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Epithelial Na+ channels (ENaCs) are activated by proteolysis of the α and γ subunits at specific sites flanking embedded inhibitory tracts. To examine the role of α subunit proteolysis in channel activation in vivo, we generated mice lacking the distal furin cleavage site in the α subunit (αF2M mice). On a normal Na+ control diet, no differences in ENaC protein abundance in kidney or distal colon were noted between wild‐type (WT) and αF2M mice. Patch‐clamp analyses revealed similar levels of ENaC activity in kidney tubules, while no physiologically relevant differences in blood chemistry or aldosterone levels were detected. Male αF2M mice did exhibit diminished ENaC activity in the distal colon, as measured by amiloride‐sensitive short‐circuit current (ISC). Following dietary Na+ restriction, WT and αF2M mice had similar natriuretic and colonic ISC responses to amiloride. However, single‐channel activity was significantly lower in kidney tubules from Na+‐restricted αF2M mice compared with WT littermates. ENaC α and γ subunit expression in kidney and distal colon were also enhanced in Na+‐restricted αF2Mvs. WT mice, in association with higher aldosterone levels. These data provide evidence that disrupting α subunit proteolysis impairs ENaC activity in vivo, requiring compensation in response to Na+ restriction. Key points: The epithelial Na+ channel (ENaC) is activated by proteolytic cleavage in vitro, but key questions regarding the role of ENaC proteolysis in terms of whole‐animal physiology remain to be addressed.We studied the in vivo importance of this mechanism by generating a mouse model with a genetic disruption to a key cleavage site in the ENaC's α subunit (αF2M mice).We found that αF2M mice did not exhibit a physiologically relevant phenotype under normal dietary conditions, but have impaired ENaC activation (channel open probability) in the kidney during salt restriction.ENaC function at the organ level was preserved in salt‐restricted αF2M mice, but this was associated with higher aldosterone levels and increased expression of ENaC subunits, suggesting compensation was required to maintain homeostasis.These results provide the first evidence that ENaC α subunit proteolysis is a key regulator of channel activity in vivo. [ABSTRACT FROM AUTHOR]

Proteases activate the epithelial sodium channel (ENaC) by cleaving the large extracellular domains of the [alpha]- and [gamma]-subunits and releasing peptides with inhibitory properties. Furin and prostasin activate mouse ENaC by cleaving the [gamma]-subunit at sites flanking a 43 residue inhibitory tract ([gamma]E144-K186). To determine whether there is a minimal inhibitory region within this 43 residue tract, we generated serial deletions in the inhibitory tract of the [gamma]-subunit in channels resistant to cleavage by furin and prostasin. We found that partial or complete deletion of a short segment in the [gamma]-subunit, R158-N171, enhanced channel activity. Synthetic peptides overlapping this segment in the [gamma]-subunit further identified a key 11-met tract, R158-F168 (RFLNLIPLLVF), which inhibited wild-type ENaC expressed in Xenopus laevis oocytes, and endogenous channels in mpkCCD cells and human airway epithelia. Further studies with amino acid-substituted peptides defined residues that are required for inhibition in this key 11-mer tract. The presence of the native [gamma] inhibitory tract in ENaC weakened the intrinsic binding constant of the 11-mer peptide inhibitor, suggesting that the [gamma] inhibitory tract and the 11-mer peptide interact at overlapping sites within the channel. ion channels; channel inhibitor; prostasin; amiloride doi: 10.1152/ajprenal.00316.2010.

The X-linked disorder Lowe syndrome arises from mutations in OCRLl, a lipid phosphatase that hydrolyzes phosphatidylinositol 4,5-bisphosphate ([PIP.sub.2]). Most patients with Lowe syndrome develop proteinuria very early in life. PIP2 dynamics are known to modulate numerous steps in membrane trafficking, and it has been proposed that OCRL1 activity regulates the biogenesis or trafficking of the multiligand receptor megalin. To examine this possibility, we investigated the effects of siRNA-mediated OCRL1 knockdown on biosynthetic and postendocytic membrane traffic in canine and human renal epithelial cells. Cells depleted of OCRL1 did not have significantly elevated levels of cellular PIP2 but displayed an increase in actin comets, as previously observed in cultured cells derived from Lowe patients. Using assays to independently quantitate the endocytic trafficking of megalin and of megalin ligands, we could observe no defect in the trafficking or function of megalin upon OCRL1 knockdown. Moreover, apical delivery of a newly synthesized marker protein was unaffected. OCRL1 knockdown did result in a significant increase in secretion of the lysosomal hydrolase cathepsin D, consistent with a role for OCRL1 in membrane trafficking between the trans-Golgi network and endosomes. Together, our studies suggest that OCRL1 does not directly modulate endocytosis or postendocytic membrane traffic and that the renal manifestations observed in Lowe syndrome patients are downstream consequences of the loss of OCRL1 function. proximal tubule; Lowe syndrome; phosphatidylinositol; megalin; CLC-5; endocytosis; cathepsin D doi: 10.1152/ajprenal.00453.2009

Acute kidney injury (AKI) is a rapid decline in renal function and can occur after ischemia/reperfusion injury (IRI) to the tubular epithelia. The nuclear factor erythroid-2-related factor 2 (NRF2) pathway protects against AKI and AKI-to-chronic kidney disease (CKD) progression, but we previously demonstrated that severe IRI maladaptively reduced NRF2 activity in mice. To understand the mechanism of this response, we subjected C57BL/6J mice to unilateral kidney IRI with ischemia times that were titrated to induce mild to severe injury. Mild IRI increased NRF2 activity and was associated with renal recovery, whereas severe IRI decreased NRF2 activity and led to progressive CKD. Due to these effects of ischemia, we tested the hypothesis that hypoxia-inducible factor-1α (HIF-1α) mediates NRF2 activity. To mimic mild and severe ischemia, we activated HIF-1α in HK-2 cells in nutrient-replete or nutrient-deficient conditions. HIF-1α activation in nutrient-replete conditions enhanced NRF2 nuclear localization and activity. However, in nutrient-deficient conditions, HIF-1α activation suppressed NRF2 nuclear localization and activity. Nuclear localization was rescued with HIF-1α siRNA knockdown. Our results suggest that severe ischemic AKI leads to HIF-1α-mediated suppression of NRF2, leading to AKI-to-CKD progression. [ABSTRACT FROM AUTHOR]

Epithelial sodium channels (ENaC) are processed by proteases as they transit the biosynthetic pathway. We recently observed that furin-dependent processing of the [alpha]-subunit of ENaC at two sites within its extracellular domain is required for channel activation due to release of a 26-residue inhibitory domain. While channels with [alpha]-subunits lacking the furin sites are not cleaved and have very low activity, channels lacking the furin consensus sites as well as the tract between these sites ([alpha]D206-R231) are active. We analyzed channels with a series of deletions in the tract [alpha]D206-R231 and lacking the [alpha]-subunit furin consensus sites in Xenopus laevis oocytes. We found an eight-residue tract that, when deleted, restored channel activity to the level found in oocytes expressing wild-type ENaC. A synthetic peptide, LPHPLQRL, representing the tract [alpha]L211-L218, inhibited wild-type ENaC expressed in oocytes with an [IC.sub.50] of 0.9 [micro]M, and inhibited channels expressed in collecting duct cells and human primary airway epithelial cells with an [IC.sub.50]s of between ~50 and 100 [micro]M. Analyses of peptides with deletions within this inhibitory tract indicate that eight residues is the minimal backbone length that is required for ENaC inhibition. Analyses of 8-mer peptides with conserved and nonconserved substitutions suggest that [L.sup.1], [P.sup.2], [H.sup.3], [P.sup.4], and [L.sup.8] are required for inhibitory activity. Our findings suggest that this eight-residue tract is a key conserved inhibitory domain that provides epithelial cells with a reserve of inactive channels that can be activated as required by proteases. peptide inhibitors; proteases; furin

Vectorial [Na.sup.+] absorption across the aldosterone-sensitive distal nephron plays a key role in the regulation of extracellular fluid volume and blood pressure. Within this nephron segment, [Na.sup.+] diffuses from the urinary fluid into principal cells through an apical, amiloride-sensitive, epithelial [Na.sup.+] channel (ENaC), which is considered to be the rate-limiting step for [Na.sup.+] absorption. We have reported that increases in tubular flow rate in microperfused rabbit cortical collecting ducts (CCDs) lead to increases in net [Na.sup.+] absorption and that increases in laminar shear stress activate ENaC expressed in oocytes by increasing channel open probability. We therefore examined whether flow stimulates net [Na.sup.+] absorption ([J.sub.Na]) in CCDs by increasing channel open probability or by increasing the number of channels at the apical membrane. Both baseline and flow-stimulated [J.sub.Na], in CCDs were mediated by ENaC, as [J.sub.Na], was inhibited by benzamil. Flow-dependent increases in [J.sub.Na] were observed following treatment of tubules with reagents that altered membrane trafficking by disrupting microtubules (colchicine) or Golgi (brefeldin A). Furthermore, reducing luminal [Ca.sup.2+] concentration ([Ca.sup.2+]) or chelating intracellular [[Ca.sup.2+]] with BAPTA did not prevent the flow-dependent increase in [J.sub.Na]. Extracellular trypsin has been shown to activate ENaC by increasing channel open probability, and we observed that trypsin significantly enhanced [J.sub.Na] when tubules were perfused at a slow flow rate. However, trypsin did not further enhance [J.sub.Na] in CCDs perfused at fast flow rates. Similarly, the shear-induced increase in benzamil-sensitive [J.sub.Na] in oocytes expressing protease resistance ENaC mutants was similar to that of controls. Our results suggest the rise in [J.sub.Na] accompanying increases in luminal flow rates reflects an increase in channel open probability. epithelial sodium channel; in vitro microperfusion; protein trafficking; mechanoregulation; laminar shear; principal cell doi:10.1152/ajprenal.00514.2005

Epithelial [Na.sup.+] channels (ENaC) are inhibited by extracellular [Na.sup.+], a process referred to as [Na.sup.+] self-inhibition. We previously demonstrated that mutation of key residues within two furin cleavage consensus sites in [alpha], or one site in [gamma] blocked subunit proteolysis and inhibited channel activity when mutant channels were expressed in Xenopus laevis oocytes (Hughey RP, Bruns JB, Kinlough CL, Harkleroad KL, Tong Q, Carattino MD, Johnson JP, Stockand JD, and Kleyman TR. J Biol Chem 279: 18111-18114, 2004). Cleavage of subunits was also blocked by these mutations when expressed in Madin-Darby canine kidney cells, and both subunit cleavage and channel activity were blocked when wild-type subunits were expressed in furin-deficient Chinese hamster ovary cells. We now report that channels with mutant [alpha]-subunits lacking either one or both furin cleavage sites exhibited a marked enhancement of the [Na.sup.+] self-inhibition response, while channels with a mutant [gamma]/-subunit showed a modestly enhanced [Na.sup.+] self-inhibition response. Analysis of [Na.sup.+] self-inhibition at varying [Na.sup.+] indicates that channels containing mutant [alpha]-subunits exhibit an increased [Na.sup.+] affinity. At the single-channel level, channels with a mutant [alpha]-subunit had a low open probability ([P.sub.o]) in the presence of a high external [Na.sup.+] in the patch pipette. [P.sub.o] dramatically increased when trypsin was also present, or when a low external [Na.sup.+] was in the patch pipette. Our results suggest that furin cleavage of ENaC subunits activates the channels by relieving [Na.sup.+] self-inhibition and that activation requires that the [alpha]-subunit be cleaved twice. Moreover, we demonstrate for the first time a clear relationship between ENaC [P.sub.o] and extracellular [Na.sup.+], supporting the notion that [Na.sup.+] self-inhibition reflects a [P.sub.o] reduction due to high extracellular [Na.sup.+]. amiloride; open probability; voltage clamp; Xenopus laevis oocyte; mutagenesis

The maintenance of proper epithelial function requires efficient sorting of newly synthesized and recycling proteins to the apical and basolateral surfaces of differentiated cells. Whereas basolateral protein sorting signals are generally confined to their cytoplasmic regions, apical targeting signals have been identified that localize to luminal, transmembrane, and cytoplasmic aspects of proteins. In the past few years, both N-and O-linked glycans have been identified as apical sorting determinants. Glycan structures are extraordinarily diverse and have tremendous information potential. Moreover, because the oligosaccharides added to a given protein can change depending on cell type and developmental stage, the potential exists for altering sorting pathways by modulation of the expression pattern of enzymes involved in glycan synthesis. In this review, we discuss the evidence for glycan-mediated apical sorting along the biosynthetic pathway and present possible mechanisms by which these common and heterogeneous posttranslational modifications might function as specific sorting signals. glycosylation; epithelia; polarity; kidney; intestine

Epithelial sodium channels (ENaCs) are composed of three structurally related subunits that form a tetrameric channel. The Xenopus laevis oocyte expression system was used to identify regions within the ENaC [alpha]-subunit that confer a dominant negative phenotype on functional expression of [alpha][beta][gamma]-ENaC to define domains that have a role in subunit-subunit interactions. Coexpression of full-length mouse [alpha][beta][gamma]-ENaC with either 1) the [alpha]-subunit first membrane-spanning domain and short downstream hydrophobic domain ([alpha]-M1H1); 2) [alpha]-M1H1 and its downstream hydrophilic extracellular loop ([alpha]-M1H1-ECL); 3) the membrane-spanning domain of a control type 2 transmembrane protein (glutamyl transpeptidase; [gamma]-GT) fused to the [alpha]-ECL ([gamma]-GT[alpha]-ECL); 4) the extracellular domain of a control type 1 trans-membrane protein (Tac) fused to the [alpha]-subunit second membrane-spanning domain and short upstream hydrophobic domain (Tac-[alpha]-H2M2); or 5) the [alpha]-subunit cytoplasmic COOH terminus ([alpha]-Ct) significantly reduced amiloride-sensitive [Na.sup.+] currents in X. laevis oocytes. Functional expression of [Na.sup.+] channels was not inhibited when full-length [alpha][beta][gamma]-ENaC was coexpressed with either 1) the [alpha]-ECL lacking a signal-anchor sequence, 2) [alpha]-M1H1 and [alpha]-Ct expressed as a fusion protein, 3) full-length [gamma]-GT, or 4) full-length Tac. Furthermore, the expression of ROMK channels was not inhibited when full-length ROMK was coexpressed with either [alpha]-M1H1-ECL or [alpha]-Ct. Full-length FLAG-tagged [alpha], [beta], or [gamma]-ENaC coimmunoprecipitated with myc-tagged [alpha]-M1H1-ECL, whereas wild-type [gamma]-GT did not. These data suggest that multiple sites within the [alpha]-subunit participate in subunit-subunit interactions that are required for proper assembly of the heterooligomeric ENaC complex. amiloride-sensitive sodium channel; dominant negative mutants

Researchers have measured the transepithelial electrical resistance and isotopic water and urea fluxes for Madin-Darby canine kidney (MDCK) type I and II cells. Attention was also given to type I cells expressing MUC1, the mucin protein, in their apical membranes. It was found that the apical membrane permeability of type I cells resembles that of other barrier epithelia. Abrupt introduction of water-permeable pores with nystatin into the apical membranes of barrier epithelia gives rise to prompt increases in water permeability.

The isolation and characterization of two full-length cDNAs encoding the protein for rat renal gamma-glutamyltranspeptidase (gammaGT) were reported. Both cDNAs were used to transfect LLC-PK1 cells, a porcine polarized renal cell line. It was found that gammaGT activity is expressed only at the apical surface. Together, the results indicate that expression of the rat gammaGT in LLC-PK1 cells provides a model system to characterize the cellular mechanisms involved in apical targeting.