ENaC Degradation in A6 Cells by the Ubiquitin-Proteosome Proteolytic Pathway

Malik, Bela; Schlanger, Lynn E.; Al‐Khalili, Otor; Bao, Hui‐Fang; Yue, Gang; Price, S. Russ; Mitch, William E.; Eaton, Douglas C.

doi:10.1074/jbc.m010626200

Cited by 107 publications

(109 citation statements)

References 26 publications

(35 reference statements)

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“…However, in epithelial cells, ␤-ENaC appears to act as an auxiliary subunit to regulate ENaC activity and trafficking to the plasma membrane (20 -22). Although the data from other investigators have shown that aldosterone has no chronic effect on the expression of ␤-ENaC in rat kidney (22,23), our results have shown that aldosterone stimulates expression of ␤-ENaC in A6 cells (24). Therefore, it is possible that the ␣-ENaC channel in human B lymphocytes is regulated by aldosterone via expression of the ␤ subunit.…”

Section: Resultscontrasting

confidence: 82%

“…Another steroid, dexamethasone, is known to increase expression of ␤-and ␥-ENaC protein levels in lung epithelial cells (32). Our earlier studies also suggest that aldosterone increases the mRNA level of ␤-ENaC and the protein level of ␥-ENaC in A6 distal nephron cells (24). The concentration of aldosterone we used was high enough that it could have activated both mineralocorticoid and glucocorticoid receptors.…”

Section: Discussionmentioning

confidence: 71%

“…Recent studies have demonstrated that ENaC is regulated by Nedd4, a ubiquitin protein ligase (26 -30). In A6 cells, a proteosome inhibitor strongly increases the cellular amount of ␤-ENaC but only causes a slight increase in ␣-ENaC protein (31). These results suggest the possibility that degradation of the plasma membrane ␤-ENaC might be much faster than that of ␣-ENaC in human B lymphocytes; thus, leading to a situation in which, although there is message and protein for both ␣-and ␤-ENaC, only ␣-ENaC is present in the membrane at a high enough concentration to be the predominant channel-forming subunit.…”

Section: Discussionmentioning

confidence: 98%

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Steroids and Exogenous γ-ENaC Subunit Modulate Cation Channels Formed by α-ENaC in Human B Lymphocytes

Al‐Khalili

Ramosevac

et al. 2004

Journal of Biological Chemistry

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Previous studies using whole-cell recording methods suggest that human B lymphocytes express an amiloride-sensitive, sodium-permeable channel. The present studies aim to determine whether this channel has biophysical properties and a molecular structure related to the ␣, ␤, and ␥ subunits of the epithelial sodium channel (ENaC). Reverse transcriptase polymerase chain reaction and Northern blots showed that human B lymphocytes express messages for both ␣-and ␤-but not ␥-ENaC. Western blots showed that both ␣-and ␤-but not ␥-ENaC proteins are expressed and strongly reduced by antisense oligonucleotides. Patch clamp experiments demonstrated that lymphocyte sodium channels are not active in cell-attached patches. However, membrane stretch can activate a 21-pS nonselective cation channel. The frequency of observance of this channel was significantly reduced by antisense oligonucleotide against ␣-ENaC but not by antisense oligonucleotide against ␤-ENaC, indicating that only the ␣ subunit of ENaC is necessary to form stretch-activated cation channels. Aldosterone (1.5 M) reduced the frequency of observance of 21-pS ␣-ENaC channels and simultaneously induced the appearance of spontaneously active 10-pS channels. Antisense oligonucleotide experiments showed that this 10-pS channel is formed from ␣-and ␤-ENaC. After expression of exogenous ␥-ENaC, aldosterone again reduced the frequency of observance of the 21-pS ␣-ENaC channel but induced the appearance of a 5-pS channel, presumably a ␣␤␥-ENaC channel. In the absence of aldosterone, the ␣ subunit forms an ␣-cryptic channel that is activated by stretch, and in the presence of aldosterone, ␤ and ␣ subunits together form an active channel that is modulated by aldosterone.The superfamily of proteins to which the epithelial sodium channel (ENaC) 1 belongs generally mediates cation transport across cell membranes. ENaC, itself, is usually associated with sodium transport across the apical membrane of a variety of epithelia including the colon, lung, and kidney. Since 1994, when ENaC was initially cloned from rat colon (1), the biophysical properties and molecular structure of ENaC have been studied extensively. Several lines of evidence suggest that ENaC, including human ENaC (hENaC), is typically composed of three subunits, ␣, ␤, and ␥, and that all three subunits are required to form a functional ␣␤␥-ENaC channel complex (1-6). In heterologous expression systems, maximal expression of the hENaC channel requires co-expression of all three subunits (7, 8), and in oocytes, expression of ␣-ENaC cRNA alone produces little expression of any amiloride-sensitive currents. However, in other cell types, expression of the exogenous ␣-ENaC subunit alone can form a stretch-activated nonselective cation channel (9), and a similar nonselective cation channel has also been described in native lung epithelial alveolar type II cells (10 -12). This lung cation channel appears to be formed from ␣-ENaC alone and is equally permeable to Na ϩ and K ϩ , is sensitive to steroid hormones, and has a h...

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Section: Resultscontrasting

confidence: 82%

Section: Discussionmentioning

confidence: 71%

Section: Discussionmentioning

confidence: 98%

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Steroids and Exogenous γ-ENaC Subunit Modulate Cation Channels Formed by α-ENaC in Human B Lymphocytes

Al‐Khalili

Ramosevac

et al. 2004

Journal of Biological Chemistry

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“…Cleaved Forms of ENaC Are Found on the Cell SurfaceProteolytic processing of ENaC subunits is not restricted to degradation in lysosomes or by proteasomes (19,37), as processed subunits are clearly expressed at the plasma membrane. Jovov et al (38) previously identified trypsin-sensitive sites within the cytoplasmic carboxyl termini of the ␤-and ␥-subunits.…”

Section: N-glycans Are Processed On All Three Subunits Of Enac-mentioning

confidence: 99%

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

Hughey¹,

Mueller²,

Bruns³

et al. 2003

Journal of Biological Chemistry

248

264

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The epithelial Na ؉ channel (ENaC) is a tetramer of two ␣-, one ␤-, and one ␥-subunit, but little is known about its assembly and processing. Because co-expression of mouse ENaC subunits with three different carboxyl-terminal epitope tags produced an amiloride-sensitive sodium current in oocytes, these tagged subunits were expressed in both Chinese hamster ovary or Madin-Darby canine kidney type 1 epithelial cells for further study. When expressed alone ␣-(95 kDa), ␤-(96 kDa), and ␥-subunits (93 kDa) each produced a single band on SDS gels by immunoblotting. However, co-expression of ␣␤␥ENaC subunits revealed a second band for each subunit (65 kDa for ␣, 110 kDa for ␤, and 75 kDa for ␥) that exhibited N-glycans that had been processed to complex type based on sensitivity to treatment with neuraminidase, resistance to cleavage by endoglycosidase H, and GalNAc-independent labeling with [ 3 H]Gal in glycosylation-defective Chinese hamster ovary cells (ldlD). The smaller size of the processed ␣-and ␥-subunits is also consistent with proteolytic cleavage. By using ␣-and ␥-subunits with epitope tags at both the amino and carboxyl termini, proteolytic processing of the ␣-and ␥-subunits was confirmed by isolation of an additional epitope-tagged fragment from the amino terminus (30 kDa for ␣ and 18 kDa for ␥) consistent with cleavage within the extracellular loop. The fragments remain stably associated with the channel as shown by immunoblotting of co-immunoprecipitates, suggesting that proteolytic cleavage represents maturation rather than degradation of the channel.The amiloride-sensitive epithelial Na ϩ channel (ENaC) 1 is composed of three structurally related subunits, termed ␣-, ␤-, and ␥-ENaC. The three subunits exhibit limited amino acid sequence identity (30 -40%) but are structurally similar with two membrane-spanning domains and cytosolic amino and carboxyl termini. We and others have shown that ENaC expressed in Xenopus oocytes has a subunit stoichiometry of two ␣-, one ␤-, and one ␥-subunit (1, 2

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“…KIAA1203 UniGene Cluster Hs.16953 is expressed in kidney and contains ubiquitin carboxyl-terminal hydrolase family two domain which may function, through proteosomal pathway, as a regulator of the amiloride-sensitive epithelial Na(+) channel SCNN1, 44 whose two subunits, SCNN1B and SCNN1G are located in KIAA1203 in close proximity on 16p11.2. Three single base pair substitutions were found in the KIAA1203 gene.…”

Section: Haplotype Analysismentioning

confidence: 99%

Familial juvenile hyperuricaemic nephropathy (FJHN): linkage analysis in 15 families, physical and transcriptional characterisation of the FJHN critical region on chromosome 16p11.2 and the analysis of seven candidate genes

et al. 2003

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Familial juvenile hyperuricaemic nephropathy (FJHN) is an autosomal dominant renal disease characterised by juvenile onset of hyperuricaemia, gouty arthritis, and progressive renal failure at an early age. Recent studies in four kindreds showed linkage of a gene for FJHN to the same genomic interval on chromosome 16p11.2, where the gene for the phenotypically similar medullary cystic disease type 2 (MCKD2) has been localised. In this study we performed linkage analysis in additional 15 FJHN families. Linkage of FJHN to 16p11.2 was confirmed in six families, which suggests that, in a large proportion of FJHN kindreds, the disease is likely to be caused by a gene or genes located outside of 16p11.2. Haplotype analysis of the new and previously analysed families provided two non-overlapping critical regions on 16p11.2 -FJHN1, delimited by markers D16S499-D16S3036 and FJHN2, delimited by markers D16S412-D16S3116. Considering MCKD2 to be a distinct molecular entity, the analysis suggests that as many as three kidney disease genes may be located in close proximity on 16p11.2. From genomic databases we compiled integrated physical and transcription maps of whole critical genomic region in which 45 known genes and 129 predicted loci have been localised. We selected, analysed and found no pathogenic mutations in seven candidate genes. The linkage and haplotype analysis reported here demonstrates the genetic heterogeneity of FJHN. The report of integrated physical and mostly in-silico predicted transcription maps of the FJHN critical region provides a basis for precise experimental annotation of the current transcript map, which is essential for final identification of the FJHN gene(s).

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ENaC Degradation in A6 Cells by the Ubiquitin-Proteosome Proteolytic Pathway

Abstract: Amiloride-sensitive epithelial Na؉ channels (ENaC) are responsible for trans-epithelial Na

Cited by 107 publications

References 26 publications

Steroids and Exogenous γ-ENaC Subunit Modulate Cation Channels Formed by α-ENaC in Human B Lymphocytes

Steroids and Exogenous γ-ENaC Subunit Modulate Cation Channels Formed by α-ENaC in Human B Lymphocytes

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

Familial juvenile hyperuricaemic nephropathy (FJHN): linkage analysis in 15 families, physical and transcriptional characterisation of the FJHN critical region on chromosome 16p11.2 and the analysis of seven candidate genes

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