ABCMdb

ABCC7 p.Thr663Ala

Predicted by SNAP2:	A: N (66%), C: N (53%), D: N (72%), E: N (66%), F: D (66%), G: N (61%), H: D (59%), I: N (61%), K: N (61%), L: N (61%), M: D (59%), N: N (82%), P: N (66%), Q: N (72%), R: N (61%), S: N (87%), V: N (66%), W: D (66%), Y: D (63%),
Predicted by PROVEAN:	A: N, C: D, D: N, E: N, F: D, G: N, H: N, I: N, K: N, L: N, M: N, N: N, P: N, Q: N, R: N, S: N, V: N, W: D, Y: D,

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[hide] Cystic fibrosis transmembrane conductance regulato... J Biol Chem. 2004 May 28;279(22):23183-92. Epub 2004 Mar 26. Yan W, Samaha FF, Ramkumar M, Kleyman TR, Rubenstein RC
Cystic fibrosis transmembrane conductance regulator differentially regulates human and mouse epithelial sodium channels in Xenopus oocytes.
J Biol Chem. 2004 May 28;279(22):23183-92. Epub 2004 Mar 26., 2004-05-28 [PMID:15047694]

Abstract [show]
The cystic fibrosis transmembrane conductance regulator (CFTR), in addition to its well defined Cl- channel properties, regulates other ion channels. CFTR inhibits murine or rat epithelial Na+ channel (mENaC or rENaC) currents in many epithelial and non-epithelial cells, whereas murine or rat ENaC increases CFTR functional expression. These regulatory interactions are reproduced in Xenopus oocytes where both the open probability and surface expression of wild type CFTR Cl- channels are increased when CFTR is co-expressed with alphabetagamma mENaC, and conversely the activity of mENaC is inhibited after wild type CFTR activation. Using the Xenopus oocyte expression system, differences in functional regulatory interactions were observed when CFTR was co-expressed with either alphabetagamma mENaC or alphabetagamma human ENaC (hENaC). Co-expression of CFTR and alphabetagamma mENaC or hENaC resulted in an approximately 3-fold increase in CFTR Cl- current compared with oocytes expressing CFTR alone. Oocytes co-injected with both CFTR and mENaC or hENaC expressed an amiloride-sensitive whole cell current that was decreased compared with that observed with the injection of mENaC or hENaC alone before CFTR activation with forskolin/3-isobutyl-1-methylxanthine. CFTR activation resulted in a further 50% decrease in mENaC-mediated currents, an approximately 20% decrease in alpha-T663-hENaC-mediated currents, and essentially no change in alpha-A663-hENaC-mediated currents. Changes in ENaC functional expression correlated with ENaC surface expression by oocyte surface biotinylation experiments. Assessment of regulatory interactions between CFTR and chimeric mouse/human ENaCs suggest that the 20 C-terminal amino acid residues of alpha ENaC confer species specificity regarding ENaC inhibition by activated CFTR.

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35 We also tested the hypothesis that a naturally occurring polymorphism in the C terminus of ␣ hENaC, substitution of Ala at residue 663 for Thr (T663A), which we have recently shown to decrease the functional and surface expression of hENaC in oocytes,2 would influence regulatory interactions between CFTR and hENaC. Login to comment
79 Co-expression of CFTR and hENaC-We next assessed regulatory interactions between CFTR and hENaC as well as the potential influence of the T663A functional polymorphism of ␣ hENaC described recently by our group2 on these interactions. Login to comment
120 We recently observed that the C-terminal 20 residues (residues 650-669) of ␣ hENaC, when expressed at the C terminus of a murine/human ␣ ENaC chimera in place of the 21 C-terminal amino acids of ␣ mENaC (residues 679-699), were sufficient to confer functionality of the T663A polymorphism of ␣ hENaC.2 Given that mENaC (Fig. 1 and Ref. Login to comment
134 As our data suggest differences between the interregulation of wild type CFTR and hENaC versus wild type CFTR and mENaC, we sought to characterize regulatory interactions between ⌬F508-CFTR and hENaC as well as the potential influence of the T663A functional polymorphism on these interactions (Fig. 7). Login to comment

[hide] CFTR fails to inhibit the epithelial sodium channe... J Physiol. 2005 May 1;564(Pt 3):671-82. Epub 2005 Mar 3. Nagel G, Barbry P, Chabot H, Brochiero E, Hartung K, Grygorczyk R
CFTR fails to inhibit the epithelial sodium channel ENaC expressed in Xenopus laevis oocytes.
J Physiol. 2005 May 1;564(Pt 3):671-82. Epub 2005 Mar 3., 2005-05-01 [PMID:15746174]

Abstract [show]
The cystic fibrosis transmembrane conductance regulator (CFTR) plays a crucial role in regulating fluid secretion by the airways, intestines, sweat glands and other epithelial tissues. It is well established that the CFTR is a cAMP-activated, nucleotide-dependent anion channel, but additional functions are often attributed to it, including regulation of the epithelial sodium channel (ENaC). The absence of CFTR-dependent ENaC inhibition and the resulting sodium hyperabsorption were postulated to be a major electrolyte transport abnormality in cystic fibrosis (CF)-affected epithelia. Several ex vivo studies, including those that used the Xenopus oocyte expression system, have reported ENaC inhibition by activated CFTR, but contradictory results have also been obtained. Because CFTR-ENaC interactions have important implications in the pathogenesis of CF, the present investigation was undertaken by our three independent laboratories to resolve whether CFTR regulates ENaC in oocytes and to clarify potential sources of previously reported dissimilar observations. Using different experimental protocols and a wide range of channel expression levels, we found no evidence that activated CFTR regulates ENaC when oocyte membrane potential was carefully clamped. We determined that an apparent CFTR-dependent ENaC inhibition could be observed when resistance in series with the oocyte membrane was not low enough or the feedback voltage gain was not high enough. We suggest that the inhibitory effect of CFTR on ENaC reported in some earlier oocyte studies could be attributed to problems arising from high levels of channel expression and suboptimal recording conditions, that is, large series resistance and/or insufficient feedback voltage gain.

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184 They found less than 35% inhibition of murine ENaC by activated CFTR (their Fig. 1A), only a modest 20% inhibition for wildtype α-hENaC and no change for T663A α-hENaC, where threonine 663 (wildtype) was replaced by alanine (their Fig. 2). Login to comment

[hide] Mutations in the amiloride-sensitive epithelial so... Hum Mutat. 2009 Jul;30(7):1093-103. Azad AK, Rauh R, Vermeulen F, Jaspers M, Korbmacher J, Boissier B, Bassinet L, Fichou Y, des Georges M, Stanke F, De Boeck K, Dupont L, Balascakova M, Hjelte L, Lebecque P, Radojkovic D, Castellani C, Schwartz M, Stuhrmann M, Schwarz M, Skalicka V, de Monestrol I, Girodon E, Ferec C, Claustres M, Tummler B, Cassiman JJ, Korbmacher C, Cuppens H
Mutations in the amiloride-sensitive epithelial sodium channel in patients with cystic fibrosis-like disease.
Hum Mutat. 2009 Jul;30(7):1093-103., [PMID:19462466]

Abstract [show]
We investigated whether mutations in the genes that code for the different subunits of the amiloride-sensitive epithelial sodium channel (ENaC) might result in cystic fibrosis (CF)-like disease. In a small fraction of the patients, the disease could be potentially explained by an ENaC mutation by a Mendelian mechanism, such as p.V114I and p.F61L in SCNN1A. More importantly, a more than three-fold significant increase in incidence of several rare ENaC polymorphisms was found in the patient group (30% vs. 9% in controls), indicating an involvement of ENaC in some patients by a polygenetic mechanism. Specifically, a significantly higher number of patients carried c.-55+5G>C or p.W493R in SCNN1A in the heterozygous state, with odds ratios (ORs) of 13.5 and 2.7, respectively.The p.W493R-SCNN1A polymorphism was even found to result in a four-fold more active ENaC channel when heterologously expressed in Xenopus laevis oocytes. About 1 in 975 individuals in the general population will be heterozygous for the hyperactive p.W493R-SCNN1A mutation and a cystic fibrosis transmembrane conductance regulator (CFTR) gene that results in very low amounts (0-10%) functional CFTR. These ENaC/CFTR genotypes may play a hitherto unrecognized role in lung diseases.

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137 Since p.T663A-SCNN1A is a frequent variant, each missense mutation was introduced on the p.T663A-SCNN1A background as found in the patient. Login to comment
138 When the cis or trans configuration of the p.T663A-SCNN1A background could not be determined, the missense mutation was generated on both the p.T663-SCNN1A and p.A663-SCNN1A background. Login to comment

[hide] Upregulated expression of ENaC in human CF nasal e... J Cyst Fibros. 2008 May;7(3):197-205. Epub 2007 Sep 4. Bangel N, Dahlhoff C, Sobczak K, Weber WM, Kusche-Vihrog K
Upregulated expression of ENaC in human CF nasal epithelium.
J Cyst Fibros. 2008 May;7(3):197-205. Epub 2007 Sep 4., [PMID:17766193]

Abstract [show]
Cystic fibrosis (CF) is characterised by the absence of CFTR function resulting in a reduced Cl(-) secretion and an increase in Na+ absorption. This Na+ hyperabsorption is mediated by the human amiloride-sensitive epithelial sodium channel (ENaC), but the underlying mechanisms are still unknown. After demonstrating functional differences of the Na+ absorption in CF and non-CF epithelia in Ussing chamber experiments with human primary cultures, we compared ENaC sequences from CF and non-CF human nasal tissue (hnENaC), investigated the mRNA transcription levels via real-time PCR and studied the protein expression in Western blot analyses. We found no differences in the sequences of CF and non-CF hnENaC, but identified some polymorphisms. The real-time experiments revealed an enhanced mRNA amount of all three hnENaC subunits in CF tissue. By comparing the two groups on the protein level, we observed differences in the abundance of the Na+ channel. While the alpha- and beta-hnENaC protein amount was increased in CF tissue the gamma-hnENaC was decreased. We conclude that the Na+ hyperabsorption in CF is not caused by mutations in hnENaC, but by an increase in the transcription of the hnENaC subunits. This could be induced by a disturbed regulation of the channel in CF.

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164 We found a similar situation for Table 2 Single nucleotide polymorphisms in the α-, β- and γ-hnENaC subunits from CF and non-CF tissue and appropriate references α-ENaC Reference β-ENaC Reference γ-ENaC Reference T663A [53,54] A314G [55] R178W P502A A614S [53,55,56] A336P [53,56] F339S A350T Y369S D375G S458R [55] Fig. 2. mRNA expression data for all three hnENaC subunits of CF patients. Login to comment
214 We identified a common T663A polymorphism within the C-terminus of the hnENaC α-subunit of CF and non-CF epithelia. Login to comment
170 We found a similar situation for Table 2 Single nucleotide polymorphisms in the b1;-, b2;- and b3;-hnENaC subunits from CF and non-CF tissue and appropriate references b1;-ENaC Reference b2;-ENaC Reference b3;-ENaC Reference T663A [53,54] A314G [55] R178W P502A A614S [53,55,56] A336P [53,56] F339S A350T Y369S D375G S458R [55] Fig. 2. Login to comment
221 We identified a common T663A polymorphism within the C-terminus of the hnENaC b1;-subunit of CF and non-CF epithelia. Login to comment

[hide] Assessment of epithelial sodium channel variants i... J Cyst Fibros. 2015 Apr 17. pii: S1569-1993(15)00101-0. doi: 10.1016/j.jcf.2015.04.001. Brennan ML, Pique LM, Schrijver I
Assessment of epithelial sodium channel variants in nonwhite cystic fibrosis patients with non-diagnostic CFTR genotypes.
J Cyst Fibros. 2015 Apr 17. pii: S1569-1993(15)00101-0. doi: 10.1016/j.jcf.2015.04.001., [PMID:25900089]

Abstract [show]
PURPOSE: Several lines of evidence suggest a role for the epithelial sodium channel (ENaC) in cystic fibrosis (CF). The purpose of our study was to assess the contribution of genetic variants in the ENaC subunits (alpha, beta, gamma) in nonwhite CF patients in whom CFTR molecular testing has been non-diagnostic. METHODS: Samples were obtained from patients who were nonwhite and whose molecular CFTR testing did not identify two mutations. Sequencing of the SCNN1A, B, and G genes was performed and variants assessed for pathogenicity and association with CF using databases, protein and splice site mutation analysis software, and literature review. RESULTS: We identified four nonsynonymous amino acid variants in SCNN1A, three in SCNN1B and one in SCNN1G. There was no convincing evidence of pathogenicity. Whereas all have been reported in the dbSNP database, only p.Ala334Thr, p.Val573Ile, and p.Thr663Ala in SCNN1A, p.Gly442Val in SCNN1B and p.Gly183Ser in SCNN1G were previously reported in ENaC genetic studies of CF or CF-like patients. Synonymous substitutions were also observed but novel synonymous variants were not detected. CONCLUSION: There is no conclusive association of ENaC genetic variants with CF in nonwhite CF patients.

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44 Ethnicity # of subjectsa SCNN1 subunit Genotype/clinical characteristicsb Sequence variantc Caucasian (France) [26] 56 B Two CFTR mutations; Classic CF p.Thr313Met, p.Gly589Ser G p.Leu481Gln, p.Val546Ile Caucasian (Europe) [28] 29; 47 A One/no CFTR mutation; typical and atypical CF cases c.-760ANG, c.-717GNC, c.-68CNT, p.Pro33Pro p.Phe61Leu, p.Val114Ile, p.Leu180Leu, p.Arg181Trp, p.Ala334Thr, p.Trp493Arg, p.Thr663Ala B p.Ser82Cys, p.Pro93Pro, c.777-5TNC, p.Phe293Phe, p.Ile515Ile, p.Gly589Ser, p.Asp629Asp G p.Tyr129Tyr, p.Ile158Ile, p.Gly183Gly, p.Glu197Lys, c.1176+14ANG, c.1373+29TNC, c.1432-7GNA, p.Leu649Leu Unknown (USA) [23] 20 A No CFTR mutations p.Arg181Trp B Non-classic CF p.Glu539Lys, c.1543-2ANG African 5; 55 (Rwanda) [27] A One/no CFTR mutation; CF-like c.-28TNC, p.Val573Ile B p.Val348Met, p.Gly442Val, p.Thr577Thr, c.1346+28CNT G p.Ser212Ser, c.1176+30GNC Caucasian (Spain) [29] 10 A One/no CFTR mutation; CF and CF-like p.Val14Gly, p.Leu203Leu, p.Arg204Trp, p.Ala304Pro, p.Ala357Thr, p.Cys641Phe, c.875+35GNA B p.Phe293Phe, p.Arg563Gln, p.Pro574Pro G p.Gly183Gly Caucasian/African (France) [24] 20; 35 B One/no CFTR mutation p.Ser82Cys, p.Asn288Ser,p.Pro369Thr G Bronchiectasis p.Gly183Ser, p.Glu197Lys Caucasian (Italy) [35] 24; 15 A One/no CFTR mutation; CFTR-related disorders c.-760ANG, p.Ala334Thr, p.Thr663Ala B p.Pro93Pro, p.Phe293Phe G p.Tyr129Tyr, p.Ile158Ile, p.Gly183Gly, p.Leu649Leu, c.1176+14ANG, c.1373+29TNC, c.1432-7GNA a Listed as number of subjects per genotype (if known). Login to comment