-ENaC were identified as being critical to downregulation of ENaC activity and [P.sub.o] in response to depletion of membrane PI (4,5)[P.sub.2]. These regions of the channel played no identifiable role in a PI (3,4,5) [P.sub.3] response. Again, conserved positive-charged residues within these domains were particularly important, being necessary for exogenous PI (4,5) [P.sub.2] to increase open probability. We conclude that [beta] and [gamma] subunits bestow phosphoinositide sensitivity to ENaC with distinct regions of the channel being critical to regulation by PI (3,4,5) [P.sub.3] and PI (4,5) [P.sub.2]. This argues that these phosphoinositides occupy distinct ligandbinding sites within ENaC to modulate open probability.