Ion channels and transporters play a critical role in ion and fluid homeostasis and thus in normal animal physiology and pathology. Tight regulation of these transmembrane proteins is therefore ...essential. In recent years, many studies have focused their attention on the role of the ubiquitin system in regulating ion channels and transporters, initialed by the discoveries of the role of this system in processing of Cystic Fibrosis Transmembrane Regulator (CFTR), and in regulating endocytosis of the epithelial Na
+
channel (ENaC) by the Nedd4 family of ubiquitin ligases (mainly Nedd4-2). In this review, we discuss the role of the ubiquitin system in ER Associated Degradation (ERAD) of ion channels, and in the regulation of endocytosis and lysosomal sorting of ion channels and transporters, focusing primarily in mammalian cells. We also briefly discuss the role of ubiquitin like molecules (such as SUMO) in such regulation, for which much less is known so far.
Department of Pharmacology and Toxicology, University of Lausanne, Lausanne, Switzerland; and Program in Cell Biology, The Hospital for Sick Children, and Department of Biochemistry, University of ...Toronto, Toronto, Ontario, Canada
Ubiquitylation of membrane proteins has gained considerable interest in recent years. It has been recognized as a signal that negatively regulates the cell surface expression of many plasma membrane proteins both in yeast and in mammalian cells. Moreover, it is also involved in endoplasmic reticulum-associated degradation of membrane proteins, and it acts as a sorting signal both in the secretory pathway and in endosomes, where it targets proteins into multivesicular bodies in the lumen of vacuoles/lysosomes. In this review we discuss the progress in understanding these processes, achieved during the past several years.
Key points
High dietary potassium (K+) intake dephosphorylates and inactivates the NaCl cotransporter (NCC) in the renal distal convoluted tubule (DCT).
Using several ex vivo models, we show that ...physiological changes in extracellular K+, similar to those occurring after a K+ rich diet, are sufficient to promote a very rapid dephosphorylation of NCC in native DCT cells.
Although the increase of NCC phosphorylation upon decreased extracellular K+ appears to depend on cellular Cl− fluxes, the rapid NCC dephosphorylation in response to increased extracellular K+ is not Cl−‐dependent.
The Cl−‐dependent pathway involves the SPAK/OSR1 kinases, whereas the Cl− independent pathway may include additional signalling cascades.
A high dietary potassium (K+) intake causes a rapid dephosphorylation, and hence inactivation, of the thiazide‐sensitive NaCl cotransporter (NCC) in the renal distal convoluted tubule (DCT). Based on experiments in heterologous expression systems, it was proposed that changes in extracellular K+ concentration (K+ex) modulate NCC phosphorylation via a Cl−‐dependent modulation of the with no lysine (K) kinases (WNK)‐STE20/SPS‐1‐44 related proline‐alanine‐rich protein kinase (SPAK)/oxidative stress‐related kinase (OSR1) kinase pathway. We used the isolated perfused mouse kidney technique and ex vivo preparations of mouse kidney slices to test the physiological relevance of this model on native DCT. We demonstrate that NCC phosphorylation inversely correlates with K+ex, with the most prominent effects occurring around physiological plasma K+. Cellular Cl− conductances and the kinases SPAK/OSR1 are involved in the phosphorylation of NCC under low K+ex. However, NCC dephosphorylation triggered by high K+ex is neither blocked by removing extracellular Cl−, nor by the Cl− channel blocker 4,4′‐diisothiocyano‐2,2′‐stilbenedisulphonic acid. The response to K+ex on a low extracellular chloride concentration is also independent of significant changes in SPAK/OSR1 phosphorylation. Thus, in the native DCT, K+ex directly and rapidly controls NCC phosphorylation by Cl−‐dependent and independent pathways that involve the kinases SPAK/OSR1 and a yet unidentified additional signalling mechanism.
Key points
High dietary potassium (K+) intake dephosphorylates and inactivates the NaCl cotransporter (NCC) in the renal distal convoluted tubule (DCT).
Using several ex vivo models, we show that physiological changes in extracellular K+, similar to those occurring after a K+ rich diet, are sufficient to promote a very rapid dephosphorylation of NCC in native DCT cells.
Although the increase of NCC phosphorylation upon decreased extracellular K+ appears to depend on cellular Cl− fluxes, the rapid NCC dephosphorylation in response to increased extracellular K+ is not Cl−‐dependent.
The Cl−‐dependent pathway involves the SPAK/OSR1 kinases, whereas the Cl− independent pathway may include additional signalling cascades.
Ubiquitylation is crucial for regulating numerous cellular functions. In the kidney, ubiquitylation regulates the epithelial Na(+) channel ENaC. The importance of this process is highlighted in ...Liddle's syndrome, where mutations interfere with ENaC ubiquitylation, resulting in constitutive Na(+) reabsorption and hypertension. There is emerging evidence that NCC, involved in hypertensive diseases, is also regulated by ubiquitylation. Here, we discuss the current knowledge and recent findings in this field.
MR (mineralocorticoid receptor) antagonists are recommended for patients with resistant hypertension even when circulating aldosterone levels are not high. Although aldosterone activates MR to ...increase epithelial sodium channel (ENaC) activity, glucocorticoids also activate MR but are metabolized by 11βHSD2 (11β-hydroxysteroid dehydrogenase type 2). 11βHSD2 is expressed at increasing levels from distal convoluted tubule (DCT) through collecting duct. Here, we hypothesized that MR maintains ENaC activity in the DCT2 and early connecting tubule in the absence of aldosterone.
We studied AS (aldosterone synthase)-deficient (AS
) mice, which were backcrossed onto the same C57BL6/J strain as kidney-specific MR knockout (KS-MR
) mice. KS-MR
mice were used to compare MR expression and ENaC localization and cleavage with AS
mice.
MR was highly expressed along DCT2 through the cortical collecting duct (CCD), whereas no 11βHSD2 expression was observed along DCT2. MR signal and apical ENaC localization were clearly reduced along both DCT2 and CCD in KS-MR
mice but were fully preserved along DCT2 and were partially reduced along CCD in AS
mice. Apical ENaC localization and ENaC currents were fully preserved along DCT2 in AS
mice and were not increased along CCD after low salt. AS
mice exhibited transient Na
wasting under low-salt diet, but administration of the MR antagonist eplerenone to AS
mice led to hyperkalemia and decreased body weight with higher Na
excretion, mimicking the phenotype of MR
mice.
Our results provide evidence that MR is activated in the absence of aldosterone along DCT2 and partially CCD, suggesting glucocorticoid binding to MR preserves sodium homeostasis along DCT2 in AS
mice.
The potassium channel Kir4.1 forms the Kir4.1/Kir5.1 heterotetramer in the basolateral membrane of the distal convoluted tubule (DCT) and plays an important role in the regulation of the ...thiazide-sensitive NaCl cotransporter (NCC). Kidney-specific deletion of the ubiquitin ligase Nedd4-2 increases expression of NCC, and coexpression of Nedd4-2 inhibits Kir4.1/Kir5.1
. Whether Nedd4-2 regulates NCC expression in part by regulating Kir4.1/Kir5.1 channel activity in the DCT is unknown.
We used electrophysiology studies, immunoblotting, immunostaining, and renal clearance to examine Kir4.1/Kir5.1 activity in the DCT and NCC expression/activity in wild-type mice and mice with kidney-specific knockout of Nedd4-2, Kir4.1, or both.
Deletion of Nedd4-2 increased the activity/expression of Kir4.1 in the DCT and also, hyperpolarized the DCT membrane. Expression of phosphorylated NCC/total NCC and thiazide-induced natriuresis were significantly increased in the Nedd4-2 knockout mice, but these mice were normokalemic. Double-knockout mice lacking both Kir4.1/Kir5.1 and Nedd4-2 in the kidney exhibited increased expression of the epithelial sodium channel
-subunit, largely abolished basolateral potassium ion conductance (to a degree similar to that of kidney-specific Kir4.1 knockout mice), and depolarization of the DCT membrane. Compared with wild-type mice, the double-knockout mice displayed inhibited expression of phosphorylated NCC and total NCC and had significantly blunted thiazide-induced natriuresis as well as renal potassium wasting and hypokalemia. However, NCC expression/activity was higher in the double-knockout mice than in Kir4.1 knockout mice.
Nedd4-2 regulates Kir4.1/Kir5.1 expression/activity in the DCT and modulates NCC expression by Kir4.1-dependent and Kir4.1-independent mechanisms. Basolateral Kir4.1/Kir5.1 activity in the DCT partially accounts for the stimulation of NCC activity/expression induced by deletion of Nedd4-2.
Nedd4-2 is a ubiquitin ligase previously demonstrated to regulate endocytosis and lysosomal degradation of the epithelial Na(+) channel (ENaC) and other ion channels and transporters. Recent studies ...using Nedd4-2 knockout mice specifically in kidney or lung epithelia has revealed a critical role for this E3 ubiquitin ligase in regulating salt and fluid transport in these tissues/organs and in maintaining homeostasis of body blood pressure. Interestingly, the primary targets for Nedd4-2 may differ in these two organs: in the lung Nedd4-2 targets ENaC, and loss of Nedd4-2 leads to excessive ENaC function and to cystic fibrosis - like lung disease, whereas in the kidney, Nedd4-2 targets the Na(+)/Cl(-) cotransporter (NCC) in addition to targeting ENaC. In accord, loss of Nedd4-2 in the distal nephron leads to increased NCC abundance and function. The aldosterone-responsive kinase, Sgk1, appears to be involved in the regulation of NCC by Nedd4-2 in the kidney, similar to its regulation of ENaC. Collectively, these new findings underscore the physiological importance of Nedd4-2 in regulating epithelial salt and fluid transport and balance.
The mechanisms through which aldosterone promotes apparently opposite effects like salt reabsorption and K(+) secretion remain poorly understood. The identification, localization, and physiological ...analysis of ion transport systems in distal nephron have revealed an intricate network of interactions between several players, revealing the complex mechanism behind the aldosterone paradox. We review the mechanisms involved in differential regulation of ion transport that allow the fine tuning of salt and K(+) balance.
Gain-of-function mutations in with no lysine (K) 1 (WNK1) and WNK4 genes are responsible for familial hyperkalemic hypertension (FHHt), a rare, inherited disorder characterized by arterial ...hypertension and hyperkalemia with metabolic acidosis. More recently, FHHt-causing mutations in the Kelch-like 3-Cullin 3 (KLHL3-CUL3) E3 ubiquitin ligase complex have shed light on the importance of WNK's cellular degradation on renal ion transport. Using full exome sequencing for a 4-generation family and then targeted sequencing in other suspected cases, we have identified new missense variants in the WNK1 gene clustering in the short conserved acidic motif known to interact with the KLHL3-CUL3 ubiquitin complex. Affected subjects had an early onset of a hyperkalemic hyperchloremic phenotype, but normal blood pressure values"Functional experiments in Xenopus laevis oocytes and HEK293T cells demonstrated that these mutations strongly decrease the ubiquitination of the kidney-specific isoform KS-WNK1 by the KLHL3-CUL3 complex rather than the long ubiquitous catalytically active L-WNK1 isoform. A corresponding CRISPR/Cas9 engineered mouse model recapitulated both the clinical and biological phenotypes. Renal investigations showed increased activation of the Ste20 proline alanine-rich kinase-Na+-Cl- cotransporter (SPAK-NCC) phosphorylation cascade, associated with impaired ROMK apical expression in the distal part of the renal tubule. Together, these new WNK1 genetic variants highlight the importance of the KS-WNK1 isoform abundance on potassium homeostasis.
Aldosterone stimulation of the mineralocorticoid receptor (MR) is involved in numerous physiological responses, including Na+ homeostasis, blood pressure control, and heart failure. Aldosterone ...binding to MR promotes different post‐translational modifications that regulate MR nuclear translocation, gene expression, and finally receptor degradation. Here, we show that aldosterone stimulates rapid phosphorylation of MR via ERK1/2 in a dose‐dependent manner (from 0.1 to 10 nM) in renal epithelial cells. This phosphorylation induces an increase of MR apparent molecular weight, with a maximal upward shift of 30 kDa. Strikingly, these modifications are critical for the regulation of the MR ubiquitylation state. Indeed, we find that MR is monoubiquitylated in its basal state, and this status is sustained by the tumor suppressor gene 101 (Tsg101). Phosphorylation leads to disruption of MR/Tsg101 association and monoubiquitin removal. These events prompt polyubiquitin‐dependent destabilization of MR and degradation. Preventing MR phosphorylation by ERK1/2 inhibition or mutation of target serines affects the sequential mechanisms of MR ubiquitylation and inhibits the aldosterone‐mediated degradation. Our data provide a novel model of negative feedback of aldosterone signaling, involving sequential phosphorylation, monoubiquitin removal and subsequent polyubiquitylation/degradation of MR.—Faresse, N., Vitagliano, J.‐J., Staub, O. Differential ubiquitylation of the mineralocorticoid receptor is regulated by phosphorylation. FASEB J. 26, 4373–4382 (2012). www.fasebj.org
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