Minireview: Regulation of Epithelial Na+ Channel Trafficking

The epithelial Na+ channel (ENaC) is a pathway for Na+ transport across epithelia, including the kidney collecting duct, lung, and distal colon. ENaC is critical for Na+ homeostasis and blood pressure control; defects in ENaC function and regulation are responsible for inherited forms of hypertension and hypotension and may contribute to the pathogenesis of cystic fibrosis and other lung diseases. An emerging theme is that epithelial Na+ transport is regulated in large part through trafficking mechanisms that control ENaC expression at the cell surface. ENaC trafficking is regulated at multiple steps. Delivery of channels to the cell surface is regulated by aldosterone (and corticosteroids) and vasopressin, which increase ENaC synthesis and exocytosis, respectively. Conversely, endocytosis and degradation is controlled by a sequence located in the C terminus of α, β, and γENaC (PPPXYXXL). This sequence functions as an endocytosis motif and as a binding site for Nedd4-2, an E3 ubiquitin protein ligase that targets ENaC for degradation. Mutations that delete or disrupt this motif cause accumulation of channels at the cell surface, resulting in Liddle’s syndrome, an inherited form of hypertension. Nedd4-2 is a central convergence point for ENaC regulation by aldosterone and vasopressin; both induce phosphorylation of a common set of three Nedd4-2 residues, which blocks Nedd4-2 binding to ENaC. Thus, aldosterone and vasopressin regulate epithelial Na+ transport in part by altering ENaC trafficking to and from the cell surface.

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Oxygen regulation of the epithelial Na channel in the collecting duct

AJP Renal Physiology ◽

10.1152/ajprenal.00245.2010 ◽

2011 ◽

Vol 300 (2) ◽

pp. F412-F424 ◽

Cited By ~ 6

Author(s):

Russell F. Husted ◽

Hongyan Lu ◽

Rita D. Sigmund ◽

John B. Stokes

Keyword(s):

Kinase Activity ◽

Collecting Duct ◽

Hypoxia Inducible Factor ◽

Na Channel ◽

Mrna Levels ◽

Apical Surface ◽

Amp Kinase ◽

Na Transport ◽

Protein Levels ◽

Epithelial Na Channel

The Po2 within the kidney changes dramatically from cortex to medulla. The present experiments examined the effect of changing Po2 on epithelial Na channel (ENaC)-mediated Na transport in the collecting duct using the mpkCCD-c14 cell line. Decreasing ambient O2 concentration from 20 to 8% decreased ENaC activity by 40%; increasing O2 content to 40% increased ENaC activity by 50%. The O2 effect required several hours to develop and was not mimicked by the acid pH that developed in monolayers incubated in low-O2 medium. Corticosteroids increased ENaC activity at each O2 concentration; there was no interaction. The pathways for O2 and steroid regulation of ENaC are different since O2 did not substantially affect Sgk1, α-ENaC, Gilz, or Usp2–45 mRNA levels, genes involved in steroid-mediated ENaC regulation. The regulation of ENaC activity by these levels of O2 appears not to be mediated by changes in hypoxia-inducible factor-1α or -2α activity or a change in AMP kinase activity. Changes in O2 concentration had minimal effect on α- or γ-ENaC mRNA and protein levels; there were moderate effects on β-ENaC levels. However, 40% O2 induced substantially greater total β- and γ-ENaC on the apical surface compared with 8% O2; both subunits demonstrated a greater increase in the mature forms. The α-ENaC subunit was difficult to detect on the apical surface, perhaps because our antibodies do not recognize the major mature form. These results identify a mechanism of ENaC regulation that may be important in different regions of the kidney and in responses to changes in dietary NaCl.

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cAMP and Serum and Glucocorticoid-inducible Kinase (SGK) Regulate the Epithelial Na+Channel through Convergent Phosphorylation of Nedd4-2

Journal of Biological Chemistry ◽

10.1074/jbc.m407858200 ◽

2004 ◽

Vol 279 (44) ◽

pp. 45753-45758 ◽

Cited By ~ 164

Author(s):

Peter M. Snyder ◽

Diane R. Olson ◽

Rajesh Kabra ◽

Ruifeng Zhou ◽

Jennifer C. Steines

Keyword(s):

Pressure Control ◽

Dominant Negative ◽

Na Channel ◽

Dependent Protein Kinase ◽

Ubiquitin Protein Ligase ◽

Convergence Point ◽

Dependent Protein ◽

Epithelial Na Channel ◽

Downstream Mediator

The epithelial Na+channel (ENaC) functions as a pathway for epithelial Na+transport, contributing to Na+homeostasis and blood pressure control. Vasopressin increases ENaC expression at the cell surface through a pathway that includes cAMP and cAMP-dependent protein kinase (PKA), but the mechanisms that link PKA to ENaC are unknown. Here we found that cAMP regulates Na+transport in part by inhibiting the function of Nedd4-2, an E3 ubiquitin-protein ligase that targets ENaC for degradation. Consistent with this model, we found that cAMP inhibited Nedd4-2 by decreasing its binding to ENaC. Moreover, decreased Nedd4-2 expression (RNA interference) or overexpression of a dominant negative Nedd4-2 construct disrupted ENaC regulation by cAMP. Nedd4-2 was a substrate for phosphorylation by PKAin vitroand in cells; three Nedd4-2 residues were phosphorylated by PKA and were required for cAMP to inhibit Nedd4-2 (relative functional importance Ser-327 > Ser-221 > Thr-246). Previous work found that these residues are also phosphorylated by serum and glucocorticoid-inducible kinase (SGK), a downstream mediator by which aldosterone regulates epithelial Na+transport. Consistent with a functional interaction between these pathways, overexpression of SGK blunted ENaC stimulation by cAMP, whereas inhibition of SGK increased stimulation. Conversely, cAMP agonists decreased ENaC stimulation by SGK. The data suggest that cAMP regulates ENaC in part by phosphorylation and inhibition of Nedd4-2. Moreover, Nedd4-2 is a central convergence point for kinase regulation of Na+transport.

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H89, an inhibitor of protein kinase A (PKA), stimulates Na+ transport by translocating an epithelial Na+ channel (ENaC) in fetal rat alveolar type II epithelium

Biochemical Pharmacology ◽

10.1016/s0006-2952(03)00456-8 ◽

2003 ◽

Vol 66 (6) ◽

pp. 1083-1089 ◽

Cited By ~ 22

Author(s):

Yoshinori Marunaka ◽

Naomi Niisato

Keyword(s):

Protein Kinase ◽

Protein Kinase A ◽

Na Channel ◽

Alveolar Type ◽

Type Ii ◽

Na Transport ◽

Fetal Rat ◽

Epithelial Na Channel ◽

Kinase A

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EGF and PGE2 inhibit rabbit CCD Na+ transport by different mechanisms: PGE2 inhibits Na(+)-K+ pump

AJP Renal Physiology ◽

10.1152/ajprenal.1993.264.4.f670 ◽

1993 ◽

Vol 264 (4) ◽

pp. F670-F677 ◽

Cited By ~ 8

Author(s):

D. H. Warden ◽

J. B. Stokes

Keyword(s):

Apical Membrane ◽

Collecting Duct ◽

Basolateral Membrane ◽

Membrane Conductance ◽

Membrane Voltage ◽

Na Channel ◽

Cortical Collecting Duct ◽

Na Transport ◽

Flux Measurements ◽

Epidermal Growth

The rabbit cortical collecting duct absorbs Na+ by a transport system comprised of an apical membrane Na+ channel and a basolateral membrane Na(+)-K(+)-adenosinetriphosphatase. The rate of Na+ absorption across this epithelium is acutely inhibited by several hormones and autacoids including epidermal growth factor (EGF) and prostaglandin E2 (PGE2). We used electrophysiological analysis to determine which Na+ transport mechanism is primarily regulated in response to EGF and PGE2. We used concentrations of EGF and PGE2 that inhibited Na+ absorption to a comparable degree. We assessed the effects of these agents on Na+ transport primarily by the calculated equivalent current; the validity of this indicator was verified using simultaneous tracer flux measurements. EGF and PGE2 had different effects on the intracellular electrophysiological parameters. EGF (in the presence of a cyclooxygenase inhibitor) hyperpolarized the apical membrane voltage in a manner analogous to the Na(+)-channel blocker amiloride, reduced the transepithelial conductance, and increased the fractional resistance of the apical membrane. In comparison, PGE2 depolarized the apical membrane voltage in a manner analogous to the Na(+)-K+ pump inhibitor ouabain, and caused no significant changes in transepithelial conductance or apical membrane conductance. The finding that EGF hyperpolarized the apical membrane indicates that this agent attenuates Na+ absorption by reducing apical Na+ entry due to a decrease in the magnitude of the apical membrane Na+ conductance. In contrast, the electrophysiological changes produced by PGE2 indicate primary inhibition of the basolateral Na(+)-K+ pump following PGE2 treatment.

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Regulation of maturation and processing of ENaC subunits in the rat kidney

AJP Renal Physiology ◽

10.1152/ajprenal.00422.2005 ◽

2006 ◽

Vol 291 (3) ◽

pp. F683-F693 ◽

Cited By ~ 102

Author(s):

Zuhal Ergonul ◽

Gustavo Frindt ◽

Lawrence G. Palmer

Keyword(s):

Collecting Duct ◽

Rat Kidney ◽

Na Channel ◽

Cortical Collecting Duct ◽

Α Subunit ◽

Salt Restriction ◽

High K ◽

Electrophysiological Measurements ◽

Immature Form ◽

Epithelial Na Channel

Antibodies directed against subunits of the epithelial Na channel (ENaC) were used together with electrophysiological measurements in the cortical collecting duct to investigate the processing of the proteins in rat kidney with changes in Na or K intake. When animals were maintained on a low-Na diet for 7–9 days, the abundance of two forms of the α-subunit, with apparent masses of 85 and 30 kDa, increased. Salt restriction also increased the abundance of the β-subunit and produced an endoglycosidase H (Endo H)-resistant pool of this subunit. The abundance of the 90-kDa form of the γ-subunit decreased, whereas that of a 70-kDa form increased and this peptide also exhibited Endo H-resistant glycosylation. These changes in α- and γ-subunits were correlated with increases in Na conductance elicited by a 4-h infusion with aldosterone. Changes in all three subunits were correlated with decreases in Na conductance when Na-deprived animals drank saline for 5 h. We conclude that ENaC subunits are mainly in an immature form in salt-replete rats. With Na depletion, the subunits mature in a process that involves proteolytic cleavage and further glycosylation. Similar changes occurred in α- and γ- but not β-subunits when animals were treated with exogenous aldosterone, and in β- and γ- but not α-subunits when animals were fed a high-K diet. Changes in the processing and maturation of the channels occur rapidly enough to be involved in the daily regulation of ENaC activity and Na reabsorption by the kidney.

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Intrarenal Ghrelin Receptor Antagonism Prevents High-Fat Diet-Induced Hypertension in Male Rats

Endocrinology ◽

10.1210/en.2013-2177 ◽

2014 ◽

Vol 155 (7) ◽

pp. 2658-2666 ◽

Cited By ~ 6

Author(s):

Brandon A. Kemp ◽

Nancy L. Howell ◽

John J. Gildea ◽

Shetal H. Padia

Keyword(s):

Growth Hormone ◽

High Fat Diet ◽

Collecting Duct ◽

Male Rats ◽

Na Channel ◽

Standard Diet ◽

High Fat ◽

Ghrelin Receptor ◽

Induced Hypertension ◽

Epithelial Na Channel

Excess weight gain contributes up to 65% of the risk of primary hypertension, and the increase in blood pressure in response to high-fat diet (HFD) is preceded by significant increases in renal tubular sodium (Na+) reabsorption. In normal rats, intrarenal ghrelin infusion increases distal nephron-dependent Na+ reabsorption via activation of the intrarenal ghrelin receptor (GHSR). This study focusses on the role of intrarenal GHSR-mediated Na+ reabsorption in HFD-induced hypertension. Dahl salt-sensitive rats received standard diet or HFD for 6 weeks. Rats underwent uninephrectomy and osmotic minipump implantation for chronic intrarenal delivery of vehicle (0.25 μL/h × 28 d), selective GHSR antagonist [D-Lys-3]-growth hormone releasing peptide-6 (0.2μM/d), or GHSR inverse agonist [D-Arg1, D-Phe5, D-Trp7,9, Leu11]-substance P (SUB-P) (3.6μM/d). HFD rats with vehicle pumps had significantly increased renal GHSR expression compared with standard diet (0.092 ± 0.005 vs 0.065 ± 0.004 arbitrary units; P < .05), whereas acyl ghrelin levels were similar (16.3±6.2 vs 15.7±8.7 pg/g tissue). HFD rats with vehicle pumps became hypertensive after 2 weeks (P < .05) and showed a significant reduction in 24-hour urine Na+ before hypertension. At this time, these rats showed an increase in collecting duct α-epithelial Na+ channel, thereby providing a potential mechanism for the excess Na+ reabsorption. In contrast, HFD rats with [D-Lys-3]-growth hormone releasing peptide-6 or SUB-P pumps never became hypertensive and did not show the reduction in urine Na+. Because SUB-P blocks the constitutive, but not ghrelin-dependent, activity of the GHSR, and HFD-induced α-epithelial Na+ channel up-regulation was abolished during GHSR antagonism, these data suggest that HFD increases the constitutive activity of renal GHSR to increase Na+ reabsorption and induce hypertension in rats.

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Surface Expression of Epithelial Na Channel Protein in Rat Kidney

The Journal of General Physiology ◽

10.1085/jgp.200809989 ◽

2008 ◽

Vol 131 (6) ◽

pp. 617-627 ◽

Cited By ~ 76

Author(s):

Gustavo Frindt ◽

Zuhal Ergonul ◽

Lawrence G. Palmer

Keyword(s):

Collecting Duct ◽

Rat Kidney ◽

Surface Expression ◽

Na Channel ◽

Cortical Collecting Duct ◽

Renal Cells ◽

Patch Clamp Recording ◽

Membrane Fractionation ◽

Whole Cell Patch Clamp ◽

Epithelial Na Channel

Expression of epithelial Na channel (ENaC) protein in the apical membrane of rat kidney tubules was assessed by biotinylation of the extracellular surfaces of renal cells and by membrane fractionation. Rat kidneys were perfused in situ with solutions containing NHS-biotin, a cell-impermeant biotin derivative that attaches covalently to free amino groups on lysines. Membranes were solubilized and labeled proteins were isolated using neutravidin beads, and surface β and γENaC subunits were assayed by immunoblot. Surface αENaC was assessed by membrane fractionation. Most of the γENaC at the surface was smaller in molecular mass than the full-length subunit, consistent with cleavage of this subunit in the extracellular moiety close to the first transmembrane domains. Insensitivity of the channels to trypsin, measured in principal cells of the cortical collecting duct by whole-cell patch-clamp recording, corroborated this finding. ENaC subunits could be detected at the surface under all physiological conditions. However increasing the levels of aldosterone in the animals by feeding a low-Na diet or infusing them directly with hormone via osmotic minipumps for 1 wk before surface labeling increased the expression of the subunits at the surface by two- to fivefold. Salt repletion of Na-deprived animals for 5 h decreased surface expression. Changes in the surface density of ENaC subunits contribute significantly to the regulation of Na transport in renal cells by mineralocorticoid hormone, but do not fully account for increased channel activity.

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