Abstract
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.
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Introduction
Ion channels play a major role in regulation of the electric properties of virtually every cell. Because of their capability to allow the passage of more than 106 ions per second across a membrane, their activity and expression at the plasma membrane is tightly regulated by a plethora of different cellular mechanisms. In recent years it has become evident that the ubiquitin system plays a major role in this control [3]. The modification of target proteins with ubiquitin often (although not always) targets them for degradation by the proteasome or lysosomes [67, 177], and is now recognized as a critical mechanism for regulation of many cellular functions. Ubiquitin is a 76 amino acid long polypeptide, which becomes conjugated via an isopeptide bond on the ε amino groups of lysine. This is achieved by an enzymatic cascade, involving an E1 (ubiquitin-activating) enzyme, an E2 (ubiquitin-conjugating) enzyme, and an E3 ubiquitin-protein ligase. E3 enzymes provide the specificity to the cascade, as they recognize the relevant target proteins and promote the conjugation of ubiquitin. The process is reversible and involves the activity of deubiquitylating (DUB) enzymes. The two major E3 classes known are RING E3s that facilitate transfer of ubiquitin from an E2 to substrates, and Hect E3s, which directly bind ubiquitin and transfer it onto the substrate. In mammals, there are over 1000 E3 enzymes and over 100 DUBs. Different types of ubiquitin modifications can be generated: Ubiquitin can be conjugated as a mono-ubiquitin on a lysine residue. However, frequently poly-ubiquitin chains are generated that link ubiquitins via internal lysines present in ubiquitin. Indeed, it is now known that polyubiquitin chains are formed that can involve any of the lysines on ubiquitin (K6, K11, K27, K29, K33, K48, K63) [218, 223] and hence largely increase the amount and versatility of ubiquitylation. Moreover, a number of ubiquitin-like (UBL) proteins have been discovered that modify proteins. Similar, but not identical enzymatic cascades are involved in UBL's conjugation, with the final outcome often not resulting in protein degradation. In this review we summarize the role of the ubiquitin system and UBLs in the regulation of ion channels and transporters both during their processing and their endocytosis and sorting (Table 1).
Role of the ubiquitin system in regulating ion channels in ER/Golgi
The ERAD system
Proteins destined for the secretory pathway and plasma membrane proteins are inserted co-translationally into the endoplasmic reticulum (ER) membrane, representing approximately one third of all newly synthesized proteins [141]. In the ER, these proteins are subject to regulation by the quality control system, ensuring that proteins fold properly or multimeric complexes assemble correctly. Proteins that do not, are recognized, retrotranslocated into the cytosol, ubiquitylated and degraded by the proteasome, a process known as ER associated degradation (ERAD). As is evident, the nature of the many client proteins is very diverse (soluble proteins, mono- or polytopic membrane proteins, protein complexes), and consequently the machinery is complex. Misfolded or unassembled proteins are recognized by chaperones [143], chaperone-like lectins [29], or protein disulfide isomerase homologues [201]. They assist with folding the client proteins and release them either for ER export or for ERAD (if folding/assembly fails). Proteins are then transferred to secondary quality control machinery, which includes the ubiquitin-protein ligases Hrd1 [107] and its homologue gp78 [35]. Hrd3 and SEL1L associate with Hrd1 and contribute to ERAD substrate processing [121, 140]. Derlin-1, -2 and -3, possibly with Sec61, have been suggested to form the retrotranslocation channel in the ER membrane, and the p97-Ufd1-Npl4 complex assists with “retrotranslocating” and delivering the proteins to the proteasome [121, 145, 160, 227, 228]. Ubiquitylation may take place during or after retrotranslocation. A number of additional ubiquitin-protein ligases can participate in ERAD, including Doa10 [196], RMA1 [139], CHIP (in combination with Hsp70) [137] or SCFfbs ligases [229].
ERAD of transmembrane proteins
The quality control of transmembrane proteins in the ER is a challenging task, as these proteins have three topologically distinct regions that have to be monitored to ensure that the folding of the protein is correct: extracellular, transmembrane and intracellular domains. Consequently, it has been proposed that there are three different types of ERAD mechanisms with respect to transmembrane proteins [33, 196, 206]. ERAD-C (cytoplasmic pathways) monitors and recognizes the cytoplasmic regions of the protein (as is the case for the Cystic Fibrosis Transmembrane Regulator (CFTR) channel, where the cytoplasmic Hsp70/CHIP act; see below), whereas the ERAD-L system detects luminal folding defects (with the help of Bip, calnexin/calyculin, OS-9, XTP3-B and others). Alternatively membrane proteins and especially multimeric transmembrane protein complexes may have folding defects in the membrane-spanning region, qualifying these as ERAD-M class of molecules. These pathways have been primarily characterized in yeast [33], where different ERAD complexes with different E3 ubiquitin-protein ligases play a role. It is currently believed that at least in yeast, ERAD-C substrates require Doa10, whereas ERAD-L and ERAD-M substrates require the Hrd1p ubiquitin-protein ligase. In mammalian cells it is likely that other ligases are involved as well. It is possible that depending on the nature of the protein several of these mechanisms are implicated in parallel, as seems to be the case for CFTR (see below). There is evidence for a large number of multimeric transmembrane complexes and channels whose assembly is controlled by ER and which involve ERAD. Examples include the T-cell receptor [19, 226], the nicotinic acetylcholine receptor [38, 207], GABAA receptors [71], voltage-gated Na+ channels [174], connexin 32 [205], NMDA receptors [104, 190], K+-channels [65, 132, 133, 179, 199, 224, 231], Ca2+-channels [209], Inositol 1,4,5-triphosphate receptors [211, 219] and ENaC [103, 202]. The mechanisms by which these proteins are recognized by ERAD vary widely, including intramembrane charges [19], C-terminal PDZ binding motifs [190, 199], or positive charges in the cytoplasmic domains [231]. It is becoming clear that these events can be regulated. This is the case for KCNK3 channels, in which 14-3-3 binding to phosphorylation sites can overcome the ER retention by dibasic signals [144], the IP3 receptors which become degraded upon stimulation, an event that involves the ERAD protein SPFH2 [154], or Kv1.2 K-channel in which phosphorylation plays a role in ER exit [224]. Here, we discuss two selected examples, namely the quality control and ERAD of CFTR, which is one of the best characterized transmembrane ERAD substrate, and ENaC, which is an example of a multimeric transmembrane protein assembled in the ER (Fig. 1).
CFTR as a model for integral membrane proteins subjected to ERAD
The cystic fibrosis transmembrane regulator (CFTR) is the gene product responsible for cystic fibrosis (CF) when mutated [167]. The large interest into ERAD of this multi-membrane spanning protein is due to a mutant CFTR (CFTRΔF508), which is present approximately in 90% of all Caucasian CF patients, and which represents a folding mutant that is retained in the ER and degraded by ERAD [109]. Indeed, it was one of the first ERAD substrates to be shown to be degraded in a ubiquitin-dependant fashion by the proteasome [93, 210]. CFTR is a member of the ATP binding cassette (ABC) transporter superfamily and contains 1,480 amino acids. It is composed of two membrane spanning domains (with six transmembrane (TM) each), two cytosolic nucleotide binding domains (NBD1 and NBD2), and a regulatory R region. The assembly of this protein into a functional ion channel is complex, as the different domains have to be folded in a coordinated fashion, whereby they are thought to interact in a co- and post-translational fashion. The complexity of this process is illustrated by the fact that only 25% of the wild-type channel are properly folded and exported to the cell surface, whereas the rest is degraded via ERAD; in the case of CFTRΔF508 the entire pool is degraded by ER [37]. As with other proteins chaperones (Hsp40, Hsp70, Hsp90, αA-crystallin, calnexin) [5, 233] contribute to the recognition of CFTR by ERAD, as do lectins [147]. ERAD involves the participation of various complexes, such as Derlin-1, either to retain CFTR in the ER membrane or to serve as retrotranslocon, and promoting the interaction with the ubiquitin-protein ligase RMA1 that acts in concert with the E2 enzyme Ubc6e [139]. It is believed that RMA1 is capable of recognizing folding defects in CFTR in a co-translational fashion, whereas CHIP, another E3 ligase, appears to interact together with Hsp70 in a post-translational manner with misfolded NBD2 [137]. Other ubiquitin-protein ligases are also important, such as gp78 [139], or SCFFbs [229]. As well, the extracting activity of p97/Ufd1/Npl4 is necessary for CFTR degradation [68].
ENaC, an example of a multimeric protein controlled by ERAD
The amiloride-sensitive epithelial Na+ channel (ENaC) is composed of three subunits, αβγ, and is expressed primarily in the apical membrane of salt-absorbing epithelia, such as those in the distal nephron, distal colon and lung [47], where it participates it salt and fluid absorption (see below). It is well established that efficient functional expression of this channel at the cell surface requires all three subunits [28, 57, 87, 135, 136]. αβ and αγ dimers are also able to generate small amiloride-sensitive currents, whereas βγ channels are rather weakly expressed at the cell surface [20]. Study of such “dimeric” channels is important for both physiological and pathophysiological reasons. ENaC is highly regulated by the steroid hormone aldosterone, which positively regulates this channel. At least in the kidney, this is achieved by regulating the expression level of αENaC; without aldosterone stimulation αENaC is expressed at only very low levels; under such conditions the β and γ subunits are retained in an as yet undefined compartment [126, 134]. Aldosterone stimulation leads to αENaC expression, assembly with the βγ subunits and translocation of the channel to the plasma membrane [126]. It is not understood how and where the βγ dimmers are retained, and how they are protected from degradation. Moreover, there is a pathological condition, pseudohypoaldosteronism type I, in which mutations in the genes encoding either α-, β- or γENaC lead to inactivation of these subunits, leaving the cell with only two subunits [34]. As for many other multimeric membrane complexes it is believed that the ENaC channel subunits assemble in the ER and are exported to the plasma membrane, and that at least part of the unassembled subunits are retained in the ER and eventually degraded by ERAD. It is not clear how subunits assemble but they do so early in biosynthesis, possibly prior to glycosylation. It was shown earlier that the N-terminus of γENaC plays a crucial role in channel assembly [4], that each subunit can interact with each other to form dimers, and that multiple ENaC domains participate in its assembly [25]. Processing/assembly of ENaC is inefficient, with less than 1% of synthesized subunits reaching the plasma membrane [202, 214], suggesting that the bulk of newly synthesized proteins are eliminated by ERAD. Little is known how unassembled ENaC chains are retained in the ER, and how they are targeted for ERAD. No ER retention signal has been identified, but a C-terminal exit signal was characterized recently [103]. Beside the fact that ENaC needs to assemble all three subunits in order to be efficiently targeted to the cell surface, the evidence (sometimes indirect) for ERAD of ENaC subunits is as follows: (1) Intracellular ENaC subunits are polyubiquitylated [13, 23, 54, 90, 130, 171, 192, 234], whereas ENaC at the cell surface appears to be either mono- or multi-mono-ubiquitylated [171, 216]. (2) Proteasome, but not lysosomal inhibition, interferes with turnover of individually expressed ENaC subunits in MDCK cells, whereas the turnover of ENaC subunits in cells expressing all three subunits is inhibited by both inhibitors. This implies that there are 2 populations of ENaC subunits in such cells, assembled ones (multi-mono-ubiquitylated) that traffic to the plasma membrane and later to the lysosomes, and the unassembled ones (polyubiquitylated) that are degraded by ERAD and the proteasome [192]. (3) It has been shown that proteasome inhibition can increase ENaC activity in A6 cells, most likely by inhibiting ERAD of ENaC subunits, and increasing the level of unassembled subunits, consequently increasing the chance of channel assembly in the ER [129]. Recently, some light has been shed on the machinery that appears to be involved in ERAD. First, it has been shown that Hsp70, a stress-induced protein, and Hsc70 (a constitutively expressed Hsp70 paralogue), both cytosolic chaperone involved in ER quality control, play an important role in ENaC regulation [69]. Interestingly, when co-expressed in Xenopus laevis oocytes, Hsp70 stimulates cell surface expression of ENaC, whereas Hsc70 decreases it. This difference is not very well understood, but it suggests that Hsc70 targets ENaC for ERAD, whereas Hsp70 may promote its folding. The same group also identified αA-crystallin as a protein involved in ERAD [103]. Similar to what was done for CFTR, αENaC was expressed in yeast cells, and its turnover tested in various mutants, including Hsp26 and Hsp42 mutants, in which the protein was stabilized. It was then shown that αA-crystallin (the Hsp26 orthologue) reduces ENaC activity and cell surface expression when co-expressed in Xenopus oocytes. Evidence was provided that it was the delivery pathway to the plasma membrane (i.e. secretory pathway) that was involved and not the endocytic pathway. Recently, the expression of ENaC subunits in yeast cells has led to additional characterization of the ERAD mechanisms involved in ENaC degradation [26]. It was shown that singly expressed ENaC subunits display several defects in different compartments (cytosolic, luminal and possibly membrane), as evidenced by the observation that both Doa10 (a ubiquitin-protein ligase important for cytosolic substrates), as well as Hrd1p (luminal and membrane regions) were required for turnover of the ENaC subunits. Moreover, it was shown that luminal Hsp40 proteins (Jem1 and Scj1), but not Bip (a luminal Hsp70 chaperone), were involved in turnover and ubiquitylation of ENaC. This was further supported in Xenopus oocytes, in which the Hsp40 orthologue ERdj3 and ERdj4 inhibited amiloride-sensitive currents [26]. However, it remains to be seen if these mechanisms are also valid in more physiological cells, such as renal epithelia.
Role of the ubiquitin system in endocytosis, sorting and recycling of ion channels and transporters
The role of ubiquitin in endocytosis and sorting of transmembrane (TM) proteins has received much attention over the past 15 years, initially from work in yeast [64, 78, 82] and in mammalian cells [114, 191]. Relevant to this review, our initial observation that ENaC binds the Nedd4/Nedd4-2 family of E3 ligases [191] opened the field to many subsequent studies that have focused on the role of ubiquitin system, and in particular the Nedd4 family proteins, in the regulation of cell surface stability and endocytosis of ion channels and transporters in mammals.
Regulation of ENaC by Nedd4-2
Perhaps the best-studied example of the regulation of ion transport by ubiquitin is that of ENaC regulation by Nedd4-2. As indicated above, ENaC regulates salt and fluid absorption in epithelia of several organs, including kidney, lung and colon. The critical role of ENaC in salt and fluid absorption in these tissues/organs is underscored by the findings that loss or gain of function mutations in this channel in humans causes pseudohypoaldosteronism Type I (PHAI) or pseudoaldosteronism (Liddle syndrome- see below), respectively, diseases that are also recapitulated in mouse models bearing the same mutations [89, 120]. In the kidney, ENaC function is up-regulated by the hormone aldosterone, which binds the mineralocorticoid receptor (MR), while in the lung it is up-regulated by glucocorticoids and their cognate receptor (GR) [125]. As indicated above, ENaC is comprised of 3 subunits, αβγ [28], with a short proline-rich sequence, called the PY motif (PPxYxxØ, minimal consensus = L/PPxY), present at the cytosolic C-terminus of each subunit [173, 187, 191]. The PY motif of β or γ ENaC is deleted or mutated in Liddle syndrome [74, 75, 120, 183], a hereditary hypertension caused by increased activity and number of ENaC channels at the plasma membrane [57]. These PY motifs serve as binding sites for Nedd4 proteins [191], in particular Nedd4-2 [2, 76, 99, 188]. Nedd4-2 (Nedd4L) possesses a C2 domain that can be spliced out [92], 4 WW domains that bind PY motifs [102, 191], and an E3 ligase Hect domain. The third WW domain, present in all Nedd4-2 but absent in most Nedd4-1 proteins (except human, zebrafish and Drosophila Nedd4-1), appears to provide higher affinity interactions to PY motifs [80, 101], but both WW3 and WW4 of Nedd4-2 are essential for interaction with and regulation of ENaC [60, 92, 100]. It is curious that human Nedd4-1, which also possesses 4 WW domains including WW3, can only inhibit ENaC function upon removal of its inhibitory C2 domain [100, 185], in line with a recent demonstration of inhibition of Hect catalytic activity by the C2 domain of the Nedd4 family relative Smurf2 [217] and Nedd4-1 and -2 [208], which in the latter can be relieved by elevated Ca2+ levels. However, it should be emphasized that the data with Nedd4-1 have been acquired in heterologous expression systems; to date there is no in vivo evidence that endogenous Nedd4-1 is involved in ENaC regulation.
Given the ability of Nedd4-2 to interact with the PY motifs of ENaC, which are deleted/mutated in Liddle syndrome, it was proposed [191] and later demonstrated [2, 99] that the increased retention of channels at the plasma membrane in this disease may be caused by impaired ubiquitylation and endocytosis of the mutant channels (Fig. 2a). Indeed, ENaC's cell surface stability was shown earlier to be negatively regulated by ubiquitylation on its α and γ subunits [192]. In support, recent studies have demonstrated that Nedd4-2 can ubiquitylate ENaC at the plasma (apical) membrane [127, 216, 234]. Moreover, the increased retention of the PY motif mutant ENaCs at the plasma membrane is primarily caused by impaired internalization of the mutant channels, with some defects also observed in channel sorting and cAMP-dependent mobilization of channels from a sub-apical pool [127]. The nature of ENaC ubiquitylation is not entirely clear, but several reports recently suggested that the channel is mono or multi-mono ubiquitylated at the cell at the cell surface [171, 216, 234], a finding that is in accord with the known role of mono or oligo-ubiquitylation in endocytosis and sorting of transmembrane proteins [81]. Another group proposed that native ENaC in A6 cells is regulated by polyubiquitylation and is degraded by the proteasome [129, 130] although it needs to be established whether the polyubiquitylated pool is the one at the plasma membrane. The type of ubiquitin linkage employed is not known, but one would expect to identify at least some K63-linked ubiquitylation of ENaC, a linkage often detected in proteins destined for endosomal sorting and lysosomal degradation, and was described in transmembrane proteins that are target for Rsp5 (the yeast Nedd4 orthologue)—mediated ubiquitylation and endocytosis/sorting [63, 111, 149]. In addition to regulating cell surface stability, the PY motifs (Nedd4-2 binding sites) of ENaC also regulate its response to intracellular Na+ concentrations, since feedback inhibition by Na+ exhibited by WT ENaC is inhibited in a PY motif mutant of the channel [45, 106, 151].
The clear prediction from these findings is that the increased retention of active ENaC at the plasma membrane would increase Na+ reabsorption in the kidney, leading to hypertension, as seen in Liddle syndrome patients. Indeed, knock-in mice bearing the Liddle syndrome mutation in βENaC exhibit salt-induced hypertension, metabolic alkalosis and hypokalemia, leading to renal and cardiac hypertrophy which resembles the human Liddle disease [159]. In accord, these Liddle mice exhibit enhanced alveolar fluid clearance following pulmonary edema [162]. Interestingly, recently generated knockout (KO) mice of Nedd4-2 exhibit a similar phenotype, with salt-induced hypertension (which was inhibited by amiloride) and cardiac hypertrophy, suggesting upregulation of ENaC. Indeed, ENaC protein expression in the kidney of these mice was elevated, and rectal PD measurements indicated excessive ENaC activity also in their colon [181]. While the potential effect of loss of Nedd4-2 on lung function of these KO mice was not described, a separate study using RNAi to knockdown Nedd4-2 in rat lungs immediately after birth, which led to ~60% reduction in level of Nedd4-2, has demonstrated increased ENaC expression along with reduced extravascular lung water following knockdown [116]. This suggests that Nedd4-2 suppresses ENaC function in the lung, similar to its function in the kidney and colon.
It has long been recognized that CFTR regulates ENaC function, in most cases leading to inhibition of ENaC activity by reducing its open probability [194]. In cystic fibrosis (CF), where CFTR is defective, Na+ and fluid absorption is elevated, leading to dehydration of airway surface fluid, mucus accumulation and repeated bacterial infections [22, 46]. Recent work by Mall, Boucher and co-workers provided further support for this model, by demonstrating that overexpression of ENaC (βENaC) in mouse airways leads to a CF-like phenotype, a process that can be ameliorated by treatment of the mice with amiloride [131, 235]. An important issue that needs to be addressed is, therefore, why do Liddle syndrome patients, exhibiting excessive ENaC activity, not develop CF-like disease?
The elevated blood pressure (BP) in the Nedd4-2 KO mice suggests that mutations or variation in the NEDD4-2 gene in humans may be involved in the regulation of blood pressure [168]. In support, a recent study suggested that a cryptic splice variant (generated by the SNP rs4149601) that generates Isoform I of Nedd4-2 (NEDD4-2 A allele, aka Nedd4-2C8 [48]) is associated with hypertension in a Chinese group of patients [128]. This splice isoform, which generates an inactive protein [48], was proposed to interact with other Nedd4-2 isoforms and block their function in a dominant negative fashion, thus leading to enhanced ENaC activity [8]. Another variant, encoding NEDD4-2-P355L, was shown to be less effective at suppressing ENaC than the native protein, likely due to its enhance phosphorylation [62] (see below).
Role of Nedd4-2 in hormone-mediated regulation of ENaC
As indicated above, aldosterone and vasopressin (ADH), as well as insulin, enhance ENaC function. In search for transcripts and proteins that exhibit early response to aldosterone and glucocorticoids, several groups identified serum and glucocorticoid- regulated kinase 1 (Sgk1), a relative of Akt (PKB) [212]; Sgk1 was shown to enhance ENaC function by stimulating an increase in ENaC abundance at the plasma membrane in response to aldosterone, including in renal collecting ducts [7, 36, 126, 142, 182]. Subsequent work, initially from Staub and co-workers, demonstrated that Sgk1 phosphorylates Nedd4-2 on Ser residues (S444, S338), a process that involves a PY motif present in Sgk1 and suggests a Nedd4-2:Sgk1 interaction (most likely of low affinity [80, 163]); This phosphorylation leads to interference with the ability of Nedd4-2 to interact with ENaC, resulting in increased abundance of channels at the plasma membrane, as demonstrated in a Xenopus oocyte model system, tissue culture cells and cortical collecting ducts [41, 58, 186] (Fig. 2b). Unlike Nedd4-2, Nedd4-1 does not possess Sgk phosphorylation sites, and is not known to be regulated by aldosterone. In addition to Sgk1, it appears that cAMP-activated PKA can also phosphorylate Nedd4-2, on the same sites as those used by Sgk1, leading to increased ENaC abundance due to its inability to interact with phosphorylated Nedd4-2 [184]. Since cAMP is generated and PKA activated in response to vasopressin release and its binding to its V2 receptor, these collective results suggest that Sgk1 and PKA serve as convergent points to phosphorylate Nedd4-2 and block its ability to suppress ENaC in response to both aldosterone and vasopressin (Fig. 2b), the two major hormones regulating fluid absorption, blood volume and BP.
In addition to the regulation of Sgk/PKA/Nedd4-2 by aldosterone and vasopressin, Insulin was recently shown to increase ENaC function by activating its downstream effectors Akt1 and Sgk1, thus feeding into the same Nedd4-2 pathway and connecting cellular metabolism and growth to ENaC [112]. Likewise, Il-1β—induced cortisol production was shown to stimulate fluid absorption (ENaC activation) via Sgk-mediated Nedd4-2 inhibition in fetal guinea pigs lungs [117]. Additionally, the WNK kinase was also shown to activate Sgk1, resulting in suppression of Nedd4-2 and elevated ENaC function [222]; mutations in WNK cause PHAII, a form of hypertension. Interestingly, another player, IkappaB kinase β (IKKB), was recently shown to phosphorylate Nedd4-2 on S444 (the same site as that phosphorylated by Sgk1), leading to enhanced accumulation and function of ENaC at the plasma membrane [49]. These latter results provide another arm to the regulation of ENaC by Nedd4-2, connecting it to inflammation and the immune system (Fig. 2b).
How does phosphorylation of Nedd4-2 by Sgk/PKA prevent it from interacting with ENaC? Recent studies identified 14-3-3 proteins as binding partners to phosphorylated Nedd4-2; 14-3-3 maintains phosphorylated Nedd4-2 in a “blocked” form that cannot bind ENaC [13, 90]. Recent work also suggested that a heterodimer of the 14-3-3β:14-3-3ε is required for this interaction [118] and that these 14-3-3 isoforms are themselves induced by aldosterone [119]. The exact mechanism of this “blockage” is still unknown, since the phosphorylation sites on Nedd4-2 are located outside the WW domains that bind the PY motifs of ENaC; it is possible that a 14-3-3 dimer binds to Nedd4-2 and alters its conformation, thus masking the binding sites on its WW domains. This, however, remains speculative until the structure of Nedd4-2 in complex with the PY motifs of ENaC, in the presence or absence of 14-3-3, is solved.
While the Sgk1/Nedd4-2 pathway can lead to enhanced ENaC function, it should be kept in mind that aldosterone, vasopressin and Sgk1 can also activate ENaC independently of Nedd4-2. Evidence for this is provided by the observations that (1) aldosterone and Sgk1 can increase cell surface levels of ENaC channels bearing the Liddle syndrome mutations (which cannot properly bind Nedd4-2) [7, 9, 43, 182], (2) Cortical collecting tubules harvested from mice bearing the Liddle syndrome mutation reveal normal response to aldosterone [39], (3) ENaC activity in the colon of these Liddle mice is enhanced by aldosterone [12], and (4) ENaC was shown to be inhibited by WNK4 kinase, a process inhibited by Sgk1, which phosphorylates WNK4 [165, 166].
Interestingly, recent reports provide a connection between ENaC and the metabolic state of the cell, by demonstrating that the metabolic sensor AMP kinase (AMPK) inhibits ENaC function in a PY motif-dependent manner, by enhancing its ability to interact with Nedd4-2, thus reducing cell surface stability of the channel [6, 14, 30] (Fig. 2c); the latter effect did not involve Sgk or PKA, and overall suggests that under metabolic stress, reducing ENaC function by activated AMPK, leading to enhanced channel endocytosis, would prevent cellular Na+ overload. Likewise, Erk (part of the MAPK pathway) downregulates ENaC by phosphorylating it on Thr residues in the C termini of β and γ ENaC, thus facilitating ENaC interaction with Nedd4/Nedd4-2 [138, 180, 225], although it is not clear yet how such phosphorylation would increase ENaC: Nedd4/Nedd4-2 binding given the hydrophobic nature of WW domain:PY motif interaction [101, 102].
Two knockout models of Sgk1 have been generated and the role of Sgk1 in ENaC regulation has been evaluated in these mice. Wulff et al. found that Sgk1 (−/−) mouse displayed a salt-losing phenotype only when mice were kept under low Na+ diet. Under such conditions, ENaC was also weakly expressed, as evidenced by potential difference (PD) measurement in isolated cortical collecting ducts and by immunohistochemistry [221]. Somewhat different data were obtained by Fejes-Toth et al. Although they also found a salt-losing phenotype under low Na+ diet, they observed by in situ patch clamping CCD cells that amiloride-sensitive Na+ currents were upregulated under these conditions, suggesting that other Na+ transporters must be upregulated, including the thiazide-sensitive Na+/Cl-cotransporter [55]. Hence the precise role of Sgk1 in vivo in the kidney remains to be further characterized, and the mechanism of aldosterone regulation continues to remain obscure.
De-ubiquitylating enzymes and ENaC
The critical role of the E3 ubiquitin ligase Nedd4-2 and ubiquitylation in the regulation of ENaC endocytosis and cell surface stability prompted a search for de-ubiquitylation enzymes (DUBs) that may participate in this regulation as well. Recently, a DUB induced by aldosterone, Usp-45, was identified by Verrey and colleagues [54]. Usp-45 deubiquitylates ENaC and stimulates ENaC cell surface stability and function; this effect is dependent on the presence of the PY motifs of ENaC and on its prior ubiquitylation by Nedd4-2, but is independent of Sgk1 [54]. Another DUB that was described to regulate ENaC is the endosomal DUB, UCH-L3 [27]. UCH-L3 increases cell surface expression of ENaC by de-ubiquitylating it, presumably in an endosomal compartment, and enhancing its recycling to the plasma membrane. Given the known role of cAMP in inducing ENaC recycling [127], it would be interesting to test whether UCH-L3 participates in this process. In addition, the DUB Usp10 was recently identified as a transcript/protein whose expression is elevated following vasopressin stimulation, an increase that leads to enhanced ENaC abundance and function at the plasma membrane [23]. Usp-10 utilizes an adapter protein, SNX3, to carry out its effect on ENaC, as detailed below.
Thus, specific DUBs appear to oppose the action of Nedd4-2 (directly, or indirectly via an adapter protein), leading to increased ENaC stabilization at the plasma membrane (Fig. 3), especially in response to hormones that are released into the blood stream to promote ENaC function and Na+/fluid absorption.
Relationship between regulation of ENaC ubiquitylation and its proteolytic cleavage
Proteolytic cleavage of the ectodomain of ENaC by serine proteases leads to activation of the channel by increasing its open probability (Po) [88, 169, 203]. In particular, the α and γ subunits are the targets for such cleavage: αENaC is cleaved by Furin at two site, releasing an inhibitory peptide/tract, whereas γENaC is cleaved by Furin at one site and by anther protease, such as prostasin/CAP1, elastase or plasmin, at a second site, to promote release of the inhibitory tract [24, 31, 32, 86, 153]. It appears that the inhibitory tract provide higher affinity for Na+ [178] and may mediate the well-known self-inhibition of ENaC by extracellular(luminal) Na+ [151]; thus, removal of the inhibitory tract by proteases promotes channel activation (increased Po) by decreasing sensitivity to luminal Na+ [178]. Curiously, while proteases increase ENaC Po, their effect is connected to ubiquitylation, Nedd4-2 and Usp-45. Work from Snyder and colleagues has demonstrated that Nedd4-2 facilitates rapid endocytosis of proteolytically cleaved ENaC, leading to increased degradation of the cleaved, active channel [97]. This endocytosis was dependent on the presence of the PY motifs of ENaC. In accord, mutations of these PY motifs (mimicking those in Liddle syndrome) or mutation of the ubiquitin acceptors sites in ENaC increased the fraction of cleaved, active channels at the plasma membrane [108]. In agreement with these observation, Staub and colleagues have recently demonstrated that Usp-45—mediated de-ubiquitylation of ENaC enhances its proteolytic cleavage (on α and γ ENaC), thus stimulating its activation [171]. These data thus suggest that ubiquitylation may regulate the conformation of the extracellular domain, and likely the accessibility of protease sites for cleavage.
The strong reliance of ENaC on Nedd4-2-mediated downregulation (channel ubiquitylation and endocytosis) has been a long-standing puzzle, since intuitively it would be much faster to close the channel rather than to inernalize it when its function is no longer needed; the recent findings that protease cleavage releases a segment of the channel (the inhibitory tract), leading to irreversible opening of the channel, helps explain this puzzle, since such constitutively active channels have to be removed from the plasma membrane to stop the influx of Na+.
Regulation of endocytosis of other ion channels by Nedd4 family ubiquitin ligases
Following the discovery of ENaC: Nedd4/Nedd4-2 interactions, several groups have identified PY motifs in numerous other channels, prompting investigations into their potential regulation by Nedd4 proteins, as described below.
Voltage-gated Na+channels
Voltage-gated Na+ channels, Navs, are responsible for generating action potential in excitable cells, primarily in cardiac and neuronal tissues. They are comprised of pore-forming α subunits assembled together with regulatory β subunits [230]. These channels play a critical role in cardiac and neuronal functions, and mutations in some of them cause human diseases [230]. For example, mutations in Nav1.5 (SCN5A) that impair its inactivation in the heart lead to long QT (type 3) and Brugada syndromes [105] and inactivation of the Scn5a gene in mice leads to cardiac function defects [152]. Likewise, gain of function mutations in Nav1.1 (SCN1A) causes generalized epilepsy with febrile seizures (GEFS+) by enhancing inward Na+ current in brain neurons [52]. Of the ten known Navs, seven (Nav 1.1–1.3 and Nav1.5–1.8) possess a conserved PY motif (PPSYxxØ) at the cytosolic C termini of their α subunits [61, 170]. Much like ENaC, cell surface stability of the cardiac Nav1.5 was demonstrated to be negatively regulated by Nedd4-2, a process that requires the PY motif of this channel, as well as active Nedd4-2, suggesting channel ubiquitylation regulates its endocytosis [1, 204]. Similar results were obtained for other PY-motif containing Navs, Nav1.2, 1.3, 1.7 and 1.8 [61, 170]; while Nedd4-2 was the predominant E3 ligase involved in suppressing these Navs, WWP2 and Nedd4-1 may contribute to the regulation as well. A recent search for NEDD4-2 variants in ~250 families with photosensitive generalized epilepsy (IGE) identified several such variants, although their expression in Xenopus oocytes did not affect their ability to interact with Nav1.2, nor alter channel function [44], suggesting that the physiological significance of these variants need to be further explored.
Recently it was shown that Nav1.6 is regulated by Nedd4-2 as well. Interestingly Nedd4-2 appears to interact with 2 motifs, one of which is a PGSP motif that needs to be phosphorylated by the p38 kinase in order to interact with Nedd4-2 [66].
Voltage-gated K+channels and Ca2+ channels
Voltage-gated K+ channels comprise several subfamilies, including the KCNQ (Kv7), KCNA (Kv1), KCNB (Kv2) and others. These channels play a key role in neuronal excitability, and mutations in several of them cause neurological and cardiac impairments. For example, loss of function mutations in KCNQ2 (Kv7.2) or KCNQ3 (Kv7.3) cause epilepsy [164], while such mutations in KCNQ1 (Kv7.1) or its associated subunit KCNE1 cause Long QT (Type I) syndrome (cardiac ventricular arrhythmia) [105].
KCNQ1 possesses a PY motif (LPxYxxØ) at its intracellular C terminus, which is conserved across species and serves as binding site for human Nedd4-2, Nedd4-1, or WWP2. Accordingly, the current produced by the KCNQ1/KCNE1 channel (Iks) is reduced by overexpression of these Nedd4 family members and is suppressed by their catalytically inactive mutants, or upon mutating the PY motif of KCNQ1 [95]. These Nedd4 family members thus affect channel ubiquitylation and internalization. Likewise, the muscarinic-sensitive K+ current (M-current), mediated by heterotetrameric channels consisting of KCNQ3 associated with either KCNQ2 or KCNQ5 (i.e., KCNQ2/3 or KCNQ3/5), are regulated by Nedd4-2, ubiquitylation and Sgk1 [50, 175]. The C termini of KCNQ2 and KCNQ3 contain an atypical “PY motif” (PPxPPY). However, while Nedd4-2 can bind the C termini of these channel subunits, binding is not mediated via this motif [50]; thus, Nedd4-2 either interacts with another C-terminal sequence, or via an adapter protein. Active Nedd4-2 is required to suppress function of these channels, suggesting that ubiquitylation regulates their stability, likely at the cell surface [50]. Similar to ENaC, the regulation of KCNQ2/3 and KCNQ3/5 via Nedd4-2 can be modulated by Sgk1 mediated phosphorylation of this E3 ligase [175].
The K+ channels Kv1.3 (KCNA3) and Kv1.5 (KCNA5), which are stimulated by insulin or IGF-1, have been demonstrated to be inhibited by Nedd4-2 and up-regulated by Sgk1, which suppresses Nedd4-2, using Xenopus oocyte expression system [17, 79]. Nedd4-2 was shown to ubiquitylate Kv1.5, thus inducing its endocytosis [17]. Interestingly, while Kv4.3 (KCND3) assembled with its accessory protein KChiP, was stimulated by Sgk1, this stimulation was not mediated via Nedd4-2 and was not associated with increased cell surface stability of this channel [10].
In addition to the above-described examples, a recent proteome array screen aimed at identifying substrates for mammalian Nedd4 protein, identified other K+ channels as in vitro substrates for Nedd4-2: KCNAB1, KCNAB3 and KCNB1 (Kv2.1), with the latter two possessing PY motifs [155]. The role of these in vitro substrates needs to be examined in vivo now to test if these channels are regulated by Nedd4-2 in a physiological setting.
The same proteome array screen also identified the Ca2+ channel, CACNB1, as a substrate for Nedd4-2, an observation that was also validated in mammalian cells [155]. Interestingly, a subset of the TRP Ca2+ channel, such as TRPV4 and TRPC4, have been shown to be ubiquitylated and AIP4/Itch, a Nedd4-like protein; this ubiquitylation leads to endocytosis of these channels [213], thus suppressing their function. This AIP4—mediated ubiquitylation requires the N terminal region of the TRP channels, although a variant PY motif (GPxYxxL) present within the N-terminus was dispensable [213]. Recently, evidence was also provided that the calcium channel TRPV6 may be regulated by Nedd4-2 [232].
Collectively, these results point to a major role of Nedd4 proteins, primarily Nedd4-2, in promoting ubiquitylation and endocytosis of numerous cell surface cation channels, thus suppressing their function.
Cl− channels/transporters
Much like cation channels, several anion channels have been shown to be regulated by Nedd4 family members, with Cl- channels (ClC) family most extensively studied. The ClC family comprises both Cl− channels and Cl−/H+ antiporters [94, 215]. ClC5 is a Cl−/H+ antiporter that is expressed primarily in endosomal membranes in the proximal tubules and is responsible for endosomal acidification, a role it carries out in collaboration with V-ATPase. This acidification is required for re-absorption of small molecule proteins, minerals and vitamins by the kidney [72]. Loss of function mutations in CLC5 leads to Dent disease, a disorder characterized by excessive loss of proteins (proteinuria), calcium and phosphate in the urine, resulting in kidney stones [220]. In accord, ClC5 knockout mice exhibit proteinuria and defects in both receptor mediated and fluid phase endocytosis, resembling Dent disease in humans [156]. A carboxy terminal PY motif (PPLPPY) was identified earlier in ClC5, and shown to be required for endocytosis of the channel [176]. It was postulated that WWP2 could be mediating the endocytosis of ClC5 by interacting with its PY motif [176], although a later study proposed that Nedd4-2 is the E3 ligase involved [84]. In the latter study, Nedd4-2 was shown to regulate ClC5 ubiquitylation and albumin uptake into endosomes. Similar to ClC5, ClC2 was also shown to be regulated by Nedd4-2 [150]. ClC2, a bone fide Cl− channel, is ubiquitously expressed and is localized to the plasma membrane. Disruption of ClC2 in mice causes testicular and retina degeneration, as well as wide-spread vacuolation in the white matter of the CNS, suggesting that regulation of ionic environment via ClC2 in these cells/tissues is necessary for their normal maintenance [16, 21]. One study also suggested that the ClC2 gene is included in a chromosomal region associated with susceptibility to epilepsy [172], however, the putative role of ClC2 in this increased susceptibility needs to be further investigated. ClC2 was shown to be positively regulated by Sgk1-3, and negatively regulated by Nedd4-2, which reduces it cell surface expression [150], although a direct interaction between Nedd4-2 and ClC2 was not demonstrated in that study, nor does ClC2 possess a PY motif. ClC-Ka is another bone fide Cl− channel that along with ClC-Kb is a basolateral channel expressed in the ascending loop of Henley and regulates Na+, Cl−, and fluid absorption. In accord, knockout of Clck1 (ClC-Ka) in mice leads to severe urinary loss [200]. The ClC-Ka and ClC-Kb channels require co-expression of an accessory protein, barttin, to become functional [53]. Mutations in BSND (the barttin gene) causes a severe form of Bartter’s syndrome, characterized by severe renal salt wasting and deafness [15]. Interestingly, Barttin contains a PY motif and its mutation enhances activity of the ClC-Kb/Barttin channel [53]. It was subsequently shown that Nedd4-2 can suppress ClC-Ka/Barttin function when co-expressed in Xenopus oocytes, an effect prevented upon mutation of the PY motif in barttin [51]. Here, too, Sgk1 and Sgk3 were able to phosphorylate Nedd4-2 and block its suppression of the channel.
In addition to the ClC family, other Cl− channels appear to be regulated by Nedd4 proteins. The Tweety (TTYH) family are maxi Cl− channels that includes three members in humans, TTYH1-3, with TTYH2 and TTYH3 (but not TTYH1) activated by Ca2+ [195]. TTYH2 and TTYH3 possess a PY motif, which a recent study has suggested is involved in binding to Nedd4-2; accordingly, Nedd4-2 was shown to ubiquitylate TTYH2 and regulate its cell surface stability [77]. An additional Cl− channel, CLIC2 (intracellular Cl− channel 2), could potentially be another target for regulation by Nedd4-2, as it was identified in the above-described proteome array screen as a target for ubiquitylation by Nedd4-2 (and Nedd4-1) and possesses a PY motif [155].
The suppression of channel function by Nedd4 family members was also reported in several transporters in addition to ClC5. For example, the excitatory amino acid transporter EAAT2 (which does not contain a PY motif) was shown to be suppressed by Nedd4-2 and stimulated by Sgk1-3 or Akt [18]. Likewise, the divalent metal transporter, DMT1, was shown to be regulated by WWP2 and Nedd4-2; this was mediated by recruitment of the adapter protein Ndfip1 [59, 83], as described below. Moreover the connexin 43, which contains a PY-motif, was also demonstrated to be regulated by Nedd4-2 [115] by one group, whereas the Staub's group found that the extened PY-motif was rather part of a tyrosine-based sorting signal not regulated by Nedd4 ubiquitin-protein ligases [197].
In addition to the regulation of CFTR and its ΔF508-CFTR trafficking mutant by ERAD in the biosynthetic pathway (described above), it was recently found that “rescued” ΔF508-CFTR that is able to reach the plasma membrane exhibits a short half life there and is targeted for degradation by the E3 ligase CHIP, with the aid of several chaperones and co-chaperones [146].
Overall, it appears that cell surface stability of numerous ion channels and transporters is regulated by Nedd4 proteins, mainly Nedd4-2 (and sometimes WWP2, Nedd4-1, or AIP4). This regulation often involves the Sgk/Akt family of kinases, which phosphorylate Nedd4-2 thus preventing it from interacting with its targets. The regulation by these kinases (and others) help link Nedd4-2 and the channels its ubiquitylates to hormone and growth factors signaling (e.g. aldosterone, vasopressin, insulin), which play critical roles in maintaining homeostasis of ion and fluid composition and overall normal physiological functions of the organism.
Regulation of endocytosis of channels/transporters by other (or unknown) E3 ligases
While most studies to date have focused on the regulation of ion channels/transporters by Nedd4 family proteins, several others have described regulation of such channels by different (or still unknown) E3 ligases. For example, function of the ENaC-related channel, ASIC, is suppressed by the E3 ligase Parkin, in a process that includes the adapter protein, PICK1 [96] (see below). Another example is the water channel AQP2, which is oligo-ubiquitylated with ub(K63) on one Lys residue upon withdrawal of vasopressin/forskolin or PKC activation, which targets it for endocytosis, MVB sorting and lysosomal degradation [98]. The E3 ligase that mediates this reaction is not yet known.
In cilia of the mammalian kidney and sensory cilia in Caenorhabditis elegans, polycystin-1 (PC1, encoded by PKD1) and PC2 (TRPP2, encoded by PKD2) function together as a mechanosensory Ca2+ channel. Using C. elegans, it was recently shown that this channel complex is sorted for lysosomal degradation by the STAM-Hrs system in a process that involves mono-ubiquitylation of PC2 by as yet an unknown E3 ligase [85]. The physiological importance of these channels and their regulation is underscored by the observation that mutations in PKD1 or PKD2 cause autosomal dominant polycystic kidney disease (ADPKD), an end stage renal disease [91]. In a separate study, it was discovered that knockout of the transcription factor TAZ in mice led to increased levels of PC2 [198]; upon phosphorylation, TAZ interacts with SCFβ-Trcp to form an E3 ligase complex, which then targets PC2 for degradation by the proteasome [198]. It is unlikely that the TAZ:SCFβ-Trcp complex (and polyubiquitylation) is also involved in targeting of the PC1:PC2 channel for sorting by STAM-Hrs and lysosomal degradation, and thus suggests that regulation of this Ca2+ channel is mediated by more than one E3 ligase. In addition to regulation by E3 ligases, a role for DUBs was also described for the regulation of TRP channels. The TRPA1 channel, which responds to cold temp and pungent compounds, was shown to associate with the DUB CYLD, which is a tumor suppressor; this leads to de-ubiquitylation of TRPA1 and its stabilization, thus connecting TRPA1 to oncogenesis [193].
Analogous to regulation of several K+ channels by Nedd4 family members (described above), a recent paper describes the suppression of ROMK1 (Kir1.1) by the RING E3 ligase POSH [123]. POSH binds the N terminus of ROMK1, leading to channel ubiquitylation via the POSH-RING domain and endocytosis via a clathrin-independent pathway [123].
Ubiquitin-mediated regulation of ion channels and transporters via adapter proteins
In most of the examples described above, Nedd4-2 (or its family relatives) was able to directly interact with its target channel/transporter, especially in those cases where these transmembrane proteins possess a PY motif that can bind the WW domains of Nedd4 proteins (eg in ENaC, several Navs, K+ channels and ClC5). In other cases, such interactions may be indirect, via adapter proteins, which themselves possess PY motifs (Fig. 4). This has been observed earlier in yeast TM proteins that are regulated by Rsp5, the S. Cerevisae orthologue of Nedd4/Nedd4-2. For example, Bul1 and Bul2 serve as adapters to connect between Rsp5 and the general amino acid permease, Gap1, thus facilitating Rsp5-mediated ubiquitylation of Gap1 and its subsequent endocytosis from the PM or sorting from the Golgi to vacuoles [189]. Likewise, several arrestin related proteins (ARTs) were recently identified as PY motif-containing adapters that connect between Rsp5 and numerous PM proteins (mostly transporters), thus promoting Rsp5-mediated ubiquitylation and endocytosis of these proteins [122]; for reasons not yet clear, the adapter proteins themselves are target for Rsp5-mediated ubiquitylation [73, 122], which is required for their function. An interesting example, also conserved in mammals, is the adapter Bsd2. One function of this ER protein is to regulate ubiquitin-mediated trafficking to the PM of the metal ion transporters, Smf1 and Smf2, in yeast [124]. The mammalian homologues of Bsd2 are Ndfip1 (N4WBP5) and Ndfip2 (MDWBP5a), and are PY motif-containing adapters for Nedd4 proteins that reside in intracellular vesicles. Similar to Bsd2 in yeast, they serve as adapters to connect between WWP2 and the divalent metal transporter DMT1 in mammals [59]. DMT1 is responsible for iron uptake in enterocytes and its release from vesicles in hepatocytes. Ndfip1&2 facilitate ubiquitylation of DMT1 by WWP2, resulting in degradation of this metal transporter (Fig. 4A). Accordingly, knockout of Ndfip1 in mice leads to increased DMT1 activity and hepatic iron accumulation [59], suggesting a key role for this adapter in regulating iron homeostasis. More recently, Ndfip1 was shown to protect human neurons from iron and cobalt toxicity, and knockdown or knockout of this adapter resulted in accumulation of these metals in neurons [83]; In this case, Nfdip1 serves as an adapter to connect neuronal DMT1 to Nedd4-2.
As indicated above, the vasopressin-inducible DUB, Usp-10, enhances ENaC abundance and function at the plasma membrane [23]. Here, however, Usp10 does not directly de-ubiquitylate ENaC, but rather a third partner, sorting nexin 3 (SNX3), a protein that when co-expressed with ENaC increases cell-surface stability of the channel. Thus, Usp-10 mediated de-ubiquitylation of SNX3 leads to increased stability of SNX3, which in turn promotes ENaC stabilization [23]. (Fig. 4B).
ASIC, the acid—sensing ion channel that is expressed in neurons, belongs to the ENaC/degenerin family, but lacks the C-terminal region present in ENaC that contains a PY motif (and thus does not interact with Nedd4-2). Instead, ASIC binds PICK1, a PDZ—containing protein that normally potentiates ASIC function following PKC stimulation. PICK1 is a substrate for Parkin, a RING finger E3 ligase, which mono-ubiquitylates PICK1; this mono-ubiquitylation (and thus protection from proteasomal degradation) appears to block the ability of PICK1 to potentiate ASIC [96] (Fig. 4C). In accord, loss of parkin in hippocampal neurons by its knockout in mice leads to enhanced ASIC activation, thus potentially promoting neuro-degeneration, as seen in Parkinson's disease [96].
Regulation of ion channels by UBLs (SUMO)
As indicated in the “Introduction” section, posttranslational modification by UBLs has emerged as an important factor in the regulation of function of numerous cellular proteins. One of the better-studied UBLs is SUMO. Several recent studies have focused on the role of SUMOylation in regulating ion channels.
The Ca2+ channel TRPM4, expressed in cardiac Purkinje fibers, was recently demonstrated to harbor a mutation, E17K, which is associated with progressive familial heart block type I (PFHB1), an autosomal dominant disease; the mutant channel exhibits an increased channel SUMOylation and accumulation at the plasma membrane, leading to enhanced function of this channel [110]. It was proposed that such enhanced SUMOylation prevents channel ubiquitylation and endocytosis, although this hypothesis was not experimentally tested (i.e., ubiquitylation of the mutant vs. WT channel was not investigated).
Several K+ channels were also reported to be regulated by SUMOylation. The K2P1 (TWIK1) leak channel was previously shown to be a weak (or inactive) channel [70, 113, 148]. A report by Goldstein and colleagues then demonstrated that this channel is maintained in an inactive state by SUMOylation on one specific Lys (K274); upon de-SUMOylation by SENP-1, the channel becomes active, exhibiting K+ selectivity and sensitivity to extracellular pH [161]. A subsequent paper challenged these findings, suggesting that the K274 residue may not be involved in SUMOylation of the channel [56]. Recently it has been shown that one SUMO is sufficient to silence the K2P1 channel [157]. More recently, other studies provided evidence for SUMO modification of other K+ channels. Kv1.5, which mediates the I(kur) depolarizing current in human atrial myocytes, was shown to be directly SUMOylated by Ubc9 (the E2 for SUMOylation reaction) at two sites [11]; removal of this SUMO conjugation by SENP2 or by mutating the SUMOylation sites led to hyperpolarizing shift in voltage dependence of steady-state inactivation of the channel. The voltage-dependent channel Kv2.1 regulates pancreatic beta cell excitability and insulin secretion. Recent work demonstrated that SUMOylation of Kv1.5 inhibits its native current by accelerating channel inactivation and inhibiting recovery from inactivation, resulting in decreased firing frequency [40]. These effects were augmented by overexpression of Ubc9 and alleviated by SENP1. It remains to be investigated how exactly (by what mechanisms) does SUMOylation of K+ channels, such as Kv1.5 or Kv2.1, regulate their biophysical properties and activity.
It is curious that KChAP, which was originally described as a chaperone that increases protein expression of several K+ channels, was later identified as PIAS3β, a SUMO E3 ligase [158]. So far it is not known if/how SUMOylation may affect the chaperone activity of KChAP/PIAS3β towards these channels.
Thus, relative to ubiquitylation of ion channels, much less is known about the mechanisms by which SUMOylation regulates function of such channels, nor is it known (yet) whether modifications by other UBLs can affect ion channel function.
Summary/perspectives
Over the past 15 years much has been learned about the role of the ubiquitin system in regulating ion channels and transporters, particularly during their processing in the ER and during endocytosis or sorting to MVBs and the lysosome. Much of the work has focused on the Nedd4 family of E3 ligases, and more recently on other E3 ligases, DUBs and several adapter proteins. In spite of these great advances, there are critical outstanding issues that need to be addressed:
-
(a)
To date, many studies demonstrating regulation of endocytosis of ion channels and transporters by E3s, especially Nedd4 family members, have been carried out by overexpression in Xenopus oocytes or tissue culture cells. It is essential that these initial studies are validated in vivo in the relevant physiological tissues. In that regard, recent advances in studying the role of Nedd4-2 in regulating ENaC in mouse kidney (and lung) represent important advances, and hopefully will be followed by similar studies on other ion channels/transporters.
-
(b)
So far, in most studies the nature of the ubiquitin attachments to the ion channels/transporter, especially at the plasma membrane, is not clear. For example, are the channels/transporters mono-, oligo- or poly ubiquitylated? Are the ubiquitins attached utilize K63, K48 (or others) linkages? Accordingly, is the cargo channel/transporter targeted to the lysosome, proteasome or both? Likewise, very little is known about the functions of UBLs in regulating ion transport, nor the role of other modifications, such as acetylation.
-
(c)
As detailed in the review, there are numerous diseases that are associated with malfunction of some of the ion channels described, including defects that involve the ubiquitin system. To develop drugs that targets these defects, it would be useful to solve the structure of the E3 ligase involved in complex with their cognate channel/transporter, or at least the binding interface of these binding partners. Given the recent advances in solving structures of transmembrane proteins, this difficult task may now become a reality. Such an approach could lead to the development of compounds that block or enhance these interactions in a specific manner, which could have an important therapeutic value.
References
Abriel H, Kamynina E, Horisberger JD, Staub O (2000) Regulation of the cardiac voltage-gated Na+ channel (H1) by the ubiquitin-protein ligase Nedd4. FEBS Lett 466:377–380
Abriel H, Loffing J, Rebhun JF, Pratt JH, Schild L, Horisberger JD, Rotin D, Staub O (1999) Defective regulation of the epithelial Na+ channel by Nedd4 in Liddle’s syndrome. J Clin Invest 103:667–673
Abriel H, Staub O (2005) Ubiquitylation of ion channels. Physiol Bethesda 20:398–407
Adams CM, Snyder PM, Welsh MJ (1997) Interactions between subunits of the human epithelial sodium channel. J Biol Chem 272:27295–27300
Ahner A, Nakatsukasa K, Zhang H, Frizzell RA, Brodsky JL (2007) Small heat-shock proteins select deltaF508-CFTR for endoplasmic reticulum-associated degradation. Mol Biol Cell 18:806–814
Almaca J, Kongsuphol P, Hieke B, Ousingsawat J, Viollet B, Schreiber R, Amaral MD, Kunzelmann K (2009) AMPK controls epithelial Na(+) channels through Nedd4-2 and causes an epithelial phenotype when mutated. Pflugers Arch 458:713–721
Alvarez de la Rosa D, Zhang P, Naray-Fejes-Toth A, Fejes-Toth G, Canessa CM (1999) The serum and glucocorticoid kinase sgk increases the abundance of epithelial sodium channels in the plasma membrane of Xenopus oocytes. J Biol Chem 274:37834–37839
Araki N, Umemura M, Miyagi Y, Yabana M, Miki Y, Tamura K, Uchino K, Aoki R, Goshima Y, Umemura S, Ishigami T (2008) Expression, transcription, and possible antagonistic interaction of the human Nedd4L gene variant: implications for essential hypertension. Hypertension 51:773–777
Auberson M, Hoffmann-Pochon N, Vandewalle A, Kellenberger S, Schild L (2003) Epithelial Na+ channel mutants causing Liddle’s syndrome retain ability to respond to aldosterone and vasopressin. Am J Physiol Ren Physiol 285:F459–F471
Baltaev R, Strutz-Seebohm N, Korniychuk G, Myssina S, Lang F, Seebohm G (2005) Regulation of cardiac shal-related potassium channel Kv 4.3 by serum- and glucocorticoid-inducible kinase isoforms in Xenopus oocytes. Pflugers Arch 450:26–33
Benson MD, Li QJ, Kieckhafer K, Dudek D, Whorton MR, Sunahara RK, Iniguez-Lluhi JA, Martens JR (2007) SUMO modification regulates inactivation of the voltage-gated potassium channel Kv1.5. Proc Natl Acad Sci USA 104:1805–1810
Bertog M, Cuffe JE, Pradervand S, Hummler E, Hartner A, Porst M, Hilgers KF, Rossier BC, Korbmacher C (2008) Aldosterone responsiveness of the epithelial sodium channel (ENaC) in colon is increased in a mouse model for Liddle’s syndrome. J Physiol 586:459–475
Bhalla V, Daidie D, Li H, Pao AC, LaGrange LP, Wang J, Vandewalle A, Stockand JD, Staub O, Pearce D (2005) Serum- and glucocorticoid-regulated kinase 1 regulates ubiquitin ligase neural precursor cell-expressed, developmentally down-regulated protein 4–2 by inducing interaction with 14-3-3. Mol Endocrinol 19:3073–3084
Bhalla V, Oyster NM, Fitch AC, Wijngaarden MA, Neumann D, Schlattner U, Pearce D, Hallows KR (2006) AMP-activated kinase inhibits the epithelial Na+ channel through functional regulation of the ubiquitin ligase Nedd4-2. J Biol Chem 281:26159–26169
Birkenhager R, Otto E, Schurmann MJ, Vollmer M, Ruf EM, Maier-Lutz I, Beekmann F, Fekete A, Omran H, Feldmann D, Milford DV, Jeck N, Konrad M, Landau D, Knoers NV, Antignac C, Sudbrak R, Kispert A, Hildebrandt F (2001) Mutation of BSND causes Bartter syndrome with sensorineural deafness and kidney failure. Nat Genet 29:310–314
Blanz J, Schweizer M, Auberson M, Maier H, Muenscher A, Hubner CA, Jentsch TJ (2007) Leukoencephalopathy upon disruption of the chloride channel ClC-2. J Neurosci 27:6581–6589
Boehmer C, Laufer J, Jeyaraj S, Klaus F, Lindner R, Lang F, Palmada M (2008) Modulation of the voltage-gated potassium channel Kv1.5 by the SGK1 protein kinase involves inhibition of channel ubiquitination. Cell Physiol Biochem 22:591–600
Boehmer C, Palmada M, Rajamanickam J, Schniepp R, Amara S, Lang F (2006) Post-translational regulation of EAAT2 function by co-expressed ubiquitin ligase Nedd4-2 is impacted by SGK kinases. J Neurochem 97:911–921
Bonifacino JS, Cosson P, Klausner RD (1990) Colocalized transmembrane determinants for ER degradation and subunit assembly explain the intracellular fate of TCR chains. Cell 63:503–513
Bonny O, Chraibi A, Loffing J, Jaeger NF, Grunder S, Horisberger JD, Rossier BC (1999) Functional expression of a pseudohypoaldosteronism type I mutated epithelial Na+ channel lacking the pore-forming region of its alpha subunit. J Clin Invest 104:967–974
Bosl MR, Stein V, Hubner C, Zdebik AA, Jordt SE, Mukhopadhyay AK, Davidoff MS, Holstein AF, Jentsch TJ (2001) Male germ cells and photoreceptors, both dependent on close cell-cell interactions, degenerate upon ClC-2 Cl(-) channel disruption. EMBO J 20:1289–1299
Boucher RC (2007) Airway surface dehydration in cystic fibrosis: pathogenesis and therapy. Annu Rev Med 58:157–170
Boulkroun S, Ruffieux-Daidie D, Vitagliano JJ, Poirot O, Charles RP, Lagnaz D, Firsov D, Kellenberger S, Staub O (2008) Vasopressin-inducible ubiquitin-specific protease 10 increases ENaC cell surface expression by deubiquitylating and stabilizing sorting nexin 3. Am J Physiol Ren Physiol 295:F889–F900
Bruns JB, Carattino MD, Sheng S, Maarouf AB, Weisz OA, Pilewski JM, Hughey RP, Kleyman TR (2007) Epithelial Na+ channels are fully activated by furin- and prostasin-dependent release of an inhibitory peptide from the gamma-subunit. J Biol Chem 282:6153–6160
Bruns JB, Hu B, Ahn YJ, Sheng S, Hughey RP, Kleyman TR (2003) Multiple epithelial Na+ channel domains participate in subunit assembly. Am J Physiol Ren Physiol 285:F600–F609
Buck TM, Kolb AR, Boyd CR, Kleyman TR, Brodsky JL (2010) The endoplasmic reticulum-associated degradation of the epithelial sodium channel requires a unique complement of molecular chaperones. Mol Biol Cell 21:1047–1058
Butterworth MB, Edinger RS, Ovaa H, Burg D, Johnson JP, Frizzell RA (2007) The deubiquitinating enzyme UCH-L3 regulates the apical membrane recycling of the epithelial sodium channel. J Biol Chem 282:37885–37893
Canessa CM, Schild L, Buell G, Thorens B, Gautschi I, Horisberger JD, Rossier BC (1994) Amiloride-sensitive epithelial Na+ channel is made of three homologous subunits. Nature 367:463–467
Caramelo JJ, Parodi AJ (2007) How sugars convey information on protein conformation in the endoplasmic reticulum. Semin Cell Dev Biol 18:732–742
Carattino MD, Edinger RS, Grieser HJ, Wise R, Neumann D, Schlattner U, Johnson JP, Kleyman TR, Hallows KR (2005) Epithelial sodium channel inhibition by AMP-activated protein kinase in oocytes and polarized renal epithelial cells. J Biol Chem 280:17608–17616
Carattino MD, Hughey RP, Kleyman TR (2008) Proteolytic processing of the epithelial sodium channel gamma subunit has a dominant role in channel activation. J Biol Chem 283:25290–25295
Carattino MD, Passero CJ, Steren CA, Maarouf AB, Pilewski JM, Myerburg MM, Hughey RP, Kleyman TR (2008) Defining an inhibitory domain in the alpha-subunit of the epithelial sodium channel. Am J Physiol Ren Physiol 294:F47–F52
Carvalho P, Goder V, Rapoport TA (2006) Distinct ubiquitin-ligase complexes define convergent pathways for the degradation of ER proteins. Cell 126:361–373
Chang SS, Grunder S, Hanukoglu A, Rosler A, Mathew PM, Hanukoglu I, Schild L, Lu Y, Shimkets RA, Nelson-Williams C, Rossier BC, Lifton RP (1996) Mutations in subunits of the epithelial sodium channel cause salt wasting with hyperkalaemic acidosis, pseudohypoaldosteronism type 1. Nat Genet 12:248–253
Chen B, Mariano J, Tsai YC, Chan AH, Cohen M, Weissman AM (2006) The activity of a human endoplasmic reticulum-associated degradation E3, gp78, requires its Cue domain, RING finger, and an E2-binding site. Proc Natl Acad Sci USA 103:341–346
Chen SY, Bhargava A, Mastroberardino L, Meijer OC, Wang J, Buse P, Firestone GL, Verrey F, Pearce D (1999) Epithelial sodium channel regulated by aldosterone-induced protein sgk. Proc Natl Acad Sci USA 96:2514–2519
Cheng SH, Gregory RJ, Marshall J, Paul S, Souza DW, White GA, O'Riordan CR, Smith AE (1990) Defective intracellular transport and processing of CFTR is the molecular basis of most cystic fibrosis. Cell 63:827–834
Christianson JC, Green WN (2004) Regulation of nicotinic receptor expression by the ubiquitin-proteasome system. EMBO J 23:4156–4165
Dahlmann A, Pradervand S, Hummler E, Rossier BC, Frindt G, Palmer LG (2003) Mineralocorticoid regulation of epithelial Na+ channels is maintained in a mouse model of Liddle’s syndrome. Am J Physiol Ren Physiol 285:F310–F318
Dai XQ, Kolic J, Marchi P, Sipione S, Macdonald PE (2009) SUMOylation regulates Kv2.1 and modulates pancreatic beta-cell excitability. J Cell Sci 122:775–779
Debonneville C, Flores SY, Kamynina E, Plant PJ, Tauxe C, Thomas MA, Munster C, Chraibi A, Pratt JH, Horisberger JD, Pearce D, Loffing J, Staub O (2001) Phosphorylation of Nedd4-2 by Sgk1 regulates epithelial Na(+) channel cell surface expression. EMBO J 20:7052–7059
Debonneville C, Staub O (2004) Participation of the ubiquitin-conjugating enzyme UBE2E3 in Nedd4-2-dependent regulation of the epithelial Na+ channel. Mol Cell Biol 24:2397–2409
Diakov A, Korbmacher C (2004) A novel pathway of epithelial sodium channel activation involves a serum- and glucocorticoid-inducible kinase consensus motif in the C terminus of the channel’s alpha-subunit. J Biol Chem 279:38134–38142
Dibbens LM, Ekberg J, Taylor I, Hodgson BL, Conroy SJ, Lensink IL, Kumar S, Zielinski MA, Harkin LA, Sutherland GR, Adams DJ, Berkovic SF, Scheffer IE, Mulley JC, Poronnik P (2007) NEDD4-2 as a potential candidate susceptibility gene for epileptic photosensitivity. Genes Brain Behav 6:750–755
Dinudom A, Harvey KF, Komwatana P, Young JA, Kumar S, Cook DI (1998) Nedd4 mediates control of an epithelial Na+ channel in salivary duct cells by cytosolic Na+. Proc Natl Acad Sci USA 95:7169–7173
Donaldson SH, Boucher RC (2007) Sodium channels and cystic fibrosis. Chest 132:1631–1636
Duc C, Farman N, Canessa CM, Bonvalet JP, Rossier BC (1994) Cell-specific expression of epithelial sodium channel alpha, beta, and gamma subunits in aldosterone-responsive epithelia from the rat: localization by in situ hybridization and immunocytochemistry. J Cell Biol 127:1907–1921
Dunn DM, Ishigami T, Pankow J, von Niederhausern A, Alder J, Hunt SC, Leppert MF, Lalouel JM, Weiss RB (2002) Common variant of human NEDD4L activates a cryptic splice site to form a frameshifted transcript. J Hum Genet 47:665–676
Edinger RS, Lebowitz J, Li H, Alzamora R, Wang H, Johnson JP, Hallows KR (2009) Functional regulation of the epithelial Na+ channel by IkappaB kinase-beta occurs via phosphorylation of the ubiquitin ligase Nedd4-2. J Biol Chem 284:150–157
Ekberg J, Schuetz F, Boase NA, Conroy SJ, Manning J, Kumar S, Poronnik P, Adams DJ (2007) Regulation of the voltage-gated K(+) channels KCNQ2/3 and KCNQ3/5 by ubiquitination. Novel role for Nedd4-2. J Biol Chem 282:12135–12142
Embark HM, Bohmer C, Palmada M, Rajamanickam J, Wyatt AW, Wallisch S, Capasso G, Waldegger P, Seyberth HW, Waldegger S, Lang F (2004) Regulation of CLC-Ka/barttin by the ubiquitin ligase Nedd4-2 and the serum- and glucocorticoid-dependent kinases. Kidney Int 66:1918–1925
Escayg A, MacDonald BT, Meisler MH, Baulac S, Huberfeld G, An-Gourfinkel I, Brice A, LeGuern E, Moulard B, Chaigne D, Buresi C, Malafosse A (2000) Mutations of SCN1A, encoding a neuronal sodium channel, in two families with GEFS + 2. Nat Genet 24:343–345
Estevez R, Boettger T, Stein V, Birkenhager R, Otto E, Hildebrandt F, Jentsch TJ (2001) Barttin is a Cl- channel beta-subunit crucial for renal Cl- reabsorption and inner ear K + secretion. Nature 414:558–561
Fakitsas P, Adam G, Daidie D, van Bemmelen MX, Fouladkou F, Patrignani A, Wagner U, Warth R, Camargo SM, Staub O, Verrey F (2007) Early aldosterone-induced gene product regulates the epithelial sodium channel by deubiquitylation. J Am Soc Nephrol 18:1084–1092
Fejes-Toth G, Frindt G, Naray-Fejes-Toth A, Palmer LG (2008) Epithelial Na+ channel activation and processing in mice lacking SGK1. Am J Physiol Ren Physiol 294:F1298–F1305
Feliciangeli S, Bendahhou S, Sandoz G, Gounon P, Reichold M, Warth R, Lazdunski M, Barhanin J, Lesage F (2007) Does sumoylation control K2P1/TWIK1 background K + channels? Cell 130:563–569
Firsov D, Schild L, Gautschi I, Merillat AM, Schneeberger E, Rossier BC (1996) Cell surface expression of the epithelial Na channel and a mutant causing Liddle syndrome: a quantitative approach. Proc Natl Acad Sci USA 93:15370–15375
Flores SY, Loffing-Cueni D, Kamynina E, Daidie D, Gerbex C, Chabanel S, Dudler J, Loffing J, Staub O (2005) Aldosterone-induced serum and glucocorticoid-induced kinase 1 expression is accompanied by Nedd4-2 phosphorylation and increased Na + transport in cortical collecting duct cells. J Am Soc Nephrol 16:2279–2287
Foot NJ, Dalton HE, Shearwin-Whyatt LM, Dorstyn L, Tan SS, Yang B, Kumar S (2008) Regulation of the divalent metal ion transporter DMT1 and iron homeostasis by a ubiquitin-dependent mechanism involving Ndfips and WWP2. Blood 112:4268–4275
Fotia AB, Dinudom A, Shearwin KE, Koch JP, Korbmacher C, Cook DI, Kumar S (2003) The role of individual Nedd4-2 (KIAA0439) WW domains in binding and regulating epithelial sodium channels. FASEB J 17:70–72
Fotia AB, Ekberg J, Adams DJ, Cook DI, Poronnik P, Kumar S (2004) Regulation of neuronal voltage-gated sodium channels by the ubiquitin-protein ligases Nedd4 and Nedd4-2. J Biol Chem 279:28930–28935
Fouladkou F, Alikhani-Koopaei R, Vogt B, Flores SY, Malbert-Colas L, Lecomte MC, Loffing J, Frey FJ, Frey BM, Staub O (2004) A naturally occurring human Nedd4-2 variant displays impaired ENaC regulation in Xenopus laevis oocytes. Am J Physiol Ren Physiol 287:F550–F561
Galan JM, Haguenauer-Tsapis R (1997) Ubiquitin lys63 is involved in ubiquitination of a yeast plasma membrane protein. EMBO J 16:5847–5854
Galan JM, Moreau V, Andre B, Volland C, Haguenauer-Tsapis R (1996) Ubiquitination mediated by the Npi1p/Rsp5p ubiquitin-protein ligase is required for endocytosis of the yeast uracil permease. J Biol Chem 271:10946–10952
Gao Y, Chotoo CK, Balut CM, Sun F, Bailey MA, Devor DC (2008) Role of S3 and S4 transmembrane domain charged amino acids in channel biogenesis and gating of KCa2.3 and KCa3.1. J Biol Chem 283:9049–9059
Gasser A, Cheng X, Gilmore ES, Tyrrell L, Waxman SG, Dib-Hajj SD (2010) Two Nedd4-binding motifs underlie modulation of sodium channel Nav1.6 by p38 MAPK. J Biol Chem 285:26149−26161
Glickman MH, Ciechanover A (2002) The ubiquitin-proteasome proteolytic pathway: destruction for the sake of construction. Physiol Rev 82:373–428
Gnann A, Riordan JR, Wolf DH (2004) Cystic fibrosis transmembrane conductance regulator degradation depends on the lectins Htm1p/EDEM and the Cdc48 protein complex in yeast. Mol Biol Cell 15:4125–4135
Goldfarb SB, Kashlan OB, Watkins JN, Suaud L, Yan W, Kleyman TR, Rubenstein RC (2006) Differential effects of Hsc70 and Hsp70 on the intracellular trafficking and functional expression of epithelial sodium channels. Proc Natl Acad Sci USA 103:5817–5822
Goldstein SA, Wang KW, Ilan N, Pausch MH (1998) Sequence and function of the two P domain potassium channels: implications of an emerging superfamily. J Mol Med 76:13–20
Gorrie GH, Vallis Y, Stephenson A, Whitfield J, Browning B, Smart TG, Moss SJ (1997) Assembly of GABAA receptors composed of alpha1 and beta2 subunits in both cultured neurons and fibroblasts. J Neurosci 17:6587–6596
Gunther W, Piwon N, Jentsch TJ (2003) The ClC-5 chloride channel knock-out mouse - an animal model for Dent’s disease. Pflugers Arch 445:456–462
Gupta R, Kus B, Fladd C, Wasmuth J, Tonikian R, Sidhu S, Krogan NJ, Parkinson J, Rotin D (2007) Ubiquitination screen using protein microarrays for comprehensive identification of Rsp5 substrates in yeast. Mol Syst Biol 3:116
Hansson JH, Nelson-Williams C, Suzuki H, Schild L, Shimkets R, Lu Y, Canessa C, Iwasaki T, Rossier B, Lifton RP (1995) Hypertension caused by a truncated epithelial sodium channel gamma subunit: genetic heterogeneity of Liddle syndrome. Nat Genet 11:76–82
Hansson JH, Schild L, Lu Y, Wilson TA, Gautschi I, Shimkets R, Nelson-Williams C, Rossier BC, Lifton RP (1995) A de novo missense mutation of the beta subunit of the epithelial sodium channel causes hypertension and Liddle syndrome, identifying a proline-rich segment critical for regulation of channel activity. Proc Natl Acad Sci USA 92:11495–11499
Harvey KF, Dinudom A, Cook DI, Kumar S (2001) The Nedd4-like protein KIAA0439 is a potential regulator of the epithelial sodium channel. J Biol Chem 276:8597–8601
He Y, Hryciw DH, Carroll ML, Myers SA, Whitbread AK, Kumar S, Poronnik P, Hooper JD (2008) The ubiquitin-protein ligase Nedd4-2 differentially interacts with and regulates members of the Tweety family of chloride ion channels. J Biol Chem 283:24000–24010
Hein C, Springael JY, Volland C, Haguenauer-Tsapis R, Andre B (1995) NPl1, an essential yeast gene involved in induced degradation of Gap1 and Fur4 permeases, encodes the Rsp5 ubiquitin-protein ligase. Mol Microbiol 18:77–87
Henke G, Maier G, Wallisch S, Boehmer C, Lang F (2004) Regulation of the voltage gated K + channel Kv1.3 by the ubiquitin ligase Nedd4-2 and the serum and glucocorticoid inducible kinase SGK1. J Cell Physiol 199:194–199
Henry PC, Kanelis V, O'Brien MC, Kim B, Gautschi I, Forman-Kay J, Schild L, Rotin D (2003) Affinity and specificity of interactions between Nedd4 isoforms and the epithelial Na + channel. J Biol Chem 278:20019–20028
Hicke L, Dunn R (2003) Regulation of membrane protein transport by ubiquitin and ubiquitin-binding proteins. Annu Rev Cell Dev Biol 19:141–172
Hicke L, Riezman H (1996) Ubiquitination of a yeast plasma membrane receptor signals its ligand-stimulated endocytosis. Cell 84:277–287
Howitt J, Putz U, Lackovic J, Doan A, Dorstyn L, Cheng H, Yang B, Chan-Ling T, Silke J, Kumar S, Tan SS (2009) Divalent metal transporter 1 (DMT1) regulation by Ndfip1 prevents metal toxicity in human neurons. Proc Natl Acad Sci USA 106:15489–15494
Hryciw DH, Ekberg J, Lee A, Lensink IL, Kumar S, Guggino WB, Cook DI, Pollock CA, Poronnik P (2004) Nedd4-2 functionally interacts with ClC-5: involvement in constitutive albumin endocytosis in proximal tubule cells. J Biol Chem 279:54996–55007
Hu J, Wittekind SG, Barr MM (2007) STAM and Hrs down-regulate ciliary TRP receptors. Mol Biol Cell 18:3277–3289
Hughey RP, Bruns JB, Kinlough CL, Harkleroad KL, Tong Q, Carattino MD, Johnson JP, Stockand JD, Kleyman TR (2004) Epithelial sodium channels are activated by furin-dependent proteolysis. J Biol Chem 279:18111–18114
Hughey RP, Bruns JB, Kinlough CL, Kleyman TR (2004) Distinct pools of epithelial sodium channels are expressed at the plasma membrane. J Biol Chem 279:48491–48494
Hughey RP, Carattino MD, Kleyman TR (2007) Role of proteolysis in the activation of epithelial sodium channels. Curr Opin Nephrol Hypertens 16:444–450
Hummler E, Vallon V (2005) Lessons from mouse mutants of epithelial sodium channel and its regulatory proteins. J Am Soc Nephrol 16:3160–3166
Ichimura T, Yamamura H, Sasamoto K, Tominaga Y, Taoka M, Kakiuchi K, Shinkawa T, Takahashi N, Shimada S, Isobe T (2005) 14-3-3 proteins modulate the expression of epithelial Na+ channels by phosphorylation-dependent interaction with Nedd4-2 ubiquitin ligase. J Biol Chem 280:13187–13194
Igarashi P, Somlo S (2002) Genetics and pathogenesis of polycystic kidney disease. J Am Soc Nephrol 13:2384–2398
Itani OA, Campbell JR, Herrero J, Snyder PM, Thomas CP (2003) Alternate promoters and variable splicing lead to hNedd4-2 isoforms with a C2 domain and varying number of WW domains. Am J Physiol Ren Physiol 285:F916–F929
Jensen TJ, Loo MA, Pind S, Williams DB, Goldberg AL, Riordan JR (1995) Multiple proteolytic systems, including the proteasome, contribute to CFTR processing. Cell 83:129–135
Jentsch TJ (2008) CLC chloride channels and transporters: from genes to protein structure, pathology and physiology. Crit Rev Biochem Mol Biol 43:3–36
Jespersen T, Membrez M, Nicolas CS, Pitard B, Staub O, Olesen SP, Baro I, Abriel H (2007) The KCNQ1 potassium channel is down-regulated by ubiquitylating enzymes of the Nedd4/Nedd4-like family. Cardiovasc Res 74:64–74
Joch M, Ase AR, Chen CX, MacDonald PA, Kontogiannea M, Corera AT, Brice A, Seguela P, Fon EA (2007) Parkin-mediated monoubiquitination of the PDZ protein PICK1 regulates the activity of acid-sensing ion channels. Mol Biol Cell 18:3105–3118
Kabra R, Knight KK, Zhou R, Snyder PM (2008) Nedd4-2 induces endocytosis and degradation of proteolytically cleaved epithelial Na+ channels. J Biol Chem 283:6033–6039
Kamsteeg EJ, Hendriks G, Boone M, Konings IB, Oorschot V, van der Sluijs P, Klumperman J, Deen PM (2006) Short-chain ubiquitination mediates the regulated endocytosis of the aquaporin-2 water channel. Proc Natl Acad Sci USA 103:18344–18349
Kamynina E, Debonneville C, Bens M, Vandewalle A, Staub O (2001) A novel mouse Nedd4 protein suppresses the activity of the epithelial Na+ channel. FASEB J 15:204–214
Kamynina E, Tauxe C, Staub O (2001) Distinct characteristics of two human Nedd4 proteins with respect to epithelial Na(+) channel regulation. Am J Physiol Ren Physiol 281:F469–F477
Kanelis V, Bruce MC, Skrynnikov NR, Rotin D, Forman-Kay JD (2006) Structural determinants for high-affinity binding in a Nedd4 WW3* domain-Comm PY motif complex. Structure 14:543–553
Kanelis V, Rotin D, Forman-Kay JD (2001) Solution structure of a Nedd4 WW domain-ENaC peptide complex. Nat Struct Biol 8:407–412
Kashlan OB, Mueller GM, Qamar MZ, Poland PA, Ahner A, Rubenstein RC, Hughey RP, Brodsky JL, Kleyman TR (2007) Small heat shock protein alphaA-crystallin regulates epithelial sodium channel expression. J Biol Chem 282:28149–28156
Kato A, Rouach N, Nicoll RA, Bredt DS (2005) Activity-dependent NMDA receptor degradation mediated by retrotranslocation and ubiquitination. Proc Natl Acad Sci USA 102:5600–5605
Keating MT, Sanguinetti MC (2001) Molecular and cellular mechanisms of cardiac arrhythmias. Cell 104:569–580
Kellenberger S, Gautschi I, Rossier BC, Schild L (1998) Mutations causing Liddle syndrome reduce sodium-dependent downregulation of the epithelial sodium channel in the Xenopus oocyte expression system. J Clin Invest 101:2741–2750
Kikkert M, Doolman R, Dai M, Avner R, Hassink G, van Voorden S, Thanedar S, Roitelman J, Chau V, Wiertz E (2004) Human HRD1 is an E3 ubiquitin ligase involved in degradation of proteins from the endoplasmic reticulum. J Biol Chem 279:3525–3534
Knight KK, Olson DR, Zhou R, Snyder PM (2006) Liddle’s syndrome mutations increase Na + transport through dual effects on epithelial Na+ channel surface expression and proteolytic cleavage. Proc Natl Acad Sci USA 103:2805–2808
Kopito RR (1999) Biosynthesis and degradation of CFTR. Physiol Rev 79:S167–S173
Kruse M, Schulze-Bahr E, Corfield V, Beckmann A, Stallmeyer B, Kurtbay G, Ohmert I, Brink P, Pongs O (2009) Impaired endocytosis of the ion channel TRPM4 is associated with human progressive familial heart block type I. J Clin Invest 119:2737–2744
Lauwers E, Jacob C, Andre B (2009) K63-linked ubiquitin chains as a specific signal for protein sorting into the multivesicular body pathway. J Cell Biol 185:493–502
Lee IH, Dinudom A, Sanchez-Perez A, Kumar S, Cook DI (2007) Akt mediates the effect of insulin on epithelial sodium channels by inhibiting Nedd4-2. J Biol Chem 282:29866–29873
Lesage F, Guillemare E, Fink M, Duprat F, Lazdunski M, Romey G, Barhanin J (1996) TWIK-1, a ubiquitous human weakly inward rectifying K + channel with a novel structure. EMBO J 15:1004–1011
Levkowitz G, Klapper LN, Tzahar E, Freywald A, Sela M, Yarden Y (1996) Coupling of the c-Cbl protooncogene product to ErbB-1/EGF-receptor but not to other ErbB proteins. Oncogene 12:1117–1125
Leykauf K, Salek M, Bomke J, Frech M, Lehmann WD, Durst M, Alonso A (2006) Ubiquitin protein ligase Nedd4 binds to connexin43 by a phosphorylation-modulated process. J Cell Sci 119:3634–3642
Li T, Koshy S, Folkesson HG (2007) Involvement of {alpha}ENaC and Nedd4-2 in the conversion from lung fluid secretion to fluid absorption at birth in the rat as assayed by RNA interference analysis. Am J Physiol Lung Cell Mol Physiol 293:L1069–L1078
Li T, Koshy S, Folkesson HG (2009) IL-1beta-induced cortisol stimulates lung fluid absorption in fetal guinea pigs via SGK-mediated Nedd4-2 inhibition. Am J Physiol Lung Cell Mol Physiol 296:L527–L533
Liang X, Butterworth MB, Peters KW, Walker WH, Frizzell RA (2008) An obligatory heterodimer of 14-3-3beta and 14-3-3epsilon is required for aldosterone regulation of the epithelial sodium channel. J Biol Chem 283:27418–27425
Liang X, Peters KW, Butterworth MB, Frizzell RA (2006) 14-3-3 isoforms are induced by aldosterone and participate in its regulation of epithelial sodium channels. J Biol Chem 281:16323–16332
Lifton RP, Gharavi AG, Geller DS (2001) Molecular mechanisms of human hypertension. Cell 104:545–556
Lilley BN, Ploegh HL (2005) Multiprotein complexes that link dislocation, ubiquitination, and extraction of misfolded proteins from the endoplasmic reticulum membrane. Proc Natl Acad Sci USA 102:14296–14301
Lin CH, MacGurn JA, Chu T, Stefan CJ, Emr SD (2008) Arrestin-related ubiquitin-ligase adaptors regulate endocytosis and protein turnover at the cell surface. Cell 135:714–725
Lin DH, Yue P, Pan CY, Sun P, Zhang X, Han Z, Roos M, Caplan M, Giebisch G, Wang WH (2009) POSH stimulates the ubiquitination and the clathrin-independent endocytosis of ROMK1 channels. J Biol Chem 284:29614–29624
Liu XF, Supek F, Nelson N, Culotta VC (1997) Negative control of heavy metal uptake by the Saccharomyces cerevisiae BSD2 gene. J Biol Chem 272:11763–11769
Loffing J, Korbmacher C (2009) Regulated sodium transport in the renal connecting tubule (CNT) via the epithelial sodium channel (ENaC). Pflugers Arch 458:111–135
Loffing J, Zecevic M, Feraille E, Kaissling B, Asher C, Rossier BC, Firestone GL, Pearce D, Verrey F (2001) Aldosterone induces rapid apical translocation of ENaC in early portion of renal collecting system: possible role of SGK. Am J Physiol Ren Physiol 280:F675–F682
Lu C, Pribanic S, Debonneville A, Jiang C, Rotin D (2007) The PY motif of ENaC, mutated in Liddle syndrome, regulates channel internalization, sorting and mobilization from subapical pool. Traffic 8:1246–1264
Luo F, Wang Y, Wang X, Sun K, Zhou X, Hui R (2009) A functional variant of NEDD4L is associated with hypertension, antihypertensive response, and orthostatic hypotension. Hypertension 54:796–801
Malik B, Schlanger L, Al-Khalili O, Bao HF, Yue G, Price SR, Mitch WE, Eaton DC (2001) Enac degradation in A6 cells by the ubiquitin-proteosome proteolytic pathway. J Biol Chem 276:12903–12910
Malik B, Yue Q, Yue G, Chen XJ, Price SR, Mitch WE, Eaton DC (2005) Role of Nedd4-2 and polyubiquitination in epithelial sodium channel degradation in untransfected renal A6 cells expressing endogenous ENaC subunits. Am J Physiol Ren Physiol 289:F107–F116
Mall M, Grubb BR, Harkema JR, O'Neal WK, Boucher RC (2004) Increased airway epithelial Na + absorption produces cystic fibrosis-like lung disease in mice. Nat Med 10:487–493
Manganas LN, Akhtar S, Antonucci DE, Campomanes CR, Dolly JO, Trimmer JS (2001) Episodic ataxia type-1 mutations in the Kv1.1 potassium channel display distinct folding and intracellular trafficking properties. J Biol Chem 276:49427–49434
Manganas LN, Wang Q, Scannevin RH, Antonucci DE, Rhodes KJ, Trimmer JS (2001) Identification of a trafficking determinant localized to the Kv1 potassium channel pore. Proc Natl Acad Sci USA 98:14055–14059
May A, Puoti A, Gaeggeler HP, Horisberger JD, Rossier BC (1997) Early effect of aldosterone on the rate of synthesis of the epithelial sodium channel alpha subunit in A6 renal cells. J Am Soc Nephrol 8:1813–1822
McDonald FJ, Price MP, Snyder PM, Welsh MJ (1995) Cloning and expression of the beta- and gamma-subunits of the human epithelial sodium channel. Am J Physiol 268:C1157–C1163
McNicholas CM, Canessa CM (1997) Diversity of channels generated by different combinations of epithelial sodium channel subunits. J Gen Physiol 109:681–692
Meacham GC, Patterson C, Zhang W, Younger JM, Cyr DM (2001) The Hsc70 co-chaperone CHIP targets immature CFTR for proteasomal degradation. Nat Cell Biol 3:100–105
Michlig S, Harris M, Loffing J, Rossier BC, Firsov D (2005) Progesterone down-regulates the open probability of the amiloride-sensitive epithelial sodium channel via a Nedd4-2-dependent mechanism. J Biol Chem 280:38264–38270
Morito D, Hirao K, Oda Y, Hosokawa N, Tokunaga F, Cyr DM, Tanaka K, Iwai K, Nagata K (2008) Gp78 cooperates with RMA1 in endoplasmic reticulum-associated degradation of CFTRDeltaF508. Mol Biol Cell 19:1328–1336
Mueller B, Lilley BN, Ploegh HL (2006) SEL1L, the homologue of yeast Hrd3p, is involved in protein dislocation from the mammalian ER. J Cell Biol 175:261–270
Nakatsukasa K, Brodsky JL (2008) The recognition and retrotranslocation of misfolded proteins from the endoplasmic reticulum. Traffic 9:861–870
Naray-Fejes-Toth A, Canessa C, Cleaveland ES, Aldrich G, Fejes-Toth G (1999) sgk is an aldosterone-induced kinase in the renal collecting duct. Effects on epithelial na+ channels. J Biol Chem 274:16973–16978
Nishikawa SI, Fewell SW, Kato Y, Brodsky JL, Endo T (2001) Molecular chaperones in the yeast endoplasmic reticulum maintain the solubility of proteins for retrotranslocation and degradation. J Cell Biol 153:1061–1070
O'Kelly I, Butler MH, Zilberberg N, Goldstein SA (2002) Forward transport. 14-3-3 binding overcomes retention in endoplasmic reticulum by dibasic signals. Cell 111:577–588
Oda Y, Okada T, Yoshida H, Kaufman RJ, Nagata K, Mori K (2006) Derlin-2 and Derlin-3 are regulated by the mammalian unfolded protein response and are required for ER-associated degradation. J Cell Biol 172:383–393
Okiyoneda T, Barriere H, Bagdany M, Rabeh WM, Du K, Hohfeld J, Young JC, Lukacs GL (2010) Peripheral protein quality control removes unfolded CFTR from the plasma membrane. Science 329:805–810
Okiyoneda T, Harada K, Takeya M, Yamahira K, Wada I, Shuto T, Suico MA, Hashimoto Y, Kai H (2004) Delta F508 CFTR pool in the endoplasmic reticulum is increased by calnexin overexpression. Mol Biol Cell 15:563–574
Orias M, Velazquez H, Tung F, Lee G, Desir GV (1997) Cloning and localization of a double-pore K channel, KCNK1: exclusive expression in distal nephron segments. Am J Physiol 273:F663–F666
Paiva S, Vieira N, Nondier I, Haguenauer-Tsapis R, Casal M, Urban-Grimal D (2009) Glucose-induced ubiquitylation and endocytosis of the yeast Jen1 transporter: role of lysine 63-linked ubiquitin chains. J Biol Chem 284:19228–19236
Palmada M, Dieter M, Boehmer C, Waldegger S, Lang F (2004) Serum and glucocorticoid inducible kinases functionally regulate ClC-2 channels. Biochem Biophys Res Commun 321:1001–1006
Palmer LG, Sackin H, Frindt G (1998) Regulation of Na+ channels by luminal Na+ in rat cortical collecting tubule. J Physiol 509(Pt 1):151–162
Papadatos GA, Wallerstein PM, Head CE, Ratcliff R, Brady PA, Benndorf K, Saumarez RC, Trezise AE, Huang CL, Vandenberg JI, Colledge WH, Grace AA (2002) Slowed conduction and ventricular tachycardia after targeted disruption of the cardiac sodium channel gene Scn5a. Proc Natl Acad Sci USA 99:6210–6215
Passero CJ, Mueller GM, Rondon-Berrios H, Tofovic SP, Hughey RP, Kleyman TR (2008) Plasmin activates epithelial Na+ channels by cleaving the gamma subunit. J Biol Chem 283:36586–36591
Pearce MM, Wang Y, Kelley GG, Wojcikiewicz RJ (2007) SPFH2 mediates the endoplasmic reticulum-associated degradation of inositol 1, 4, 5-trisphosphate receptors and other substrates in mammalian cells. J Biol Chem 282:20104–20115
Persaud A, Alberts P, Amsen EM, Xiong X, Wasmuth J, Saadon Z, Fladd C, Parkinson J, Rotin D (2009) Comparison of substrate specificity of the ubiquitin ligases Nedd4 and Nedd4-2 using proteome arrays. Mol Syst Biol 5:333
Piwon N, Gunther W, Schwake M, Bosl MR, Jentsch TJ (2000) ClC-5 Cl− -channel disruption impairs endocytosis in a mouse model for Dent’s disease. Nature 408:369–373
Plant LD, Dementieva IS, Kollewe A, Olikara S, Marks JD, Goldstein SA (2010) One SUMO is sufficient to silence the dimeric potassium channel K2P1. Proc Natl Acad Sci USA 107:10743–10748
Pourrier M, Schram G, Nattel S (2003) Properties, expression and potential roles of cardiac K+ channel accessory subunits: MinK, MiRPs, KChIP, and KChAP. J Membr Biol 194:141–152
Pradervand S, Wang Q, Burnier M, Beermann F, Horisberger JD, Hummler E, Rossier BC (1999) A mouse model for Liddle’s syndrome. J Am Soc Nephrol 10:2527–2533
Rabinovich E, Kerem A, Frohlich KU, Diamant N, Bar-Nun S (2002) AAA-ATPase p97/Cdc48p, a cytosolic chaperone required for endoplasmic reticulum-associated protein degradation. Mol Cell Biol 22:626–634
Rajan S, Plant LD, Rabin ML, Butler MH, Goldstein SA (2005) Sumoylation silences the plasma membrane leak K+ channel K2P1. Cell 121:37–47
Randrianarison N, Escoubet B, Ferreira C, Fontayne A, Fowler-Jaeger N, Clerici C, Hummler E, Rossier BC, Planes C (2007) beta-Liddle mutation of the epithelial sodium channel increases alveolar fluid clearance and reduces the severity of hydrostatic pulmonary oedema in mice. J Physiol 582:777–788
Rauh R, Dinudom A, Fotia AB, Paulides M, Kumar S, Korbmacher C, Cook DI (2006) Stimulation of the epithelial sodium channel (ENaC) by the serum- and glucocorticoid-inducible kinase (Sgk) involves the PY motifs of the channel but is independent of sodium feedback inhibition. Pflugers Arch 452:290–299
Reid CA, Berkovic SF, Petrou S (2009) Mechanisms of human inherited epilepsies. Prog Neurobiol 87:41–57
Ring AM, Cheng SX, Leng Q, Kahle KT, Rinehart J, Lalioti MD, Volkman HM, Wilson FH, Hebert SC, Lifton RP (2007) WNK4 regulates activity of the epithelial Na+ channel in vitro and in vivo. Proc Natl Acad Sci USA 104:4020–4024
Ring AM, Leng Q, Rinehart J, Wilson FH, Kahle KT, Hebert SC, Lifton RP (2007) An SGK1 site in WNK4 regulates Na+ channel and K+ channel activity and has implications for aldosterone signaling and K+ homeostasis. Proc Natl Acad Sci USA 104:4025–4029
Riordan JR (2005) Assembly of functional CFTR chloride channels. Annu Rev Physiol 67:701–718
Rossier BC, Schild L (2008) Epithelial sodium channel: mendelian versus essential hypertension. Hypertension 52:595–600
Rossier BC, Stutts MJ (2009) Activation of the epithelial sodium channel (ENaC) by serine proteases. Annu Rev Physiol 71:361–379
Rougier JS, van Bemmelen MX, Bruce MC, Jespersen T, Gavillet B, Apotheloz F, Cordonier S, Staub O, Rotin D, Abriel H (2005) Molecular determinants of voltage-gated sodium channel regulation by the Nedd4/Nedd4-like proteins. Am J Physiol Cell Physiol 288:C692–C701
Ruffieux-Daidie D, Poirot O, Boulkroun S, Verrey F, Kellenberger S, Staub O (2008) Deubiquitylation regulates activation and proteolytic cleavage of ENaC. J Am Soc Nephrol 19:2170–2180
Sander T, Schulz H, Saar K, Gennaro E, Riggio MC, Bianchi A, Zara F, Luna D, Bulteau C, Kaminska A, Ville D, Cieuta C, Picard F, Prud'homme JF, Bate L, Sundquist A, Gardiner RM, Janssen GA, de Haan GJ, Kasteleijn-Nolst-Trenite DG, Bader A, Lindhout D, Riess O, Wienker TF, Janz D, Reis A (2000) Genome search for susceptibility loci of common idiopathic generalised epilepsies. Hum Mol Genet 9:1465–1472
Schild L, Lu Y, Gautschi I, Schneeberger E, Lifton RP, Rossier BC (1996) Identification of a PY motif in the epithelial Na channel subunits as a target sequence for mutations causing channel activation found in Liddle syndrome. EMBO J 15:2381–2387
Schmidt JW, Catterall WA (1986) Biosynthesis and processing of the alpha subunit of the voltage-sensitive sodium channel in rat brain neurons. Cell 46:437–444
Schuetz F, Kumar S, Poronnik P, Adams DJ (2008) Regulation of the voltage-gated K(+) channels KCNQ2/3 and KCNQ3/5 by serum- and glucocorticoid-regulated kinase-1. Am J Physiol Cell Physiol 295:C73–C80
Schwake M, Friedrich T, Jentsch TJ (2001) An internalization signal in ClC-5, an endosomal Cl-channel mutated in dent’s disease. J Biol Chem 276:12049–12054
Schwartz AL, Ciechanover A (2008) Targeting proteins for destruction by the ubiquitin system: implications for human pathobiology. Annu Rev Pharmacol Toxicol 49:73−96
Sheng S, Carattino MD, Bruns JB, Hughey RP, Kleyman TR (2006) Furin cleavage activates the epithelial Na+ channel by relieving Na+ self-inhibition. Am J Physiol Ren Physiol 290:F1488–F1496
Shi G, Nakahira K, Hammond S, Rhodes KJ, Schechter LE, Trimmer JS (1996) Beta subunits promote K+ channel surface expression through effects early in biosynthesis. Neuron 16:843–852
Shi H, Asher C, Chigaev A, Yung Y, Reuveny E, Seger R, Garty H (2002) Interactions of beta and gamma ENaC with Nedd4 can be facilitated by an ERK-mediated phosphorylation. J Biol Chem 277:13539–13547
Shi PP, Cao XR, Sweezer EM, Kinney TS, Williams NR, Husted RF, Nair R, Weiss RM, Williamson RA, Sigmund CD, Snyder PM, Staub O, Stokes JB, Yang B (2008) Salt-sensitive hypertension and cardiac hypertrophy in mice deficient in the ubiquitin ligase Nedd4-2. Am J Physiol Ren Physiol 295:F462–F470
Shigaev A, Asher C, Latter H, Garty H, Reuveny E (2000) Regulation of sgk by aldosterone and its effects on the epithelial Na(+) channel. Am J Physiol Ren Physiol 278:F613–F619
Shimkets RA, Warnock DG, Bositis CM, Nelson-Williams C, Hansson JH, Schambelan M, Gill JR Jr, Ulick S, Milora RV, Findling JW et al (1994) Liddle’s syndrome: heritable human hypertension caused by mutations in the beta subunit of the epithelial sodium channel. Cell 79:407–414
Snyder PM, Olson DR, Kabra R, Zhou R, Steines JC (2004) cAMP and serum and glucocorticoid-inducible kinase (SGK) regulate the epithelial Na(+) channel through convergent phosphorylation of Nedd4-2. J Biol Chem 279:45753–45758
Snyder PM, Olson DR, McDonald FJ, Bucher DB (2001) Multiple WW domains, but not the C2 domain, are required for inhibition of the epithelial Na+ channel by human Nedd4. J Biol Chem 276:28321–28326
Snyder PM, Olson DR, Thomas BC (2002) Serum and glucocorticoid-regulated kinase modulates Nedd4-2-mediated inhibition of the epithelial Na+ channel. J Biol Chem 277:5–8
Snyder PM, Price MP, McDonald FJ, Adams CM, Volk KA, Zeiher BG, Stokes JB, Welsh MJ (1995) Mechanism by which Liddle’s syndrome mutations increase activity of a human epithelial Na+ channel. Cell 83:969–978
Snyder PM, Steines JC, Olson DR (2004) Relative contribution of Nedd4 and Nedd4-2 to ENaC regulation in epithelia determined by RNA interference. J Biol Chem 279:5042–5046
Soetens O, De Craene JO, Andre B (2001) Ubiquitin is required for sorting to the vacuole of the yeast general amino acid permease, Gap1. J Biol Chem 276:43949–43957
Standley S, Roche KW, McCallum J, Sans N, Wenthold RJ (2000) PDZ domain suppression of an ER retention signal in NMDA receptor NR1 splice variants. Neuron 28:887–898
Staub O, Dho S, Henry P, Correa J, Ishikawa T, McGlade J, Rotin D (1996) WW domains of Nedd4 bind to the proline-rich PY motifs in the epithelial Na+ channel deleted in Liddle’s syndrome. EMBO J 15:2371–2380
Staub O, Gautschi I, Ishikawa T, Breitschopf K, Ciechanover A, Schild L, Rotin D (1997) Regulation of stability and function of the epithelial Na+ channel (ENaC) by ubiquitination. EMBO J 16:6325–6336
Stokes A, Wakano C, Koblan-Huberson M, Adra CN, Fleig A, Turner H (2006) TRPA1 is a substrate for de-ubiquitination by the tumor suppressor CYLD. Cell Signal 18:1584–1594
Stutts MJ, Rossier BC, Boucher RC (1997) Cystic fibrosis transmembrane conductance regulator inverts protein kinase A-mediated regulation of epithelial sodium channel single channel kinetics. J Biol Chem 272:14037–14040
Suzuki M, Mizuno A (2004) A novel human Cl(-) channel family related to Drosophila flightless locus. J Biol Chem 279:22461–22468
Swanson R, Locher M, Hochstrasser M (2001) A conserved ubiquitin ligase of the nuclear envelope/endoplasmic reticulum that functions in both ER-associated and Matalpha2 repressor degradation. Genes Dev 15:2660–2674
Thomas MA, Zosso N, Scerri I, Demaurex N, Chanson M, Staub O (2003) A tyrosine-based sorting signal is involved in connexin43 stability and gap junction turnover. J Cell Sci 116:2213–2222
Tian Y, Kolb R, Hong JH, Carroll J, Li D, You J, Bronson R, Yaffe MB, Zhou J, Benjamin T (2007) TAZ promotes PC2 degradation through a SCFbeta-Trcp E3 ligase complex. Mol Cell Biol 27:6383–6395
Tiffany AM, Manganas LN, Kim E, Hsueh YP, Sheng M, Trimmer JS (2000) PSD-95 and SAP97 exhibit distinct mechanisms for regulating K(+) channel surface expression and clustering. J Cell Biol 148:147–158
Uchida S, Marumo F (2000) Severely impaired urine-concentrating ability in mice lacking the CLC-K1 chloride channel. Exp Nephrol 8:361–365
Ushioda R, Hoseki J, Araki K, Jansen G, Thomas DY, Nagata K (2008) ERdj5 is required as a disulfide reductase for degradation of misfolded proteins in the ER. Science 321:569–572
Valentijn JA, Fyfe GK, Canessa CM (1998) Biosynthesis and processing of epithelial sodium channels in Xenopus oocytes. J Biol Chem 273:30344–30351
Vallet V, Chraibi A, Gaeggeler HP, Horisberger JD, Rossier BC (1997) An epithelial serine protease activates the amiloride-sensitive sodium channel. Nature 389:607–610
van Bemmelen MX, Rougier JS, Gavillet B, Apotheloz F, Daidie D, Tateyama M, Rivolta I, Thomas MA, Kass RS, Staub O, Abriel H (2004) Cardiac voltage-gated sodium channel Nav1.5 is regulated by Nedd4-2 mediated ubiquitination. Circ Res 95:284–291
VanSlyke JK, Deschenes SM, Musil LS (2000) Intracellular transport, assembly, and degradation of wild-type and disease-linked mutant gap junction proteins. Mol Biol Cell 11:1933–1946
Vashist S, Ng DT (2004) Misfolded proteins are sorted by a sequential checkpoint mechanism of ER quality control. J Cell Biol 165:41–52
Wanamaker CP, Christianson JC, Green WN (2003) Regulation of nicotinic acetylcholine receptor assembly. Ann NY Acad Sci 998:66–80
Wang J, Peng Q, Lin Q, Childress C, Carey D, Yang W (2010) Calcium activates Nedd4 E3 ubiquitin ligases by releasing the C2 domain-mediated auto-inhibition. J Biol Chem 285:12279–12288
Wang Y, Fu X, Gaiser S, Kottgen M, Kramer-Zucker A, Walz G, Wegierski T (2007) OS-9 regulates the transit and polyubiquitination of TRPV4 in the endoplasmic reticulum. J Biol Chem 282:36561–36570
Ward CL, Omura S, Kopito RR (1995) Degradation of CFTR by the ubiquitin-proteasome pathway. Cell 83:121–127
Webster JM, Tiwari S, Weissman AM, Wojcikiewicz RJ (2003) Inositol 1, 4, 5-trisphosphate receptor ubiquitination is mediated by mammalian Ubc7, a component of the endoplasmic reticulum-associated degradation pathway, and is inhibited by chelation of intracellular Zn2+. J Biol Chem 278:38238–38246
Webster MK, Goya L, Ge Y, Maiyar AC, Firestone GL (1993) Characterization of sgk, a novel member of the serine/threonine protein kinase gene family which is transcriptionally induced by glucocorticoids and serum. Mol Cell Biol 13:2031–2040
Wegierski T, Hill K, Schaefer M, Walz G (2006) The HECT ubiquitin ligase AIP4 regulates the cell surface expression of select TRP channels. EMBO J 25:5659–5669
Weisz OA, Wang JM, Edinger RS, Johnson JP (2000) Non-coordinate regulation of endogenous epithelial sodium channel (ENaC) subunit expression at the apical membrane of A6 cells in response to various transporting conditions. J Biol Chem 275:39886–39893
Wellhauser L, D'Antonio C, Bear CE (2010) ClC transporters: discoveries and challenges in defining the mechanisms underlying function and regulation of ClC-5. Pflugers Arch 460:543−557
Wiemuth D, Ke Y, Rohlfs M, McDonald FJ (2007) Epithelial sodium channel (ENaC) is multi-ubiquitinated at the cell surface. Biochem J 405:147–155
Wiesner S, Ogunjimi AA, Wang HR, Rotin D, Sicheri F, Wrana JL, Forman-Kay JD (2007) Autoinhibition of the HECT-type ubiquitin ligase Smurf2 through its C2 domain. Cell 130:651–662
Winget JM, Mayor T The diversity of ubiquitin recognition: hot spots and varied specificity. Mol Cell 38:627–635
Wojcikiewicz RJ, Xu Q, Webster JM, Alzayady K, Gao C (2003) Ubiquitination and proteasomal degradation of endogenous and exogenous inositol 1, 4, 5-trisphosphate receptors in alpha T3—1 anterior pituitary cells. J Biol Chem 278:940–947
Wrong OM, Norden AG, Feest TG (1994) Dent’s disease; a familial proximal renal tubular syndrome with low-molecular-weight proteinuria, hypercalciuria, nephrocalcinosis, metabolic bone disease, progressive renal failure and a marked male predominance. QJM 87:473–493
Wulff P, Vallon V, Huang DY, Volkl H, Yu F, Richter K, Jansen M, Schlunz M, Klingel K, Loffing J, Kauselmann G, Bosl MR, Lang F, Kuhl D (2002) Impaired renal Na(+) retention in the sgk1-knockout mouse. J Clin Invest 110:1263–1268
Xu BE, Stippec S, Chu PY, Lazrak A, Li XJ, Lee BH, English JM, Ortega B, Huang CL, Cobb MH (2005) WNK1 activates SGK1 to regulate the epithelial sodium channel. Proc Natl Acad Sci USA 102:10315–10320
Xu P, Duong DM, Seyfried NT, Cheng D, Xie Y, Robert J, Rush J, Hochstrasser M, Finley D, Peng J (2009) Quantitative proteomics reveals the function of unconventional ubiquitin chains in proteasomal degradation. Cell 137:133–145
Yang JW, Vacher H, Park KS, Clark E, Trimmer JS (2007) Trafficking-dependent phosphorylation of Kv1.2 regulates voltage-gated potassium channel cell surface expression. Proc Natl Acad Sci USA 104:20055–20060
Yang LM, Rinke R, Korbmacher C (2006) Stimulation of the epithelial sodium channel (ENaC) by cAMP involves putative ERK phosphorylation sites in the C termini of the channel’s beta- and gamma-subunit. J Biol Chem 281:9859–9868
Yang M, Omura S, Bonifacino JS, Weissman AM (1998) Novel aspects of degradation of T cell receptor subunits from the endoplasmic reticulum (ER) in T cells: importance of oligosaccharide processing, ubiquitination, and proteasome-dependent removal from ER membranes. J Exp Med 187:835–846
Ye Y, Meyer HH, Rapoport TA (2003) Function of the p97-Ufd1-Npl4 complex in retrotranslocation from the ER to the cytosol: dual recognition of nonubiquitinated polypeptide segments and polyubiquitin chains. J Cell Biol 162:71–84
Ye Y, Shibata Y, Yun C, Ron D, Rapoport TA (2004) A membrane protein complex mediates retro-translocation from the ER lumen into the cytosol. Nature 429:841–847
Yoshida Y, Chiba T, Tokunaga F, Kawasaki H, Iwai K, Suzuki T, Ito Y, Matsuoka K, Yoshida M, Tanaka K, Tai T (2002) E3 ubiquitin ligase that recognizes sugar chains. Nature 418:438–442
Yu FH, Catterall WA (2003) Overview of the voltage-gated sodium channel family. Genome Biol 4:207
Zerangue N, Schwappach B, Jan YN, Jan LY (1999) A new ER trafficking signal regulates the subunit stoichiometry of plasma membrane K(ATP) channels. Neuron 22:537–548
Zhang W, Na T, Wu G, Jing H, Peng JB (2010) Downregulation of intestinal apical calcium entry channel TRPV6 by ubiquitin E3 ligase NEDD4-2. J Biol Chem (in press)
Zhang Y, Nijbroek G, Sullivan ML, McCracken AA, Watkins SC, Michaelis S, Brodsky JL (2001) Hsp70 molecular chaperone facilitates endoplasmic reticulum-associated protein degradation of cystic fibrosis transmembrane conductance regulator in yeast. Mol Biol Cell 12:1303–1314
Zhou R, Patel SV, Snyder PM (2007) Nedd4-2 catalyzes ubiquitination and degradation of cell surface ENaC. J Biol Chem 282:20207–20212
Zhou Z, Treis D, Schubert SC, Harm M, Schatterny J, Hirtz S, Duerr J, Boucher RC, Mall MA (2008) Preventive but not late amiloride therapy reduces morbidity and mortality of lung disease in betaENaC-overexpressing mice. Am J Respir Crit Care Med 178:1245–1256
Acknowledgements
Work from the authors’ laboratories described in this review was funded by the Canadian Institute of Health Research and the Canadian CF Foundation (to DR), and the Swiss National Science Foundation (to OS).
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Rotin, D., Staub, O. Role of the ubiquitin system in regulating ion transport. Pflugers Arch - Eur J Physiol 461, 1–21 (2011). https://doi.org/10.1007/s00424-010-0893-2
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DOI: https://doi.org/10.1007/s00424-010-0893-2