A Missense Mutation in the Extracellular Domain of Αenac

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A Missense Mutation in the Extracellular Domain of aENaC Causes Liddle Syndrome

† † † Mahdi Salih,* Ivan Gautschi, Miguel X. van Bemmelen, Michael Di Benedetto, ‡ † Alice S. Brooks, Dorien Lugtenberg,§ Laurent Schild, and Ewout J. Hoorn*

Departments of *Internal Medicine and ‡Clinical Genetics, Erasmus Medical Center, Rotterdam, The Netherlands; †Département de Pharmacologie et de Toxicologie, Université de Lausanne, Lausanne, Switzerland; and §Department of Human Genetics, Radboud University Medical Center, Nijmegen, The Netherlands

ABSTRACT Liddle syndrome is an autosomal dominant form of hypokalemic hypertension due to mutations in the b-or g-subunit of the epithelial sodium channel (ENaC). Here, we describe a family with Liddle syndrome due to a mutation in aENaC. The proband was referred because of resistant hypokalemic hypertension, suppressed renin and aldosterone, and no mutations in the genes encoding b-orgENaC. Exome sequencing revealed a heterozygous, nonconservative T.C single-nucleotide mutation in aENaC that substituted Cys479 with Arg (C479R). C479 is a highly conserved residue in the extracellular domain of ENaC and likely involved in a disulfide bridge with the partner cysteine C394. In oocytes, the C479R and C394S mutations resulted in similar twofold increases in amiloride-sensitive ENaC current. Quantification of mature cleaved aENaC in membrane fractions showed that the number of channels did not increase with these mutations. Trypsin, which increases open probability of the channel by proteolytic cleavage, resulted in significantly higher currents in the wild type than in C479R or C394S mutants. In summary, a mutation in the extracellular domain of aENaC causes Liddle syndrome by increasing intrinsic channel activity. This mechanism differs from that of the b-andg-mutations, which result in an increase in channel density at the cell surface. This mutation may explain other cases of patients with resistant hypertension and also provides novel insight into ENaC activation, which is relevant for kidney sodium reabsorption and salt-sensitive hypertension.

J Am Soc Nephrol 28: 3291–3299, 2017. doi: https://doi.org/10.1681/ASN.2016111163

Hypertension is one of the most common noncom- sodium channel (ENaC).6 In 1963, Liddle et al.7 municable disorders worldwide and a major risk reported a “familial renal disorder simulating factor for stroke, myocardial infarction, heart fail- primary aldosteronism but with negligible aldosterone ure, and ESRD.1 Primary or essential hypertension secretion.” Liddle syndrome or pseudoaldosteron- is a complex genetic trait that is also inﬂuenced by ism (OMIM 177200) is now known as an autosomal other risk factors, such as dietary sodium and po- dominant form of salt-sensitive hypertension that is tassium intake, obesity, and diabetes.2,3 In contrast, further characterized by suppressed plasma renin monogenic forms of hypertension are very rare but and aldosterone, hypokalemia, and metabolic have been instrumental in revealing the molecular pathways contributing to primary hypertension.4 The majority of these pathways point toward a Received November 2, 2016. Accepted June 4, 2017. role for increased sodium reabsorption by the kid- Published online ahead of print. Publication date available at neys, especially in the aldosterone-sensitive distal www.jasn.org. 5 nephron. Indeed, several monogenic forms of hy- Correspondence: Dr. Ewout J. Hoorn, PO Box 2040, Room pertension are caused by mutations increasing so- H-438, 3000 CA Rotterdam, The Netherlands. Email: e.j.hoorn@ dium reabsorption in this segment through the erasmusmc.nl sodium chloride cotransporter or the epithelial Copyright © 2017 by the American Society of Nephrology

J Am Soc Nephrol 28: 3291–3299, 2017 ISSN : 1046-6673/2811-3291 3291 BASIC RESEARCH www.jasn.org alkalosis.8 The syndrome was linked to mutations in the exomes as proxy for variant allele frequencies in the general SCNN1B or SCNN1G gene, encoding the b-org-subunit of population (seven times heterozygously in .100,000 alleles; ENaC.9,10 Mutations in SCNN1B or SCNN1G delete or modify Exome Aggregation Consortium). The ENaC blocker triam- the intracellular PY motifs in ENaC in such a way that Nedd4–2 terene normalized BP and serum potassium in the proband. fails to ubiquitylate the channel, leading to a retention of active Genotyping of the five siblings also identified the novel C479R ENaC at the cell surface.11,12 Here, we report a family with mutation in subject II-4. The mutation segregated with sup- Liddle syndrome due to a gain of function mutation in the pressed plasma renin and aldosterone but not with hyperten- extracellular domain of the a-subunit of ENaC (SCNN1A) sion (Figure 1C). Whole-exome sequencing in the proband did that predominantly increases channel open probability (Po) not identify additional mutations to explain the hypertensive but not channel surface density. trait in this family. Subject II-4 had mild hypertension (average ambulatory BP of 138/88 mmHg) that was sensitive to sodium chloride supplementation (145/91 mmHg) and also improved RESULTS with triamterene (121/71 mmHg). In a standardized diuretic test, the natriuretic response to triamterene in the proband Clinical and Genetic Characteristics of a Novel ENaC and II-4 was in the high range or increased compared with the Mutation response in healthy volunteers (Figure 1D).13 Thus, two siblings The proband was referred because of resistant hypertension, (the proband and II-4) show a clinical picture compatible with hypokalemia, metabolic alkalosis, and suppressed levels of Liddle syndrome and carry the C479R missense mutation. plasma renin and aldosterone. Despite a positive family his- tory for hypertension (Figure 1A), no mutations in SCNN1B C479 Is Located in the Extracellular Domain of ENaC or SCNN1G were identified. Diagnostic exome sequencing The ultimate proof of Liddle syndrome, however, is the dem- revealed a novel heterozygous, nonconservative T.Csingle- onstration that the mutation results in a gain of function of nucleotide mutation that results in the substitution of cyste- ENaC. The DNAvariant that encodes the C479R mutant has so ine 479 to arginine (C479R) in aENaC [c.1435T.C(p. far never been described. The C479 is a highly conserved Cys (Cys479Arg))] (Figure 1B). The mutation is reported at a residue that belongs to the second cysteine-rich domain very low frequency in a large database collecting .60,000 (CRD2) of the extracellular domain of ENaC that is likely involved in disulfide bridges.14 The human aENaC subunit (haENaC) C479 is conserved among not only the ENaC subunits and ENaC homologs but also the Acid- Sensing Ion Channel 1 (ASIC1) orthologs (Figure 2A). The crystal structure of chickenASIC1revealsthatthecASIC1 C366 forms a disulfide bond with another highly conserved cysteine, C291 in CRD2, that corresponds in haENaC to a disulfide bond between C479 and the C394 (Figure 2B).15 Therefore, we analyzed not only the consequences of the C479R mutation on hE- NaC function in Xenopus laevis oocytes but also, the functional effects of the mutation of the partner Cys C394S involved in the disulfide bond. In addition, because C479 is a highly conserved Cys, we performed a similar functional analysis of the corre- Figure 1. The novel aENaC mutation is characterized clinically by hypertension, sup- sponding Cys mutations C507S and pressed plasma renin and aldosterone, and an exaggerated natriuretic response to an C422S in rat aENaC. ENaC blocker. (A) Pedigree showing three generations of the family with Liddle syn- fi drome. Generation II was analyzed by genotyping and biochemical pro ling. The arrow C479R Increases ENaC Current in indicates the proband. (B) Sequence chromatogram. (C) The C479R mutation segregated Oocytes with suppressed plasma renin and aldosterone but not with hypertension. Renin and aldosterone were measured in the absence of interfering drugs. Dashed lines represent Both C479R and C394S result in a similar lower limits of normal. HT, hypertension; NT, normotension; WT, wild type. (D) Results of approximately twofold increase in amilor- a standardized diuretic test showing the natriuretic response to a single dose of the ENaC ide-sensitive ENaC current (Figure 3A). blocker triamterene in the proband and subject II-4 in comparison with healthy volun- These results strongly suggest that the teers.13 *Proband; #subject II-4. channel gain of function is due to the

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extracellular domain of rat ENaC results in a channel gain of function.14 Furthermore both C479Arg and C507Ser substitutions in haENaC and rat aENaC subunit have comparable stimulatory effects on ENaC current, indicating that the effect does not depend on the substituting amino acid. To- gether, these observations support the idea that the disruption of the C479-C394 disul- ﬁde bridge by the C479R substitution is likely the primary cause of the observed gain of function in ENaC.

Surface Density of C479R Channel Mutant The increase in ENaC activity due to the C479R mutation can result from an increase in channel Po,single-channelcon- ductance, or the number of channels at the cell surface. To test the latter possibility, we analyzed on Western blot the cleaved (CL) forms of the wild type and C479R a- and gENaC subunits (Figure 4, A and B), expressed in the whole oocyte, at the cell surface (Figure 4, C and D, Supplemental Figure 1) and analyzed urinary extracellular vesicles (Supplemental Figure 2).16 It is now well es- tablished that the CL forms of a and g represent the mature ENaC subunits that are incorporated in the functional channel complex present at the cell surface.17 The full length (FL) of aENaC (93 kD) was detected in oocytes expressing a-subunit alone or abgENaC wild type and C479R mutant Figure 2. C479 is a highly conserved Cys that forms a disulfide bond with C394. (A) (Figure 4A). The CL form of aENaC (69 Sequence comparison of haENaC and rat aENaC (SCAA), bENaC (SCAB), and gENaC (SCAG) subunit isoforms with human hASIC1 and chicken cASIC1. (B) Crystal structure kD) was detected only for the abgENaC of a cASIC1 subunit with the disulfide bonds in the extracellular domain labeled in wild type and the aC479Rbg mutant. The green for the first cysteine-rich domain (CRD1) and yellow for CRD2. The Cys366 (red) CL form of g-subunit (76 kD) was detected corresponding to Cys479 in the human aENaC (hSCAA) makes a disulfide bond with for both abgENaC wild type and Cys291 (purple) corresponding to C394 in hSCAA (inset). aC479Rbg mutant. Quantification of the intensities of the CL forms of a- and g-subunits from the aC479Rbg ENaC complex disruption of the disulfide bond between the two Cys. Because relative to those of a and g in the abgENaC complex did the proband is heterozygous for the C479R mutation, we rep- not show any significant difference. Subsequently, cell surface licated this condition in vitro by coinjecting ENaC wild type expression of ENaC wild type and mutant was assessed by and C479R in a 1:1 ratio and observed still a significant in- biotinylation of surface proteins and followed by affinity pu- crease in ENaC current, but this effect was reduced by one half rification on Neutravidin-agarose beads. The representative compared with C479R expressed alone (Figure 3B). To pro- immunoblot in Figure 4C shows that, in oocytes expressing vide further evidence for a gain of function due to the disrup- comparable amounts of aENaC wild type and mutant under tion of the disulfide bridge, we tested the corresponding their FL and CL forms (Figure 4C, left panel) (CL, 69 kD; FL, mutations in rat aENaC subunit and found that the C507S 97 kD), the CL band is the main detected form at the cell and C422S mutations have comparable stimulatory effects on surface at similar amounts for both channel types (Figure ENaC activity as C479R and C394S in haENaC (Figure 3C). 4C, right panel). We quantified the FL + CL band intensities These results are consistent with previous observations that in total membrane and cell surface fractions of wild-type and disruption of particular disulfide bonds in the CRD2 of the C479R mutant ENaC-expressing oocytes (Figure 4D). The

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Figure 3. aENaC C479R is a gain of function mutation. (A) Amiloride-sensitive current increase of aC479R ENaC mutant and the Cys partner, aC394S, mutant. Current values were normalized for the average INa of the wild-type (wt) control obtained in oocytes of each independent batch (n$4). Bars represent mean6SD for haENaC wt (n=225), haENaC C479R (n=231), and haENaC C394S (n=18). (B) Normalized amiloride-sensitive current values as in A with haENaC wt (n=14) and haENaC C479R expressed alone (n=13) or together with haENaC wt at a cRNA weight ratio of 1:1 (n=13). (C) Normalized amiloride-sensitive current values for rat aENaC wt (n=12), C507S (n=12), and C422S (n=12) corresponding to C479R and C394S in the human aENaC sequence. *P , 0.05. data obtained from four independent experiments show that, to 0.27. An alternative but less likely explanation is that the for a comparable expression of aENaC wild type and C479R C479R and the C394S mutations decrease the efficiency of mutant, the amounts of wild-type and C479R mutant ENaC ENaC cleavage by trypsin, despite a gain of function effect of functional channels at the cell surface are similar. Similarly, the the mutation. An alternative explanation for the lower sensi- aENaC C479R mutation did increase gENaC levels at the cell tivity to trypsin of the C479R and C394S mutants is an appar- surface of injected oocytes (Supplemental Figure 1). On the basis ent saturable expression of the ENaC current because of the of these data, we can conclude that the approximately twofold limited capacity of the oocyte to face large inward Na+ cur- 19 higher ENaC current measured for the haC479R mutant is not rents. The latter possibility was, however, excluded in exper- correlated with an increase in the mature channel density at the iments testing the effect of trypsin on wild-type, C479R, and cell surface. Consistently, no differences in the abundance of CL C394S hENaC at different levels of current expression by in- aENaC in urinary extracellular vesicles were detected in the two jecting increasing amounts of cRNAs ranging from 0.1 to 10 subjects carrying the mutation (Supplemental Figure 2).16 ng per oocyte (Figure 5C). The slope of this linear relation was approximately twofold lower for the ENaC mutants than for Intrinsic Activity of C479R Mutant the wild type. This lower response to trypsin for the gain of Totest the possibility of a gain of function C479R mutation due function ENaC mutants is consistent with a higher intrinsic to an increase in channel Po, we used trypsin, which proteo- activity of the channel with a higher Po.Wethenverified these lytically cleaves ENaC and activates the channel by increasing observations with the corresponding Cys mutations, C507S 18 the Po. We reasoned that, if the C479R channel mutant has a and C422S, of rat ENaC and also included the rat bENaC Liddle higher Po than the ENaC wild type, then it should show a mutant Y618A (Figure 6). Y618A disrupts the PY motif and higher current but a lower sensitivity to activation by trypsin. increases the number of active channels at the cell surface.20 The magnitude of the currents in the absence of trypsin was Wild-type ENaC and the Y618A mutant show a significantly significantly higher for the C479R and C394S mutants com- higher response to trypsin compared with the C507S and pared with the wild type (2.1- and 2.4-fold increases, respec- C422S mutants, despite the fact that the Y618A baseline current tively). After trypsin treatment, the currents expressed by the was significantly higher than that of the wild type and the Cys mutant forms were no longer different from those of ENaC mutants (Figure 6A). The linear relationship between currents wild type, although a trend toward higher currents was ob- with or without trypsin was similar for wild-type and Y618C rat served for the mutants (Figure 5A, Supplemental Figure 3 ENaC but approximately twofold smaller for the C507S and shows original traces). Shown in Figure 5B for individual oo- C422S rat ENaC mutants (Figure 6B). Taken together, these cytes, the wild-type hENaC has a significantly lower current results suggest that the ENaC gain of function mutations than either of the mutants, but the trypsin-induced increase in C479R and C507S are essentially due to an increased intrinsic current is higher than that for the C479R and C394S mutants. channel activity. This smaller effect of trypsin shown for the C479R and C394S gain of function mutants is consistent with a higher basal Po.It is interesting to note that, even at high ENaC baseline currents, DISCUSSION such as those observed for the mutants, the trypsin effect pla- teaued at a twofold increase in INa. This suggests that the Here, we report a mutation in aENaC associated with Liddle baseline Po of C479R and C394S is below or equal to 0.5, syndrome. Our functional investigations of the C479R mutation whereas the baseline Po of wild-type hENaC is below or equal show that this Cys mutation in the second cysteine-rich domain

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Figure 4. C479R does not increase channel surface density. (A) Anti-HA tag (red; left panel) and anti-gENaC (green; right panel) Western blot analysis of Triton-soluble fractions from Xenopus oocytes noninjected (n.i.) or injected with cRNAs for ha-HA alone or with b-andgENaC cRNAs together with either the wild type (wt) or C479R mutant ha-HA. (B) The intensities of the bands corresponding to CL ha-andhgENaC were normalized to the amount of 2,2,2-Trichloroethanol–labeled total protein obtained for each lane on the blot. The ratios between the thus-calculated values for ha-andhgENaC in the hawtbgENaC and those for haC479RbgENaC are shown in the graph. Data correspond to mean6SEM (ten blots from seven independent experiments); differences are NS. (C, right panel) Anti-HA immunoblot analysis of neutravidin-bound fractions isolated from control (2) or cell surface biotinylated (+)

Xenopus oocytes n.i. or injected with either awt-HA/b/g or aC479R-HA/b/g cRNAs. (C, left panel) Inputs corresponding to 1% of the Triton-soluble preparations from biotinylated oocytes used in the pull-down experiments. (D) Values (mean6SD) of CL + FL band intensities of inputs or neutravidin-bound fractions corresponding to experiments shown in C. Results from four blots with samples of four independent experiments. Differences are NS. of the extracellular domain of aENaC is a gain of function mu- increase in amiloride-sensitive current without changing tation. Our data are consistent with a higher intrinsic ENaC channel surface expression. Furthermore, mutation of the activity due to an increase in Po, likely resulting from the disrup- partner cysteine involved in the disulfide bridge (aC7S) had tion of the disulfide bonds between Cys479 and Cys394. The the same effects. Of interest, mutations of the cysteines in the ENaC channel opener trypsin induces a lower response for the extracellular cysteine-rich domains can result in either chan- gain of function mutants haC479R and raC507S, consistent with a nel loss or gain of function. Indeed, mutation of the human higher channel Po. This functional effect of the disruption of the Cys133 into a tyrosine causes the mirror image of Liddle syn- Cys479-Cys394 disulfide bond is conserved in both rat and hu- drome, pseudohypoaldosteronism type 1, a severe salt-losing man ENaC channel isoforms. Our data are remarkably consis- syndrome in neonates.14,21 tent with the mutational analysis of cysteine-rich domains of The majority of the previously reported Liddle mutations ENaC previously reported by Firsov et al.14 They showed that affect the intracellular PY motif of b-orgENaC, impairing the aC16S mutation in rat aENaC (corresponding to the C479R channel degradation by Nedd4–2.9,10 A mutation in aENaC and C507S mutations in our work) resulted in a threefold causing Liddle syndrome has only been reported once

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previously and was also located in the PY motif.22 Therefore, this report on an aENaC mutation causing Liddle syndrome contains two novel aspects, including the location in the extracellular domain and the effect on Po rather than surface expression. Of note, aENaC gain of function mutations have been identified previously in patients with cystic fibrosis–like symptoms, but it is not known whether these mutations also caused hypertension.23,24 In addition, some of the mutations in a-orbENaC causing atypical cystic fibrosis were located 24,25 outside the PY motif and increased channel Po. A gain of function mutation (N530) in the putative extracellular domain of gENaC causing Liddle syndrome has also been reported previously.26 However, structural models of ENaC on the basis of the crystal structure of the homolog ASIC1 predict that the N530 residue is located in the second transmembrane a-helix of gENaC and is not located in the extracellular domain of the channel. Finally, single-nucleotide polymorphisms of the aENaC gene locus correlated with salt-sensitive hypertension in one Chinese study.27 Of interest, the proband had the classic Liddle syndrome phenotype, whereas his sibling had a milder degree of salt- sensitive hypertension and no hypokalemia. Such differences between affected family members, including the absence of hypokalemia, have been observed previously.28 The pheno- typic differences between the proband and II-4 suggest vari- able expressivity, possibly related to sex, environmental factors, or genetic modifiers. Alternatively, the proband and his hypertensive siblings without the mutation (subjects II-2, II-3, and II-6) (Figure 1A) may have an additional genetic variant predisposing to hypertension, although we did not identify this using whole-exome sequencing. Thus, in this family, the mutation segregated with suppressed renin and aldosterone but not hypertension. In addition, our family seemed to have a milder phenotype compared with previous Liddle kin- dreds.9,10,28 This may also relate to the molecular mechanism of the aENaC mutation. In vivo ENaC channel Po determined by the patch clamp technique ranged from 0.3 to 0.7.29 In Xenopus oocytes, ENaC 19 Po under comparable conditions was estimated to be 0.3. Assuming that trypsin stabilizes ENaC in the open conforma- Figure 5. Lower trypsin sensitivity of C479R and C394S than tion with a Po of one, then the slope current relations for the wild type aENaC suggests higher intrinsic channel activity. (A) ENaC wild type and mutants suggest that, under our experi- Amiloride-sensitive current (microampere) of hENaC wild-type mental conditions, the Po of wild-type ENaC is around 0.25 (wt; n=17), haENaC C479R (n=17), and haENaC C394S (n=18) and that the Po of the mutants is around 0.5. Such an increase 2 in the absence ( ) or presence (+) of trypsin. The magnitude of the in Po, estimated from the trypsin response, represents the currents in the absence of trypsin was significantly higher for C479R main component of the higher ENaC current expressed by and C394S compared with wt (P,0.01 by one-way ANOVA), the C479R and the related mutants. Such a gain of function whereas the currents in oocytes expressing the mutant forms were no resulting from an increased Po is certainly limited by the longer significantly higher after trypsin treatment. (B) Relationship between baseline INa in the absence of trypsin and fold increase in INa after the addition of trypsin in wt and mutant human ENaC

(C479R and C394S). Current values for a single oocyte (filled symbols) best fit values for the slopes hawt (4.08; 95% confidence interval, and means6SD (open symbols) are shown (P,0.01 by ANOVA). (C) 3.82 to 4.35), haC479R (2.28; 95% confidence interval, 2.14 to 2.42), Correlation between current INa (microampere) values in the absence and haC394S (2.23; 95% confidence interval, 2.03 to 2.42). Sup- 1 and presence of trypsin. Linear regression analysis gives the following plemental Figure 3 shows original traces. INa, Na current.

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the cell surface may potentially enhance sodium transport to a much greater extent, because the capacity of the membrane surface to accommodate a high density of ENaC channels is less restricted than the capacity of ENaC to increase in its Po. Along this line, we observed that the Y618A mutation, affecting channel interaction with Nedd4–2, is more efficient than C507S in increasing ENaC- mediated current (Figure 6). Although an increase in single-channel conductance could theoretically explain the C479R channel gain of function, we know from previous structure-function studies on ENaC that such mutations are restricted to the second transmembrane domain, a region that par- ticipates in the ion channel pore and selec- tivity filter.30 In summary, we report a mutation in aENaC associated with Liddle syndrome. This represents the firstENaCgainoffunc- tion mutation in the extracellular domain causing a higher intrinsic channel activity, a mechanism different from the previously reported mutations in b or g associated with Liddle syndrome.9,10 This study raises the question about the necessity of genotyping patients with unexplained cases of hypertension associated with suppressed plasma renin and aldosterone and a poor response to standard antihypertensive therapy. Further- more, this mutation provides novel insight into ENaC activation and potentially, distal nephron sodium reabsorption and salt- sensitive hypertension.

CONCISE METHODS Figure 6. Rat aENaC mutations C507S and C422S are also less sensitive to trypsin than wild type and the bENaC Liddle mutation Y618C. (A) Relationship between the Additional information can be found in Supple- baseline INa in the absence of trypsin and the fold increase in INa after the addition of mental Material. trypsin for wild-type (wt) rat ENaC, two aENaC mutants (C507S and C422S), and the fi bENaC Liddle mutant Y618A. Current values for a single oocyte ( lled symbols) and Studies in Patients 6 , means SD (open symbols) are shown (P 0.01 by ANOVA). (B) Correlation between The patient studies were performed in accor- current INa values (microampere) in the absence and presence of trypsin. Linear re- dance with the Declaration of Helsinki. All pa- gression analysis gives best fit values for the slopes corresponding to ra /rb/rg, wt tients provided written informed consent, and ra /rb/rg,ra /rb/rg,andra /rb/rg , and the values were 4.07 (95% con- C507S C422S wt Y618A the ethics committee approved the study. fidence interval, 3.92 to 4.23), 2.09 (95% confidence interval, 2.0 to 2.18), 1.92 (95% confidence interval, 1.74 to 2.09), and 4.04 (95% confidence interval, 3.79 to 4.30), Plasma renin concentration and plasma aldo- 1 respectively. INa, Na current. sterone were measured by enzyme-kinetic assay and LC/MS, respectively. These measure- functional characteristics of the channels. Because ENaC likely ments were performed in the absence of interfering drugs (renin- never functions physiologically with a Po of one, a gain of angiotensinsysteminhibitors ordiuretics).Thetriamterene testwas function due to an increased channel gating would reasonably on the basis of the thiazide test.31 Results were compared with a not exceed three- to fourfold. In contrast, Liddle gain of func- historic cohort of healthy subjects receiving 100 mg triamterene tion mutation resulting from an increased ENaC retention at in a similar setting.13

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Exome Sequencing 5%–15% acrylamide gradient minigels supplemented with 0.5% The analyses of the exome data were divided into two steps: the renal (vol/vol) of 2,2,2-Trichloroethanol for subsequent in-gel fluores- gene panel analysis and the exome analysis. In the renal gene panel cence labeling of proteins.34 Total protein per lane was assessed den- analysis, an in silico enrichment of genes associated with genetic renal sitometrically from these images using ImageJ. Band intensities were disorders was performed (version: DGD141114). After the patient assessed with the Odyssey v2.1 software and normalized with the consented for the second step, exome analysis, likely pathogenic var- amount of total 2,2,2-Trichloroethanol–labeled protein in the corre- iants in all coding genes were analyzed. Exome sequencing was per- sponding lanes. formed using an Illumina HiSeq2000TM sequencer at BGI-Europe (Copenhagen, Denmark). Read alignment to the human reference Analyses of Cell Surface Biotinylated Fractions genome (GrCH37/hg19) and variant calling were performed at BGI- Control or injected oocytes (approximately 25 per condition) were Europe using BWA and GATK software, respectively. Variant anno- incubated for 15 minutes on ice in 1 ml Biotinylation buffer. Biotiny- tation was performed using a custom designed in-house annotation lated fractions were isolated from ENaC-enriched fractions that had and variant prioritization pipeline. been purified as described before.35 Neutravidin-bound fractions and Triton-soluble fractions (1% of total) were resolved by SDS- Site-Directed Mutagenesis and Expression in PAGE, transferred to nitrocellulose membranes, and blocked as de- X. laevis Oocytes scribed before. To account for nonspecific binding to Neutravidin Mutant forms of the wild type of human a-, b-, and gENaC subunits beads, band intensity values from control, nonbiotinylated samples had been cloned in the pBSK(+)_Xglob vector.32 In these vectors, the were subtracted from the values of the corresponding biotinylated ENaC cDNAs are flanked by sequences corresponding to the 59 and 39 samples. noncoding stretches of Xenopus b-globin, which boosts protein ex- 33 pression when injected into Xenopus oocytes. Plasmids suitable for Statistical Analyses in vitro transcription of wild-type and mutant forms of the haENaC Statistical analyses were performed using GraphPad Prism software were generated as described in Supplemental Material. All constructs (GraphPad Software). Differences between groups were assessed by were verified by sequencing. Subsequently, healthy stage 5 and 6 X. one-way ANOVA and the Tukey post hoc test. P,0.05 was considered laevis oocytes were pressure injected with mixes containing equal statistically significant. amounts of cRNAs encoding a-, b-, and g-subunits for a total of (unless stated otherwise) 1 or 3 ng cRNA for human and rat ENaC, respectively. Mutant and control cRNAs were prepared in parallel, ACKNOWLEDGMENTS and greater than or equal to three independent batches of cRNA were used. We thank the proband and his family members who participated in this study and the referring physician Dr. Arie T.J. Lavrijssen. We also Electrophysiology thank Usha Musterd-Bhaggoe for technical support and Dr. Jan Electrophysiologic measurements were made 24–32 hours after in- Loffing (University of Zürich) for epithelial sodium channel anti- jection with the standard two-electrode voltage clamp technique bodies. using a TEV-200A voltage clamp amplifier (Dagan, Minneapolis, This study was supported by Swiss National Science Founda- MN), an ITC-16 digitizer interface (Instrutech Corp., Elmont, tion grant 310030_135378 (to L.S.) and Dutch Kidney Foundation NY), and the PatchMaster data acquisition and analysis package grant KSP-14OK19 (to E.J.H.). (HEKA Elektronik Dr. Schulze GmbH, Ludwigshafen/Rhein, Ger- many). The amiloride-sensitive currents were measured in the presence of 10 mM of this blocker adjusted in a separate solution. Inward DISCLOSURES Na+ currents were generated by switching from the amiloride-containing perfusion solution to that without amiloride. In the experi- None. ment with proteases, the oocytes were perfused for 2 minutes with the amiloride-free solution supplemented with 2 mg/ml trypsin (Sigma- REFERENCES Aldrich Chemie GmbH, Buchs, Switzerland). All INa values were normalized in all experiments to the mean of the amiloride-sensitive fi currents measured for wild-type ENaC with the same oocyte batch. 1. Poulter NR, Prabhakaran D, Caul eld M: Hypertension. Lancet 386: 801–812, 2015 2. Mente A, O’Donnell MJ, Rangarajan S, McQueen MJ, Poirier P, Isolation of ENaC-Enriched Fractions Wielgosz A, Morrison H, Li W, Wang X, Di C, Mony P, Devanath A, To isolate membrane fractions, 15–30 oocytes were disrupted by pi- Rosengren A, Oguz A, Zatonska K, Yusufali AH, Lopez-Jaramillo P, petting in 1.5 ml membrane isolation buffer followed by centrifuga- Avezum A, Ismail N, Lanas F, Puoane T, Diaz R, Kelishadi R, Iqbal R, tion through cell shredders. The membrane pellets obtained after 30 Yusuf R, Chifamba J, Khatib R, Teo K, Yusuf S; PURE Investigators: As- 3 sociation of urinary sodium and potassium excretion with blood pres- minutes of centrifugation at 20,000 g (4°C) were resuspended in sure. N Engl J Med 371: 601–611, 2014 membrane solubilization solution. Proteins in samples were resolved 3. Padmanabhan S, Caulfield M, Dominiczak AF: Genetic and molecular along with prestained molecular weight markers by SDS-PAGE on aspects of hypertension. Circ Res 116: 937–959, 2015

3298 Journal of the American Society of Nephrology J Am Soc Nephrol 28: 3291–3299, 2017 www.jasn.org BASIC RESEARCH

4. Lifton RP, Gharavi AG, Geller DS: Molecular mechanisms of human the epithelial sodium channel (ENaC) found in Liddle’s syndrome hypertension. Cell 104: 545–556, 2001 [Abstract]. Acta Physiol 198: P-TUE-59, 2010 5. Meneton P, Loffing J, Warnock DG: Sodium and potassium handling by 23. Azad AK, Rauh R, Vermeulen F, Jaspers M, Korbmacher J, Boissier B, the aldosterone-sensitive distal nephron: The pivotal role of the distal and Bassinet L, Fichou Y, des Georges M, Stanke F, De Boeck K, Dupont L, connecting tubule. Am J Physiol Renal Physiol 287: F593–F601, 2004 Balascáková M, Hjelte L, Lebecque P, Radojkovic D, Castellani C, 6. Scheinman SJ, Guay-Woodford LM, Thakker RV, Warnock DG: Genetic Schwartz M, Stuhrmann M, Schwarz M, Skalicka V, de Monestrol I, disorders of renal electrolyte transport. NEnglJMed340: 1177–1187, 1999 Girodon E, Férec C, Claustres M, Tümmler B, Cassiman JJ, Korbmacher 7. Liddle GW, Bledsoe T, Coppage WS: A familial renal disorder simu- C, Cuppens H: Mutations in the amiloride-sensitive epithelial sodium lating primary aldosteronism but with negligible aldosterone secretion. channel in patients with cystic fibrosis-like disease. Hum Mutat 30: Trans Assoc Am Phys 76: 199–213, 1963 1093–1103, 2009 8. Gennari FJ, Hussain-Khan S, Segal A: An unusual case of metabolic 24. Rauh R, Diakov A, Tzschoppe A, Korbmacher J, Azad AK, Cuppens H, alkalosis: A window into the pathophysiology and diagnosis of this Cassiman JJ, Dötsch J, Sticht H, Korbmacher C: A mutation of the common acid-base disturbance. Am J Kidney Dis 55: 1130–1135, 2010 epithelial sodium channel associated with atypical cystic fibrosis in- 9. Hansson JH, Nelson-Williams C, Suzuki H, Schild L, Shimkets R, Lu Y, creases channel open probability and reduces Na+ self inhibition. J Canessa C, Iwasaki T, Rossier B, Lifton RP: Hypertension caused by a Physiol 588: 1211–1225, 2010 truncated epithelial sodium channel gamma subunit: Genetic hetero- 25. Rauh R, Soell D, Haerteis S, Diakov A, Nesterov V, Krueger B, Sticht H, geneity of Liddle syndrome. Nat Genet 11: 76–82, 1995 Korbmacher C: A mutation in the b-subunit of ENaC identified in a 10. Shimkets RA, Warnock DG, Bositis CM, Nelson-Williams C, Hansson patient with cystic fibrosis-like symptoms has a gain-of-function effect. JH, Schambelan M, Gill JR, Ulick S, Milora RV, Findling JW, Canessa Am J Physiol Lung Cell Mol Physiol 304: L43–L55, 2013 CM, Rossier BC, Lifton RP: Liddle’s syndrome: Heritable human hy- 26. Hiltunen TP, Hannila-Handelberg T, Petäjäniemi N, Kantola I, Tikkanen pertension caused by mutations in the beta subunit of the epithelial I, Virtamo J, Gautschi I, Schild L, Kontula K: Liddle’ssyndromeassoci- sodium channel. Cell 79: 407–414, 1994 ated with a point mutation in the extracellular domain of the epithelial 11. Abriel H, Loffing J, Rebhun JF, Pratt JH, Schild L, Horisberger JD, Rotin sodium channel gamma subunit. J Hypertens 20: 2383–2390, 2002 D, Staub O: Defective regulation of the epithelial Na+ channel by 27. Xu H, Li NF, Hong J, Zhang L, Zhou L, Li T, Ou Yang WJ, Cheng QY,: Nedd4 in Liddle’s syndrome. J Clin Invest 103: 667–673, 1999 [Relationship between four single nucleotide polymorphisms of epi- 12. Schild L, Canessa CM, Shimkets RA, Gautschi I, Lifton RP, Rossier BC: A thelial sodium channel alpha subunit gene and essential hypertension mutation in the epithelial sodium channel causing Liddle disease in- of Kazakhs in Xinjiang.] Zhongguo Yi Xue Ke Xue Yuan Xue Bao 31: creases channel activity in the Xenopus laevis oocyte expression sys- 740–745, 2009 tem. Proc Natl Acad Sci U S A 92: 5699–5703, 1995 28. Botero-Velez M, Curtis JJ, Warnock DG: Brief report: Liddle’ssyn- 13. Möhrke W, Knauf H, Mutschler E: Pharmacokinetics and pharmacody- drome revisited–A disorder of sodium reabsorption in the distal namics of triamterene and hydrochlorothiazide and their combination tubule. N Engl J Med 330: 178–181, 1994 in healthy volunteers. Int J Clin Pharmacol Ther 35: 447–452, 1997 29. Nesterov V, Dahlmann A, Krueger B, Bertog M, Loffing J, Korbmacher 14. Firsov D, Robert-Nicoud M, Gruender S, Schild L, Rossier BC: Muta- C: Aldosterone-dependent and -independent regulation of the epi- tional analysis of cysteine-rich domains of the epithelium sodium thelial sodium channel (ENaC) in mouse distal nephron. Am J Physiol channel (ENaC). Identification of cysteines essential for channel ex- Renal Physiol 303: F1289–F1299, 2012 pression at the cell surface. JBiolChem274: 2743–2749, 1999 30. Kellenberger S, Gautschi I, Pfister Y, Schild L: Intracellular thiol-medi- 15. Jasti J, Furukawa H, Gonzales EB, Gouaux E: Structure of acid-sensing ated modulation of epithelial sodium channel activity. JBiolChem280: ion channel 1 at 1.9 A resolution and low pH. Nature 449: 316–323, 7739–7747, 2005 2007 31. Colussi G, Bettinelli A, Tedeschi S, De Ferrari ME, Syrén ML, Borsa N, 16. Salih M, Fenton RA, Zietse R, Hoorn EJ: Urinary extracellular vesicles as Mattiello C, Casari G, Bianchetti MG: A thiazide test for the diagnosis of markers to assess kidney sodium transport. Curr Opin Nephrol Hy- renal tubular hypokalemic disorders. Clin J Am Soc Nephrol 2: 454– pertens 25: 67–72, 2016 460, 2007 17. Frindt G, Ergonul Z, Palmer LG: Surface expression of epithelial Na 32. Dirlewanger M, Huser D, Zennaro MC, Girardin E, Schild L, channel protein in rat kidney. J Gen Physiol 131: 617–627, 2008 Schwitzgebel VM: A homozygous missense mutation in SCNN1A is re- 18. Kleyman TR, Carattino MD, Hughey RP: ENaC at the cutting edge: sponsible for a transient neonatal form of pseudohypoaldosteronism Regulation of epithelial sodium channels by proteases. JBiolChem type 1. Am J Physiol Endocrinol Metab 301: E467–E473, 2011 284: 20447–20451, 2009 33. Krieg PA, Melton DA: Formation of the 39 end of histone mRNA by post- 19. Anantharam A, Tian Y, Palmer LG: Open probability of the epithelial transcriptional processing. Nature 308: 203–206, 1984 sodium channel is regulated by intracellular sodium. JPhysiol574: 34. Ladner CL, Yang J, Turner RJ, Edwards RA: Visible fluorescent de- 333–347, 2006 tection of proteins in polyacrylamide gels without staining. Anal Bio- 20. Tamura H, Schild L, Enomoto N, Matsui N, Marumo F, Rossier BC: chem 326: 13–20, 2004 Liddle disease caused by a missense mutation of beta subunit of the 35. Michlig S, Harris M, Loffing J, Rossier BC, Firsov D: Progesterone down- epithelial sodium channel gene. JClinInvest97: 1780–1784, 1996 regulates the open probability of the amiloride-sensitive epithelial 21. Chang SS, Grunder S, Hanukoglu A, Rösler A, Mathew PM, Hanukoglu I, sodium channel via a Nedd4-2-dependent mechanism. J Biol Chem Schild L, Lu Y, Shimkets RA, Nelson-Williams C, Rossier BC, Lifton RP: 280: 38264–38270, 2005 Mutations in subunits of the epithelial sodium channel cause salt wasting with hyperkalaemic acidosis, pseudohypoaldosteronism type 1. Nat Genet 12: 248–253, 1996 22. Goehl K, Haerteis S, Nelson-Williams C, Lifton RP, Korbmacher C, Rauh This article contains supplemental material online at http://jasn.asnjournals. R: Functional characterization of a novel mutation in the a-subunit of org/lookup/suppl/doi:10.1681/ASN.2016111163/-/DCSupplemental.

J Am Soc Nephrol 28: 3291–3299, 2017 aENaC Mutation and Liddle 3299 1

Supplemental Material: Supplemental Figures1–3and Complete Methods

(Second revision – revisions highlighted in red font)

A Missense Mutation in the Extracellular Domain

of Alpha ENaC Causes Liddle Syndrome

Mahdi Salih, Ivan Gautschi, Miguel X. van Bemmelen, Michael Di Benedetto,

Alice S. Brooks, Dorien Lugtenberg, Laurent Schild, Ewout J. Hoorn

Supplemental Material – Salih et al.

Supplemental Figure 1. αENaC C479R mutation does not result in increased γENaC levels at the cell surface of injected oocytes.

A B

Legend: After probing with anti-HA antibody (see Figure 4), blots were re-blocked and analyzed with anti-γENaC antibody as described for lysates in the Materials and Methods section. (A) Right panel, immunoblot analysis of Neutravidin-bound fractions isolated from control (-) or cell-surface biotinylated (+) Xenopus oocytes, non-injected (n.i.), or injected with either αwt-HA/β/γ or αC479R-HA/β/γ cRNAs. Left panel. Inputs corresponding to 1% of the

Triton-soluble preparations from biotinylated oocytes used in the pull-down experiments. (B)

Values (mean  SEM) of CL+FL band intensities of inputs or of Neutravidin-bound fractions corresponding to experiments shown in A. Results from three blots with samples of three independent experiments. Differences are non-significant (N.S.).

Supplemental Material – Salih et al.

Supplemental Figure 2.αENaC in human urinary extracellular vesicles.

Legend: αENaC in urinary extracellular vesicles isolated from the reported family. Urinary extracellular vesicles were collected in the absence of interfering medication (no renin- angiotensin inhibitors or diuretics). A human kidney (HK) sample was included as positive control and showed the expected bands at 100 kDa (full-length αENaC) and ~30 kDa (cleaved

αENaC). It is unclear what the low intensity band at ~45 kDa represents. Of these bands, only the

30 kDa band was detected in human urinary extracellular vesicles. Although no firm conclusions can be drawn based on the small number of individuals,no clear difference in the abundance of this 30 kDa band was visible between the two subjects carrying the C479R mutation (II-5 and II-

4) compared to those without the mutation (II-1, II-2, II-3, and II-6) and a healthy control (HC).

II-5T and II-4T are urinary extracellular vesicles isolated after the standardized diuretic test with triamterene (T).Of note, an additional band at ~55 kDa was visible in some of the subjects.

Supplemental Material – Salih et al.

Although this band could represent partially or atypically cleaved αENaC, it could also be nonspecific, as it was also vaguely visible in kidneys of αENaC -/- mice.1

Supplemental Material – Salih et al.

Supplemental Figure 3. Original current traces for the different ENaC types in the presence or absence of trypsin.

Supplemental Material – Salih et al.

Legend Supplemental Figure 3: Three original traces from hENaCwt, C479R, and C394S in the absence (left) or presence (right) of trypsin. Results from independent experiments; please note the current and time scales.

Supplemental Material – Salih et al.

Complete Methods

Studies in patients

All subjects provided written informed consent for diagnostic exome sequencing or genotyping.

Our medical ethics committee approved the isolation of urinary extracellular vesicles (MEC-

2015-204, see below for details). Plasma renin concentration and plasma aldosterone were measured by enzyme-kinetic assay and LC/MS, respectively. These measurements were performed in the absence of interfering drugs (renin-angiotensin system inhibitors or diuretics).

All routine serum and urinary measurements were determined using the Cobas 8000 modular analyzer series (Roche, Basel, Switzerland). Ambulatory blood pressure measurements were performed with the ultralite 90217A (Spacelabs Healthcare, Snoqualmie, USA). Salt-sensitivity was tested by providing sodium chloride tablets (6 g/day). The triamterene test was based on the thiazide test.2 Briefly, after an overnight fast, patients were invited to drink tap water (10 ml/kg body wt) to facilitate spontaneous voiding. After four basal clearances, 100 mg of triamterene was administered orally and hourly clearances were performed for six hours. Results were compared to a historic cohort of healthy subjects receiving 100 mg triamterene in a similar setting.3

Exome sequencing

The analysis of the exome data was divided into two steps: the renal gene panel analysis and the exome analysis. In the renal gene panel analysis, an in silico enrichment of genes associated with genetic renal disorders was performed (version: DGD141114). After the patient consented for the second step, exome analysis, likely pathogenic variants in all coding genes were analyzed.

Exome sequencing was performed using a Illumina HiSeq2000TM sequencer at BGI-Europe

(Copenhagen, Denmark). Read alignment to the human reference genome (GrCH37/hg19) and

Supplemental Material – Salih et al.

8 variant calling was performed at BGI using BWA and GATK software, respectively. Variant annotation was performed using a custom designed in-house annotation and variant prioritization pipeline.

Urinary extracellular vesicles

Previously we showed that increased or reduced abundances of renal epithelial transport proteins

(e.g., in Gitelman or Gordon syndrome) are reflected in urinary extracellular vesicles.4, 5 To address whether the novel mutation reported in our family is characterized by increased αENaC abundance, we isolated and analyzed urinary extracellular vesicles, as reported previously.5

Briefly, all urine samples were treated with a protease inhibitor (Complete Protease Inhibitor

Tablet, Roche Diagnostics, Mannheim, Germany) before storage at -80°C. Urinary exosomes were isolated using a 2-step centrifugation process. First, urine was centrifuged at 17,000 x g for

15 minutes at 37°C to remove whole cell membranes and other high density particles.

Dithiothreitol was used to disrupt the Tamm-Horsfall polymeric network. Subsequently, the samples were subjected to ultracentrifugation at 200,000 x g for 105 minutes at 25°C (Beckman

L8-70M ultracentrifuge, Rotor 45 Ti). The pellet that formed during ultracentrifugation was suspended in isolation buffer (10 mM triethanolamine, 250 mM sucrose, pH 7.6). Finally, the suspended pellets were solubilized in Laemmli buffer for Western blot analysis. Western blotting of the urinary exosomes was performed as described previously.4, 5 The antibody against αENaC was a kind gift by Dr. J. Löffing (Institute of Anatomy, University of Zürich, Switzerland) and binds the N-terminal tail. For the patient study, the amount of sample loaded during Western blotting was normalized by the urinary creatinine concentration. CD9 was also blotted as urinary extracellular vesicle marker (R&D systems, Minneapolis, USA).

Supplemental Material – Salih et al.

Site-directed mutagenesis, RNA in vitro transcription, and expression in Xenopus laevis oocytes

Mutant forms of the wild-type forms of human α-, β-, and γENaC subunits had been cloned in the pBSK(+)_Xglob vector.6 In these vectors, the ENaC cDNAs are flanked by sequences corresponding to the 5' and 3' non-coding stretches of Xenopus β-globin, which boosts protein expression when injected into Xenopus oocytes.7 Plasmids suitable for in vitro transcription of wild-type and mutant forms of the human αENaC subunit (hαENaC) were generated as follows.

Using the NEBuilder HiFi DNA Assembly Cloning Kit (New England Biolabs, Ipswich, MA), we replaced the SacII-SacII fragment of pBSK(+)_hαENaC by a synthetic DNA strand (Eurofins

Genomics GmbH, Ebersberg, Germany) including the 15-20 base pairs overlapping the termini of the digested plasmid, the 3' Xglobin UTR, but lacking the polyA-encoding stretch. A similar synthetic stand, containing the required T>C change was used to generate hαENaC_C479R mutant. The thus generated pBSK(+)_hαENaC_Δ(pA) plasmids were subsequently used to construct vectors containing the PolyA-encoding stretch by subcloning their EcoRI-SpeI inserts

(comprising both 5’ and 3’ UTRs, and the αENaC ORFs) into pSDEasy(SB)Δ(SmaI-EcoRV) (a version of pSDEasy(SB) containing one single EcoRI recognition site in the multiple cloning stretch), leading to pSD(Xglob)_hαENaCwt and pSD(Xglob)_hαENaC(C479R). To generate vectors for wildtype and C479R mutant hαENaC containing a C-terminal triple-HA-tag

(3x(YPYDVPDYA)), we used synthetic DNA strands corresponding to the Bpu10I-BamHI inserts of the previously generated vectors, flanked by 15-18 bp stretches overlapping the ends of the digested vectors, and with the sequence encoding the extended HA-tag in-frame with and preceding the stop codon. A novel HpaI/HincII site (GTT/AAC) was generated as well by introducing an additional Adenosine base shortly after the stop codon to facilitate the

Supplemental Material – Salih et al.

10 identification of positive clones. Cys394Ser and Cys479Ser mutants of hαENaC were generated by replacing the SmaI-MluNI insert of pSD(Xglob)_hαENaCwt by the corresponding synthetic

DNA fragments with the required mutated sequences to introduce either a Cys394Ser or a

Cys479Ser conversion. The synthetic DNA fragments contain as well a silent mutation resulting in a novel, unique XbaI site, and comprise flanking sequences for annealing with the ends of the digested plasmid. Plasmids with wildtype and mutant forms of rat ENaC subunits have been described elsewhere.8 All constructs were verified by sequencing.

In vitro transcription of cRNAs pSDEasy vectors containing hαENaC cDNAs were linearized with BglII. pBSK(+)_Xglob vectors containing hβ-, and hγENaC cDNAs were linearized with NotI. Capped cRNAs were in vitro synthesized using either the SP6 (pSDEasy) or T7 RNA polymerase (pBSK(+)). Healthy stage V and VI Xenopus laevis oocytes were pressure-injected with mixes containing equal amounts of cRNAs encoding α-, β-, and γ-subunits for a total of (unless stated otherwise) 1 or 3 ng cRNA for human and rat ENaC, respectively. Oocytes were kept at 19°C in a solution containing (in mM) 85 NaCl, 1 KCl, 2.4 NaHCO3, 0.82 MgSO4, 0.41 CaCl2, 0.33 Ca(NO3)2, 10

HEPES, and 4.08 NaOH.

Electrophysiology

Electrophysiological measurements were made 24-32 h after injection with the standard two- electrode voltage clamp technique, using a TEV-200A voltage clamp amplifier (Dagan,

Minneapolis, MN), an ITC-16 digitizer interface (Instrutech Corp. Elmont, NY), and the

PatchMaster data acquisition and analysis package (HEKA Elektronik Dr. Schulze GmbH,

Supplemental Material – Salih et al.

Ludwigshafen/Rhein, Germany). The two electrodes contained a 1 M KCl solution. All electrophysiological experiments were performed at room temperature (22°C). The holding potential was −80 mV. The composition of the perfusion solution was (in mM) 120 NaCl, 2.5

KCl, 1.8 CaCl2, and 10 HEPES. The amiloride-sensitive currents were measured in the presence of 10 μM of this blocker adjusted in a separate solution. Inward Na+ currents were generated by switching from the amiloride-containing perfusion solution to that without amiloride. In the experiment with proteases, the oocytes were perfused for 2 min with the amiloride-free solution supplemented with 2 μg/ml trypsin (Sigma-Aldrich Chemie GmbH, Buchs, Switzerland). All INa values were normalized in all experiments to the mean of the amiloride-sensitive currents measured for wildtype ENaC with the same oocyte batch.

Isolation of ENaC-enriched fractions

To isolate membrane fractions, 15-30 oocytes were disrupted by pipetting in 1.5 ml of membrane isolation buffer (in mM): 50 Tris/HCl (pH 7.0 at room temperature), 150 NaCl, 5 MgCl2, 1 DTT, followed by centrifugation through cell shredders (Macherey and Nagel, Oensingen,

Switzerland). The membrane pellets obtained after 30 min centrifugation at 20,000 g (4°C) were resuspended in membrane solubilization solution (25 μl per oocyte) containing (in mM): 50

Tris/HCl (pH 7.0 at RT), 150 NaCl, 1 DTT and 1% (v/v) Triton X100. Membrane proteins were solubilized by 45-60 min incubation on an orbital shaker at 4°C, and centrifuged for 12 min as before. The Triton-soluble fractions thus obtained were mixed with 5x Sample buffer (30 mM

DTT final concentration) and heated for 20-30 min at 37°C.

Supplemental Material – Salih et al.

Western blot analysis of Triton-soluble fractions

Proteins in samples were resolved, along with pre-stained molecular weight markers (peqGold

Protein Marker V, Peqlab Biotechnologie GmbH, Erlangen, Germany #27-2210), by SDS-PAGE on 5–15% acrylamide gradient minigels supplemented with 0.5% (v/v) of 2,2,2-Trichloroethanol

(Acros-Organics, Geel, Belgium) for subsequent, in-gel, fluorescently labelling of proteins.9

After electro-transfer onto nitrocellulose (Membrane Solutions, Bellevue, WA,

#MSNC020230301), the blotted, fluorescently-labelled proteins were imagined on an UV- transilluminator using an 8-bit digitizing system (Vilber Lourmat GmbH, Eberhardzell,

Germany) and data stored as non-compressed TIF images. Total protein per lane was assessed densitometrically from these images using ImageJ (ImageJ, U. S. National Institutes of Health,

Bethesda, Maryland, USA, http://imagej.nih.gov/ij/, 1997-2016). A rabbit antibody against

γENaC was a generous gift from Dr. J. Löffing's lab (Institute of Anatomy, University of Zürich,

Switzerland). Anti-HA tag mouse monoclonal antibody (clone 16B12, #HA.11) was purchased from Covance, Dedham, MA. After 1 h of blocking at room temperature in 0.1% (w/v) casein solution, the blots were incubated overnight with the primary antibody in blocking solution supplemented with 0.1% (v/v) Tween-20 (CBB-T). After three rounds of washing over 20–30 minutes in TBS, 0.1% Tween-20 (TBS-T), the blot was incubated for 1 h in the presence of

IRDye-conjugated secondary antibodies (LI-COR Biosciences GmbH, Bad Homburg, Germany):

Polyclonal goat antibody anti-rabbit IRDye 800CW (#926–32211) and goat anti-mouse IRDye

680CW (#926–68070), all diluted 1/12,000 in CBB-T. After washing in TBS-T, the blots were scanned with an Odyssey Infrared Imaging System (LI-COR Biosciences). Band intensities were assessed with the Odyssey v2.1 software and normalized with the amount of total, TCE-labelled, protein in the corresponding lanes.

Supplemental Material – Salih et al.

Analysis of cell-surface biotinylated fractions

Control or injected oocytes (~25 per condition) were incubated for 15 min on ice in 1 ml

Biotinylation buffer (in mM, Triethanolamine 10, NaCl 150mM, CaCl2 2, pH 9.5, supplemented with 1 mg/ml NHS-Sulfo-S-S-Biotin; Thermo-Scientific #21331). To have controls for nonspecific binding to Neutravidin beads, equal amounts of oocytes were incubated in the absence of biotinylation reagent. The residual reagent was quenched by replacing the biotinylation solution with 1 ml of MBS supplemented with (mM) Glycine 192, Tris/HCl 25, pH 7.5 and incubating for 5 min at 22°C. After one rinsing step in MBS, drained oocytes were stored at -

20°C. Biotinylated fractions were isolated from ENaC-enriched fractions which had been purified as described before, except for the substitution of 1 M DTT with 10 mM N-

Ethylmaleimide. 25 µl samples of each Triton-X100-soluble fraction were withdrawn and mixed with equal volumes of 2xSample buffer/DTT (50 mM final) for further analysis. The rest of the isolated fractions were adjusted to 150 mM NaCl (from a 5 M stock solution) and incubated on an orbital shaker for 5-6 h at 4°C in the presence of 25 µl (bed volume) of Neutravidin-agarose beads (Thermo Scientific #29202). Non-bound fractions were discarded, the beads were washed twice with high salt solution (in mM, Tris/HCl pH 7.5, 50; NaCl, 500; EDTA, 5; PMSF 1, and

1% (v/v) TritonX100) by incubating each time for 5 min on an orbital shaker at 22°C. Beads were rinsed once in low salt solution (in mM, Tris/HCl pH 7.5, 50; NaCl, 150; PMSF 0.5, and

0.5% (v/v) TritonX100), and resuspended in 50 µl of 2xSample buffer/DTT (50 mM final).

Bound fractions were eluted by heating for 10 min at 72°C. Triton-soluble fractions (1% of total) and Neutravidin-bound fractions were resolved by SDS-PAGE, transferred to nitrocellulose membranes, and blocked as described before. Nitrocellulose membranes were subsequently incubated overnight at 4°C in the presence of rat monoclonal anti-HA Antibody (Clone 3F10,

Supplemental Material – Salih et al.

Roche #11 867 423 001) diluted 1/1,000 in 0.1% (w/v) casein solution and, after washing, 2 hours in the presence of HRP-conjugated Goat Anti-Rat immunoglobulins (Jackson

ImmunoResearch Europe #112-035-03) diluted 1/5,000 in 1% (w/v) skimmed milk, in PBS.

After washing, HRP signal was revealed using Western Bright Sirius detection reagent

(Advansta, Menlo Park, CA, #K12043-D20) and detected using a Fusion Solo imaging system

(Vilber Lourmat, Marne-la-Vallée, France). Band intensities were measured from 16-bit gray- scale images using ImageJ software. To account for non-specific binding to Neutravidin beads, band intensity values from control, non-biotinylated samples were subtracted from the values of the corresponding, biotinylated samples.

Supplemental Material – Salih et al.

References

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Supplemental Material – Salih et al.