Epithelial Na+ channels (ENaCs) are activated by extracellular trypsin or co-expression with channel-activating proteases, although there is no direct evidence that these proteases activate ENaC cleaving the channel. We previously demonstrated α and γ subunits of cleaved during maturation near consensus sites for furin cleavage. Using site-specific mutagenesis channel subunits, expression in furin-deficient cells, furin-specific inhibitors, we now report cleavage correlates activity. Channel...

Epithelial sodium channels (ENaC) are expressed in the apical membrane of high resistance Na+ transporting epithelia and have a key role regulating extracellular fluid volume airway surface liquids. Maturation activation ENaC subunits involves furin-dependent cleavage ectodomain at two sites α subunit single site within γ subunit. We now report that serine protease prostasin further activates by inducing distal to furin site. Dual is predicted release 43-amino acid peptide. Channels with...

The epithelial Na+ channel (ENaC) is a tetramer of two α-, one β-, and γ-subunit, but little known about its assembly processing. Because co-expression mouse ENaC subunits with three different carboxyl-terminal epitope tags produced an amiloride-sensitive sodium current in oocytes, these tagged were expressed both Chinese hamster ovary or Madin-Darby canine kidney type 1 cells for further study. When alone α-(95 kDa), β-(96 γ-subunits (93 kDa) each single band on SDS gels by immunoblotting....

The kidney has an extraordinary ability to maintain stable fractional solute and fluid reabsorption over a wide range of glomerular filtration rates (GFRs). Internalization filtered low molecular weight proteins, vitamins, hormones, other small molecules is mediated by the proximal tubule (PT) multiligand receptors megalin cubilin. Changes in GFR accompanying shear stress (FSS) modulate acute changes PT ion transport thought be microvillar bending. We found that FSS also affects apical...

The proper function of the organs that make up urinary tract (kidneys, ureters, bladder, and urethra) depends on their ability to sense respond mechanical forces, including shear stress wall tension. However, we have limited understanding mechanosensors in these tissue sites which molecules are expressed. Possible candidates include stretch-activated PIEZO channels (PIEZO1 PIEZO2), been implicated mechanically regulated body functions touch sensation, proprioception, lung inflation, blood...

The mechanisms that link visceral mechanosensation to the perception of internal organ status (i.e., interoception) remain elusive. In response bladder filling, urothelium releases ATP, which is hypothesized stimulate voiding function by communicating degree fullness subjacent tissues, including afferent nerve fibers. To determine if PIEZO channels as mechanosensors in these events, we generated conditional urothelial Piezo1-, Piezo2-, and dual Piezo1/2-knockout (KO) mice. While functional...

The degenerin/epithelial Na+ channel (ENaC) superfamily is a group of structurally related ion channels that are involved in diverse biological processes, including responses to mechanical stimuli. In renal cortical collecting ducts, changes rates perfusion affect reabsorption through an amiloride-sensitive pathway, suggesting ENaC may be mechanosensitive channel. this study, we examined whether expressed oocytes regulated by laminar shear stress (LSS). A 1.8-mm (internal diameter) pipette...

Epithelial Na+ channels (ENaC) are inhibited by extracellular Na+, a process referred to as self-inhibition. We previously demonstrated that mutation of key residues within two furin cleavage consensus sites in alpha, or one site gamma, blocked subunit proteolysis and channel activity when mutant were expressed Xenopus laevis oocytes (Hughey RP, Bruns JB, Kinlough CL, Harkleroad KL, Tong Q, Carattino MD, Johnson JP, Stockand JD, Kleyman TR. J Biol Chem 279: 18111-18114, 2004). Cleavage...

The epithelial Na(+) channel (ENaC) regulates salt and water reabsorption, processes that require significant expenditure of cellular energy. To test whether the ubiquitous metabolic sensor AMP-activated kinase (AMPK) ENaC, we examined effects AMPK activation on amiloride-sensitive currents in Xenopus oocytes polarized mouse collecting duct mpkCCD(c14) cells. Microinjection expressing ENaC (mENaC) with either active protein or an activator inhibited mENaC relative to controls as measured by...

Epithelial sodium channels (ENaCs) mediate Na(+) entry across the apical membrane of high resistance epithelia that line distal nephron, airway and alveoli, colon. These are composed three homologous subunits, termed alpha, beta, gamma, which have intracellular amino carboxyl termini two membrane-spanning domains connected by large extracellular loops. Maturation ENaC subunits involves furin-dependent cleavage loops at sites within alpha subunit a single site gamma subunit. The must be...

Vectorial Na(+) absorption across the aldosterone-sensitive distal nephron plays a key role in regulation of extracellular fluid volume and blood pressure. Within this segment, diffuses from urinary into principal cells through an apical, amiloride-sensitive, epithelial channel (ENaC), which is considered to be rate-limiting step for absorption. We have reported that increases tubular flow rate microperfused rabbit cortical collecting ducts (CCDs) lead net laminar shear stress activate ENaC...

Maturation of the epithelial sodium channel (ENaC) involves furin-dependent cleavage at two extracellular sites within alpha subunit and a single site gamma subunit. Channels lacking furin processing have very low activity. We recently identified prostasin-dependent (RKRK(186)) in also demonstrated that tract D206-R231, between subunit, as well E144-K186, prostasin are inhibitory domains. ENaC by furin, subsequently prostasin, leads to stepwise increase open probability result release...

Renal denervation lowers arterial blood pressure (ABP) in multiple clinical trials and some experimental models of hypertension. These antihypertensive effects have been attributed to the removal renal afferent nerves. The purpose present study was define function, anatomy, contribution mouse sensory neurons a nerve-dependent model First, electrical stimulation nerves produced frequency-dependent increases ABP that were eliminated by ganglionic blockade. Stimulus-triggered averaging revealed...

Epithelial sodium channels (ENaC) are processed by proteases as they transit the biosynthetic pathway. We recently observed that furin-dependent processing of alpha-subunit ENaC at two sites within its extracellular domain is required for channel activation due to release a 26-residue inhibitory domain. While with alpha-subunits lacking furin not cleaved and have very low activity, consensus well tract between these (alphaD206-R231) active. analyzed series deletions in alphaD206-R231 Xenopus...

Epithelial cells are continuously exposed to mechanical forces including shear stress and stretch, although the effect these have on tight junction (TJ) organization function poorly understood. Umbrella form outermost layer of stratified uroepithelium undergo large cell shape surface area changes during bladder cycle. Here we investigated effects filling voiding umbrella TJ. We found that promoted a significant increase in length TJ ring, which was quickly reversed within 5 min voiding....

Apical large-conductance Ca(2+)-activated K(+) (BK) channels in the cortical collecting duct (CCD) mediate flow-stimulated secretion. Dietary loading for 10-14 days leads to an increase BK channel mRNA abundance, enhanced secretion microperfused CCDs, and a redistribution of immunodetectable from intracellular pool apical membrane (Najjar F, Zhou H, Morimoto T, Bruns JB, Li HS, Liu W, Kleyman TR, Satlin LM. Am J Physiol Renal 289: F922-F932, 2005). To test whether this adaptation was...

Lowe syndrome is an X-linked disorder characterized by cataracts at birth, mental retardation and progressive renal malfunction that results from loss of function the OCRL1 (oculocerebrorenal Lowe) protein. a lipid phosphatase converts phosphatidylinositol 4,5-bisphosphate to 4-phosphate. The pathogenesis patients has been suggested result alterations in membrane trafficking, but this cannot fully explain disease progression. We found knockdown zebrafish caused developmental defects...

The epithelial sodium channel (ENaC) is activated by a unique mechanism, whereby inhibitory tracts are released proteolytic cleavage within the extracellular loops of two its three homologous subunits. While furin biosynthetic pathway releases one tract from α-subunit and moderately activates channel, full activation through release second γ-subunit requires once then at distal site protease, such as prostasin, plasmin, or elastase. We now report that coexpression mouse transmembrane...

During maturation, the α- and γ-subunits of epithelial Na+ channel (ENaC) undergo proteolytic processing by furin. Cleavage γ-subunit furin at consensus site γRKRR143 subsequent cleavage a second protease distal strongly activate channel. For example, coexpression prostasin with ENaC increases both function γRKRK186 site. We generated polyclonal antibody that recognizes region 144-186 in (anti-γ43) to determine whether promotes release intervening tract between putative sites. Anti-γ43...