Marcelo D. Carattino

Epithelial Na+ channels are fully activated by furin- and prostasin-dependent release of an inhibitory peptide from the gamma-subunit.

Epithelial sodium channels (ENaC) are expressed in the apical membrane of high resistance Na+ transporting epithelia and have a key role in regulating extracellular fluid volume and the volume of airway surface liquids. Maturation and activation of ENaC subunits involves furin-dependent cleavage of the ectodomain at two sites in the α subunit and at a single site within the γ subunit. We now report that the serine protease prostasin further activates ENaC by inducing cleavage of the γ subunit at a site distal to the furin cleavage site. Dual cleavage of the γ subunit is predicted to release a 43-amino acid peptide. Channels with a γ subunit lacking this 43-residue tract have increased activity due to a high open probability. A synthetic peptide corresponding to the fragment cleaved from the γ subunit is a reversible inhibitor of endogenous ENaCs in mouse cortical-collecting duct cells and in primary cultures of human airway epithelial cells. Our results suggest that multiple proteases cleave ENaC γ subunits to fully activate the channel.

Maturation of the Epithelial Na+ Channel Involves Proteolytic Processing of the α- and γ-Subunits

The epithelial Na+ channel (ENaC) is a tetramer of two α-, one β-, and one γ-subunit, but little is known about its assembly and processing. Because co-expression of mouse ENaC subunits with three different carboxyl-terminal epitope tags produced an amiloride-sensitive sodium current in oocytes, these tagged subunits were expressed in both Chinese hamster ovary or Madin-Darby canine kidney type 1 epithelial cells for further study. When expressed alone α-(95 kDa), β-(96 kDa), and γ-subunits (93 kDa) each produced a single band on SDS gels by immunoblotting. However, co-expression of αβγENaC subunits revealed a second band for each subunit (65 kDa for α, 110 kDa for β, and 75 kDa for γ) that exhibited N-glycans that had been processed to complex type based on sensitivity to treatment with neuraminidase, resistance to cleavage by endoglycosidase H, and GalNAc-independent labeling with [3H]Gal in glycosylation-defective Chinese hamster ovary cells (ldlD). The smaller size of the processed α- and γ-subunits is also consistent with proteolytic cleavage. By using α- and γ-subunits with epitope tags at both the amino and carboxyl termini, proteolytic processing of the α- and γ-subunits was confirmed by isolation of an additional epitope-tagged fragment from the amino terminus (30 kDa for α and 18 kDa for γ) consistent with cleavage within the extracellular loop. The fragments remain stably associated with the channel as shown by immunoblotting of co-immunoprecipitates, suggesting that proteolytic cleavage represents maturation rather than degradation of the channel.

ENaC at the Cutting Edge: Regulation of Epithelial Sodium Channels by Proteases

Epithelial Na+ channels facilitate the transport of Na+ across high resistance epithelia. Proteolytic cleavage has an important role in regulating the activity of these channels by increasing their open probability. Specific proteases have been shown to activate epithelial Na+ channels by cleaving channel subunits at defined sites within their extracellular domains. This minireview addresses the mechanisms by which proteases activate this channel and the question of why proteolysis has evolved as a mechanism of channel activation.

The epithelial Na+ channel is inhibited by a peptide derived from proteolytic processing of its alpha subunit.

Epithelial sodium channels (ENaCs) mediate Na+ entry across the apical membrane of high resistance epithelia that line the distal nephron, airway and alveoli, and distal colon. These channels are composed of three homologous subunits, termed α, β, and γ, which have intracellular amino and carboxyl termini and two membrane-spanning domains connected by large extracellular loops. Maturation of ENaC subunits involves furin-dependent cleavage of the extracellular loops at two sites within the α subunit and at a single site within the γ subunit. The α subunits must be cleaved twice, immediately following Arg-205 and Arg-231, in order for channels to be fully active. Channels lacking α subunit cleavage are inactive with a very low open probability. In contrast, channels lacking both α subunit cleavage and the tract αAsp-206-Arg-231 are active when expressed in oocytes, suggesting that αAsp-206-Arg-231 functions as an inhibitor that stabilizes the channel in the closed conformation. A synthetic 26-mer peptide (α-26), corresponding to αAsp-206-Arg-231, reversibly inhibits wild-type mouse ENaCs expressed in Xenopus oocytes, as well as endogenous Na+ channels expressed in either a mouse collecting duct cell line or primary cultures of human airway epithelial cells. The IC50 for amiloride block of ENaC was not affected by the presence of α-26, indicating that α-26 does not bind to or interact with the amiloride binding site. Substitution of Arg residues within α-26 with Glu, or substitution of Pro residues with Ala, significantly reduced the efficacy of α-26. The peptide inhibits ENaC by reducing channel open probability. Our results suggest that proteolysis of the α subunit activates ENaC by disassociating an inhibitory domain (αAsp-206-Arg-231) from its effector site within the channel complex.

Proteolytic processing of the epithelial sodium channel gamma subunit has a dominant role in channel activation.

Maturation of the epithelial sodium channel (ENaC) involves furin-dependent cleavage at two extracellular sites within the α subunit and at a single extracellular site within the γ subunit. Channels lacking furin processing of the α subunit have very low activity. We recently identified a prostasin-dependent cleavage site (RKRK186) in the γ subunit. We also demonstrated that the tract α D206-R231, between the two furin cleavage sites in the α subunit, as well as the tract γ E144-K186, between the furin and prostasin cleavage sites in the γ subunit, are inhibitory domains. ENaC cleavage by furin, and subsequently by prostasin, leads to a stepwise increase in the open probability of the channel as a result of release of the α and γ subunit inhibitory tracts, respectively. We examined whether release of either theα orγ inhibitory tract has a dominant role in activating the channel. Co-expression of prostasin and either wild type channels or mutant channels lacking furin cleavage of the α subunit (αR205A,R208A,R231Aβγ) in Xenopus laevis oocytes led to increases in whole cell currents to similar levels. In an analogous manner and independent of the proteolytic processing of theα subunit, amiloride-sensitive currents in oocytes expressing channels carrying γ subunits with both a mutation in the furin cleavage site and a deletion of the inhibitory tract (αβγR143A,ΔE144-K186 and αR205A,R208A,R231AβγR143A, ΔE144-K186) were significantly higher than those from oocytes expressing wild type ENaC. When channels lacked the α and γ subunit inhibitory tracts, α subunit cleavage was required for channels to be fully active. Channels lacking both furin cleavage and the inhibitory tract in theγ subunit (αβγR143A,ΔE144-K186) showed a significant reduction in the efficacy of block by the syntheticα-26 inhibitory peptide representing the tract αD206-R231. Our data indicate that removal of the inhibitory tract from the γ subunit, in the absence ofα subunit cleavage, results in nearly full activation of the channel.

Role of proteolysis in the activation of epithelial sodium channels.

Purpose of reviewEpithelial sodium channels mediate Na+ transport across high resistance, Na+-transporting epithelia. This review describes recent findings that indicate that epithelial sodium channels are activated by the proteolytic release of inhibitory peptides from the α and γ subunits. Recent findingsNon-cleaved channels have a low intrinsic open probability that may reflect enhanced channel inhibition by external Na+ – a process referred to as Na+ self-inhibition. Cleavage at a minimum of two sites within the α or γ subunits is required to activate the channel, presumably by releasing inhibitory fragments. The extent of epithelial sodium channel proteolysis is dependent on channel residency time at the plasma membrane, as well as on the balance between levels of expression of proteases that activate epithelial sodium channels and inhibitors of these proteases. Regulated epithelial sodium channel proteolysis has been observed in rat kidney and in human airway epithelia. SummaryProteolysis of epithelial sodium channel subunits plays a key role in modulating epithelial sodium channel activity through changes in channel open probability.

Effect of aldosterone on BK channel expression in mammalian cortical collecting duct

Apical large-conductance Ca(2+)-activated K(+) (BK) channels in the cortical collecting duct (CCD) mediate flow-stimulated K(+) secretion. Dietary K(+) loading for 10-14 days leads to an increase in BK channel mRNA abundance, enhanced flow-stimulated K(+) secretion in microperfused CCDs, and a redistribution of immunodetectable channels from an intracellular pool to the apical membrane (Najjar F, Zhou H, Morimoto T, Bruns JB, Li HS, Liu W, Kleyman TR, Satlin LM. Am J Physiol Renal Physiol 289: F922-F932, 2005). To test whether this adaptation was mediated by a K(+)-induced increase in aldosterone, New Zealand White rabbits were fed a low-Na(+) (LS) or high-Na(+) (HS) diet for 7-10 days to alter circulating levels of aldosterone but not serum K(+) concentration. Single CCDs were isolated for quantitation of BK channel subunit (total, alpha-splice variants, beta-isoforms) mRNA abundance by real-time PCR and measurement of net transepithelial Na(+) (J(Na)) and K(+) (J(K)) transport by microperfusion; kidneys were processed for immunolocalization of BK alpha-subunit by immunofluorescence microscopy. At the time of death, LS rabbits excreted no urinary Na(+) and had higher circulating levels of aldosterone than HS animals. The relative abundance of BK alpha-, beta(2)-, and beta(4)-subunit mRNA and localization of immunodetectable alpha-subunit were similar in CCDs from LS and HS animals. In response to an increase in tubular flow rate from approximately 1 to 5 nl.min(-1).mm(-1), the increase in J(Na) was greater in LS vs. HS rabbits, yet the flow-stimulated increase in J(K) was similar in both groups. These data suggest that aldosterone does not contribute to the regulation of BK channel expression/activity in response to dietary K(+) loading.

Defining an inhibitory domain in the gamma subunit of the epithelial sodium channel

Proteases activate the epithelial sodium channel (ENaC) by cleaving the large extracellular domains of the α- and γ-subunits and releasing peptides with inhibitory properties. Furin and prostasin activate mouse ENaC by cleaving the γ-subunit at sites flanking a 43 residue inhibitory tract (γE144-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 γ-subunit in channels resistant to cleavage by furin and prostasin. We found that partial or complete deletion of a short segment in the γ-subunit, R158-N171, enhanced channel activity. Synthetic peptides overlapping this segment in the γ-subunit further identified a key 11-mer 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 γ inhibitory tract in ENaC weakened the intrinsic binding constant of the 11-mer peptide inhibitor, suggesting that the γ inhibitory tract and the 11-mer peptide interact at overlapping sites within the channel.

Shear stress-dependent regulation of apical endocytosis in renal proximal tubule cells mediated by primary cilia

Significance The proximal tubule (PT) of the kidney is the primary site for reabsorption of ions, solutes, and filtered low molecular weight proteins. PT cells rapidly modulate ion transport capacity in response to the fluid shear stress (FSS) that accompanies changes in glomerular filtration rate. We report here that PT cells also adjust their capacity for endocytosis in response to FSS. Apical endocytosis of the megalin–cubilin ligand albumin and of fluid phase markers is markedly increased upon exposure to FSS. Moreover, Ca2+ signaling mediated by the primary cilia on PT cells is required for this response. These studies define a novel pathway in PT cells that plays an essential role in maintaining kidney function. The kidney has an extraordinary ability to maintain stable fractional solute and fluid reabsorption over a wide range of glomerular filtration rates (GFRs). Internalization of filtered low molecular weight proteins, vitamins, hormones, and other small molecules is mediated by the proximal tubule (PT) multiligand receptors megalin and cubilin. Changes in GFR and the accompanying fluid shear stress (FSS) modulate acute changes in PT ion transport thought to be mediated by microvillar bending. We found that FSS also affects apical endocytosis in PT cells. Exposure of immortalized PT cell lines to physiologically relevant levels of FSS led to dramatically increased internalization of the megalin–cubilin ligand albumin as well as the fluid phase marker dextran. FSS-stimulated apical endocytosis was initiated between 15 and 30 min postinduction of FSS, occurred via a clathrin- and dynamin-dependent pathway, and was rapidly reversed upon removing the FSS. Exposure to FSS also caused a rapid elevation in intracellular Ca2+ [Ca2+]i, which was not observed in deciliated cells, upon treatment with BAPTA-AM, or upon inclusion of apyrase in the perfusion medium. Strikingly, deciliation, BAPTA-AM, and apyrase also blocked the flow-dependent increase in endocytosis. Moreover, addition of ATP bypassed the need for FSS in enhancing endocytic capacity. Our studies suggest that increased [Ca2+]i and purinergic signaling in response to FSS-dependent ciliary bending triggers a rapid and reversible increase in apical endocytosis that contributes to the efficient retrieval of filtered proteins in the PT.

Novel determinants of epithelial sodium channel gating within extracellular thumb domains

Activity of the epithelial Na+ channel (ENaC) is modulated by Na+ self-inhibition, an allosteric down-regulation of channel open probability by extracellular Na+. We searched for determinants of Na+ self-inhibition by analyzing changes in this inhibitory response resulting from specific mutations within the extracellular domains of mouse ENaC subunits. Mutations at γMet438 altered the Na+ self-inhibition response in a substitution-specific manner. Fourteen substitutions (Ala, Arg, Asp, Cys, Gln, Glu, His, Ile, Phe, Pro, Ser, Thr, Tyr, and Val) significantly suppressed Na+ self-inhibition, whereas three mutations (Asn, Gly, and Leu) moderately enhanced the inhibition. Met to Lys mutation did not alter Na+ self-inhibition. Mutations at the homologous site in the α subunit (G481A, G481C, and G481M) dramatically increased the magnitude and speed of Na+ self-inhibition. Mutations at the homologous βAla422 resulted in minimal or no change in Na+ self-inhibition. Low, high, and intermediate open probabilities were observed in oocytes expressing αG481Mβγ, αβγM438V, and αG481M/βγM438V, respectively. This pair of residues map to theα5 helix in the extracellular thumb domain in the chicken acid sensing ion channel 1 structure. Both residues likely reside near the channel surface because both αG481Cβγ and αβγM438C channels were inhibited by an externally applied and membrane-impermeant sulfhydryl reagent. Our results demonstrate that αGly481 and γMet438 are functional determinants of Na+ self-inhibition and of ENaC gating and suggest that the thumb domain contributes to the channel gating machinery.