Genetic Analysis of the β Subunit of the Epithelial Na+ Channel in Essential Hypertension
Abstract—Mutations of the last exon of the β subunit of the amiloride-sensitive epithelial Na+ channel (βENaC) can lead to Liddle’s syndrome, a rare monogenic form of hypertension. The objective of this study was to test whether more subtle changes of βENaC could be implicated in essential hypertension. After determination of the βENaC coding gene organization (12 exons spanning 23.5 kb), a systematic screening of the last exon of the gene was performed in 525 subjects (475 whites, 50 Afro-Caribbeans), all probands of hypertensive families. This search was extended to the remaining 11 exons in a subset of 101 probands with low-renin hypertension. Seven amino acid changes were detected: G589S, T594M, R597H, R624C, E632G (last exon), G442V, and V434M (exon 8). These genetic variants were more frequent in subjects of African origin (44%) than in whites (1%). The functional properties of the variants were analyzed in Xenopus oocytes by two independent techniques, ie, electrophysiology and 22Na+ uptake. Small but not significant differences were observed between the variants and wild type. The clinical evaluation of the family bearing the G589S variant, which provided the highest relative ENaC activity, did not show a cosegregation between the mutation and hypertension. The present study illustrates the difficulty in establishing a relation of causality between a susceptibility gene and hypertension. Furthermore, it does not favor a substantial role of the βENaC gene in essential hypertension.
The ENaC constitutes the rate-limiting step of Na+ reabsorption in many epithelial tissues, such as the renal distal tubule, distal colon, and airways, and plays a key role in the sodium-retaining properties of aldosterone. It is composed of 3 homologous subunits, entitled α, β, and γ. The structure of these subunits is characterized by the presence of 2 transmembrane domains separated by a large extracellular loop. The carboxy- and amino-terminal domains are oriented toward cytoplasm.1 Truncations or substitutive mutations of critical residues of the last exon of the β2 3 4 5 or γ6 subunit can be responsible for Liddle’s syndrome, a rare autosomal-dominant form of low-renin hypertension with hypokalemia, metabolic alkalosis, and volume expansion. All mutations described to date in families affected with Liddle’s syndrome either abolish2 6 or alter3 4 a highly conserved PY motif of the intracytoplasmic C-terminus of these subunits. In each case, a gain of function with a significant increase of the amiloride-sensitive current is observed after expression of the mutant subunits in Xenopus oocytes. Conversely, mutations in the amino-terminal end and in the extracellular loop of the ENaC result in a loss of function and are responsible for pseudohypoaldosteronism type 1, an autosomal-recessive salt-wasting disease with hyperkalemia, metabolic acidosis, and dehydration.7 8
Epidemiological, clinical, and physiological considerations favor a relationship between salt intake and blood pressure, especially in patients of African origin and/or with low plasma renin.9 Given the critical role of ENaC in sodium reabsorption, the genes coding for the 3 subunits of this channel are good candidates for salt-sensitive forms of hypertension. This study was undertaken to test the hypothesis of the existence of mutations of the last exon of βENaC in patients with familial essential hypertension belonging to the HYPERGENE data set.10 Patients were classified according to their ethnic origin, and the detection of molecular variants was extended to all the coding sequences of βENaC in a subset of 101 patients with low-renin profile.
The screening of the last exon of the βENaC gene was performed in 525 hypertensive probands previously selected in the HYPERGENE data set of hypertensive families recruited in the Broussais Hypertension Clinic.10 All patients gave informed consent to participate in this study, which was approved by the Comité de Protection de la Recherche Biomédicale de Paris-Cochin. In this data set, only patients who satisfied the following criteria were considered: established hypertension with either diastolic blood pressure ≥95 mm Hg or the presence of antihypertensive therapy, onset of hypertension before 55 years of age, body mass index ≤27 kg/m2, absence of secondary hypertension, absence of diabetes mellitus or renal insufficiency, and absence of exogenous factors (excessive alcohol consumption, estrogen use) that could interact with blood pressure. The screening of the other 11 exons of the βENaC was performed in a subset of 101 low-renin probands from the same database. Low-renin profile was defined in untreated patients by plasma active renin levels <15 pg/mL or 20 pg/mL in supine or upright position, respectively, and plasma aldosterone levels <120 pg/mL and 300 pg/mL in supine and upright position, respectively.
Biochemical and Hormonal Measurements
Plasma active renin was measured by an immunoradiometric assay11 using two monoclonal antibodies in a commercially available kit (ERIA, Diagnostics Pasteur). Plasma aldosterone was measured by radioimmunoassay as previously described (Coat-a count Aldosterone Radioimmunologie, Behring). Normal values have been previously reported.12
Determination of the Intron-Exon Boundaries of βENaC
SSCP13 of the coding sequences of the βENaC using genomic DNA extracted from leukocytes of hypertensive patients required the knowledge of the genomic organization of the βENaC. To determine the intron-exon boundaries of the gene, a strategy of amplification of each intron was used, starting from the known cDNA sequence.14 15 Pairs of primers distributed every 200 bp over the whole cDNA were designed and used to amplify selected parts of the genomic DNA. When no amplification was obtained, novel primers were designed, about 100 bp apart. Amplification was performed using the long-range PCR kit (Perkin-Elmer). The products of amplification were then run on agarose gels, the DNA bands obtained were cut from the gel, purified using a Gene Clean II kit (Bio 101), and subcloned into the plasmid pTAg (kit LigATor, R&D). Six subclones of the variant PCR product were sequenced using the method of Sanger et al.16
The search for mutations of the βENaC coding parts was performed by SSCP. The primers for the 12 exons of the βENaC were designed according to the intron-exon boundaries (Table 1⇓) to generate products less than 280 bp to maintain the maximum efficiency of the SSCP technique.13 Forward and reverse primers B1, B3, B4, and B6 were designed according to Chang et al.7 Due to the length (378 bp) of the coding part of the last exon, the pairs of primers 12A, 12B, and 12C were designed to amplify 3 overlapping segments of this exon. The forward primer 12A and reverse primer 12C are located in the last intron (nucleotides −30 to −10 before exon 12) and the 3′ untranslated region (nucleotides 2082 to 2102 of the cDNA sequence), respectively, whereas the 4 others are complementary to the coding sequences. All the other primers are located within introns including at least 10 bp of intronic sequence on either side of splice junctions to be able to detect possible variants of the intron-exon boundaries.
DNA samples (100 ng) were amplified using genomic DNA in a total volume of 20 μL containing 50 mmol/L KCl, 5 mmol/L Tris-HCl, 0.01% gelatin, 1.5 mmol MgCl2, 125 μmol/L dNTPs, 10 pmol of each unlabeled primer, 0.5 U Taq polymerase, and 0.05 μL [α-32P]dCTP (10 μCi/μL). PCR products were then diluted 5-fold in 95% formamide, 20 mmol/L EDTA, 0.05% bromophenol blue, and 0.05% xylene cyanol. After denaturation at 90°C for 4 minutes, the samples were cooled on ice before they were loaded onto a nondenaturing gel in 2 conditions: first, an MDE gel (Bioprobe Systems) prepared in 0.5× TBE electrophoresis buffer (1× TBE=90 mmol/L Tris-borate [pH 7.8], 2 mmol/L EDTA), run at room temperature for 14 hours at 300 V constant voltage; and second, a 5% polyacrylamide gel (49:1 polyacrylamide:methylene-bis-acrylamide) prepared in 0.5× TBE at 500 V, run at 30 W constant power for 3 to 4 hours. The gels were dried and autoradiographed with an intensifying screen for 12 to 24 hours.
Individual SSCP bands presenting a shift, as well as controls, were cut directly from the dried gel, suspended in 100 μL H2O, and incubated at 37°C for 1 hour. A 2-μL aliquot was subjected to enzymatic amplification in a 100-μL reaction in the same conditions. The double-stranded PCR product resulting from this amplification was directly sequenced as described by Khorona et al.17 In case of reading difficulty or ambiguity, they were subcloned into the plasmid pTAg (kit LigATor, R&D). Six subclones of the variant PCR product were sequenced using the method of Sanger et al.16 Confirmation of each rare variant was also obtained by modification of a specific enzyme restriction site: G589S (AluI), T594M (NlaIII), R597H (Fnu4HI), R624C (HgaI), E632G (HinfI).
The presence of the G442V variant in the whole set of hypertensive and control subjects was checked by allele-specific hybridization analysis: each product amplified with the B8 forward and reverse primers was denatured by NaOH 0.4N 5 minutes and transferred to a nylon membrane (Hybond N+, Amersham), then spotted in duplicate on a nylon membrane (Hybond +) and neutralized with 3 mol/L sodium acetate before cross-linking. Each membrane was therefore hybridized in PEG for 6 hours with 32P end-labeled oligonucleotide probes corresponding to the wild-type and mutant sequences (wild-type: 5′-gCACATgCCAATgCAg-3′; mutant: 5′-gCACATgACAATgCA-3′). After 2 washes at room temperature, filters were washed for 10 minutes at 48°C in a solution containing 3× SSC and 0.1% SDS. Filters were exposed for 1 hour with an intensifying screen (Kodak X-Omat).
The plasmid pEXO, designed for optimal expression of cDNAs in Xenopus oocytes18 containing the human lung βENaC cDNA, was grown in CJ 236 bacteria using a tryptone/yeast extract/NaCl/phosphate medium containing 25 μg/mL uracyl and infected with R408 helper phage at a multiplicity of 10/1 (phage/cell). Uracyl-containing single-strand DNA was precipitated using 5% PEG 6000 and 0.95 mol/L ammonium acetate and then purified using M13 purification resin (Promega). β Single-strand DNA (500 ng) and 10 pmol phosphorylated mutagenic primer were denatured together for 10 minutes at 95°C, slowly cooled to room temperature, and chilled on ice. T4 DNA polymerase (3 U), T4 DNA ligase (3 U), T4 DNA polymerase buffer, and 1 mmol/L dNTPs were added to the reaction, and the mixture was put on ice for 10 minutes, held at room temperature for 15 minutes, and then heated to 37°C for 1 hour. After heat inactivation, 2 μL of this reaction mixture was used to transform XL1 bacteria. Ampicillin-resistant colonies hybridizing with [γ32P]ATP-labeled mutagenic oligonucleotides were checked by dideoxy sequencing16 using a dye terminator kit and automatic sequencing (Applied Biosystems 373A, Perkin-Elmer).
Expression and Electrophysiological Recording in Xenopus Oocytes
Mature female Xenopus laevis were maintained at 20°C with a 12-hour light-dark cycle. Individual females were anesthetized in ice and oocyte clusters surgically removed from the ovary. Oocyte clusters were torn apart with forceps in ND96 medium containing (in mmol/L) 96 NaCl, 2 KCl, 10 HEPES, 1.8 CaCl2, at pH 7.4. Denuded oocytes were obtained by collagenase digestion (type IA, Sigma, 370 U/mL) during 2 hours at room temperature and rinsed several times in ND96. Stage 5 to 6 oocytes were selected and incubated overnight at 18°C in ND96 medium with gentamycin (50 μg/mL). Healthy oocytes were selected and injected with 50 nL of cRNA (5 to 20 ng/mL).The oocytes were incubated for 2 to 4 days after injection in ND96 medium supplemented with gentamycin and 10 μmol/L amiloride.
For current measurements, oocytes were punctured with 2 conventional microelectrodes (3 mol/L KCl) for voltage-clamp experiments (TEV-200 voltage clamp, Dagan Corporation). The specific ENaC was defined as the total current recorded at −70 mV holding potential minus the current recorded at the same holding potential in the presence of 30 μmol/L amiloride. 22Na+ uptake experiments were carried out as previously described.18 Briefly, 20 to 30 oocytes were preincubated for 15 minutes into 1 mL of Na+-free medium (in mmol/L: 96 choline chloride, 2 KCl, 10 HEPES, 1.8 CaCl2, pH 7.4). The oocytes were incubated into 200 μL of a medium containing 48 mmol/L NaCl (15 to 25 μCi/mmol, 48 mmol/L choline chloride, 2 mmol/L KCl, 10 mmol/L HEPES, 1.8 mmol/L CaCl2, pH 7.4). After 10 minutes, the oocytes were thoroughly rinsed with cold choline medium, and β emission was measured with a Tricarb 1600CA counter (Packard). The specific signal was defined as the difference between the total uptake and the uptake measured in the presence of 30 μmol/L amiloride.
Experimental Design and Statistical Analysis
To evaluate the effect of each molecular variant into Xenopus oocytes, several independent measurements were performed with at least 2 different batches of in vitro transcribed RNA. In each experiment, currents observed with one or several mutants were compared with those generated by wild type (n=2 to 10 oocytes measured for each mutant in each experiment) and normalized according to the level of expression of the wild type measured in the same experiment. Quality of the RNAs was checked by electrophoresis: only RNAs for which a perfect correlation existed between gel analyses and optical density measurement were injected into oocytes. Temperature was kept below 20°C because an increased activity was noticed above 25°C.
When a sufficient number of independent experiments had been performed (for G589S), the mean activity of the mutant was also calculated as the mean value of the activity measured during each independent experiment. This procedure provided a new set of data, in which the interoocyte variation was reduced. A 2-way ANOVA for unbalanced data was performed using the Superanova (Abacus Concepts Inc) statistical package. To correct for the non-Gaussian distribution of the Na+ currents or flux, these parameters were log transformed for the statistical analysis. A first ANOVA was conducted with an additive model using the log-transformed Na+ current or flux as the dependent variable and the type of mutation and the day of experiment as independent parameters. Both mutations and experiments influenced significantly (P<10−4) the observed current. A post hoc analysis using a bilateral Dunnett test was therefore performed, indicating whether the current or flux observed with each mutant was significantly different from that observed with the wild-type cRNA, either at the 0.05 or 0.01 level. The average increases of current and flux are expressed as a ratio between mutant and wild type.
The main features of the 525 patients of the HYPERGENE database and of the low-renin subset are shown in Table 2⇓. Most of the patients were white (90.5%), and 50 of them (9.5%) were of African origin (40 West Indies, 10 West Africa). Patients of African origin were significantly younger than those of European ancestry and had a tendency to lower blood pressure and lower treatment rate. As expected, the proportion of females in this group was slightly higher than that observed in whites (66% versus 52%), as well as the proportion of patients in the low-renin subset (22.7% versus 9.6%). These findings can reflect true differences between the 2 ethnic groups, as well as consequences of selection or random findings on this small subset of patients of African origin.
Using the PCR-based cloning strategy and the known human cDNA sequence, the overall intron-exon organization for the βENaC gene was determined for the coding region and in agreement with the GT/AG rule: it is composed of at least 12 exons separated by 11 introns encompassing approximately 23.5 kb (Table 3⇓). This strategy cannot rule out the presence of 1 or more untranslated exons in the 5′ part of the gene. Amplification of 3 random genomic DNA samples by primers located within the last part of the coding sequence (nucleotides 1883 to 1904 of the cDNA) and just before the polyA site (nucleotides 2326 to 2347 and 2491 to 2512) led to expected 465- and 630-bp-length products, respectively, ruling out the presence of an additional intron in this region. We found no evidence for a particular gene organization that could explain the 3′ truncated variant previously cloned from a human adult lung cDNA library. Since this variant was detected only by reverse transcription and not by Northern blot, we cannot eliminate a false-positive finding due to a strong secondary structure of the cDNA.14 Overall, the number of translated exons of βENaC is the same as for γENaC19 and the overall intron-exon size and organizations of both genes appear similar (A.P., X.J., unpublished data, 1998).
Detection of Genetic Variants
Exon 12 of the β subunit of the ENaC was systematically screened in 525 probands of the HYPERGENE database. Three rare polymorphisms leading to no change in amino acid sequence were detected at codons Ile 515 (ATC→ATT; n=3), Thr 577 (ACC→ACT; n=1), and Leu 628 (CTG→TTG; n=1) in 5 patients. More interestingly, 5 missense mutations were detected in 7 unrelated subjects, all in the heterozygous state: G589S (GGC→AGC), T594M (ACG→ATG), R597H (CGC→CAC), R624C (CGT→ TGT), and E632G (GAG→GGG) (Figure 1⇓). Each mutation was found in only 1 subject, except for T594M, which was found in 3 subjects of African ancestry (3 of 50=6%). One mutation, R624C, abolishes a putative protein kinase C phosphorylation site located 3′ next to the PY motif.
Two other missense mutations were found by the SSCP screening of the 11 other βENaC exons in the 101 patients with the low-renin profile. Both are located in exon 8: G442V (GGC→GTC) and V434M (GTG→ATG) (Figure 1⇑). The V434M variant was found in the heterozygous state in a single white patient. The G442V variant was found in 8 patients of African origin, also in the heterozygous state. The whole set of 525 hypertensive subjects from the HYPERGENE database was screened for variant G442V, which was found in 11 additional individuals. All except 1 were of African origin.
Clinical and Biological Features of Hypertensive Patients Bearing βENaC Variants
Clinical and biological characteristics of the corresponding hypertensive probands are shown in Table 4⇓. Some had phenotypes compatible with an in vivo effect of the mutation with a low-renin profile and a tendency to hypokalemia (see, for example, subjects bearing the G589S and the R597H variants). However, in most cases, the outpatient clinical and biological workup performed in those patients was unremarkable compared with that observed on average in this database of hypertensive patients.
Interestingly, most of these variants were observed in the subset of patients of African origin. This was especially the case for the G442V (only 1 of 19 white subjects) and T594M (0 of 3) polymorphisms. Exon 12 variants were much more frequent in subjects of African descent (8%) than in whites (0.6%, P<10−4) (Table 5⇓). Variants of exon 8 were also almost exclusively found in the African subgroup, with a frequency of 36% compared with 0.4% (P<10−4) in whites.
Functional Expression in Xenopus Oocytes
The Na+ channel activity was measured after expression of the mutant β chains in Xenopus oocytes with normal human α and γENaC. Measurements obtained after expression of βΔ, which corresponds to mutation β579del32 recently described in a new Liddle’s pedigree,5 were used as a positive control. This positive control displayed a large range of values, some corresponding to a large increase in Na+ current, others being within the range of normal values. A similar pattern was also observed for some of the mutants, especially G589S, R597H, and E632G.
The mean sodium current measured after expression of most of the mutations was either unchanged or slightly increased compared with the current obtained after expression of the wild-type chain. Using the Dunnett statistical test, statistical significant increases in Na+ current were observed only for G589S (1.47±0.11, n=30, P<0.01) and R597H (1.39±0.17, n=26, P<0.05) (Table 6⇓). A nonsignificant 1.30-fold increase was observed with T594M.
To further analyze these genetic variants, independent measurements of the amiloride-sensitive 22Na+ uptake into Xenopus oocytes expressing the different constructs were performed. Statistically significant increases were observed, not only for the positive control (1.76±0.07, P<0.01) but also for G589S (1.30±0.06, P<0.01) and R624C variants (1.35±0.08, P<0.01). The increase observed with E632G (1.36±0.12) reached only the 0.05 significance level. The results observed with other variants were not significantly different from those observed with the wild-type sequence.
On the whole, some variants displayed a statistically significant increase of Na+ current but no significant change of Na+ flux (R597H) or vice versa (R624C). A functional mutation would have been expected to increase both assays, as was indeed the case for the βΔ mutant, previously found in a Liddle’s syndrome family and disrupting the C-terminal PY motif.5 Even though we took multiple precautions to limit the variability of each functional assay (Na+ current and uptake), a large variability was observed for each mutant with both techniques. Thus, only the G589S variant, which displayed significant differences with both assays, was considered as positive. Even for this variant, the higher amiloride-sensitive 22Na+ uptake value was mainly explained by the greatly increased activity observed in only 1 of 4 independent experiments: 77±6%, 232±16%, 148±19%, and 106±7%. Accordingly, histogram analysis of the repartition of the 22Na+ uptake (Figure 2⇓) shows a main mode that superimposes with wild type and a second mode at higher values, where the contribution of the 232±16% experiment is important. These data certainly do not favor a strong stimulation of ENaC activity associated with the G589S variant. To identify more subtle phenotypes, this variant was more extensively analyzed in vivo.
Genotype-Phenotype Analysis of G589S Kindred
Seven individuals (all women) belonging to 2 generations of the same family bearing the G589S variant were evaluated (Figure 3⇓). Among them, 4 were heterozygous G589S, all hypertensives. The proband bearing the G589S mutation had a mild hypokalemia (3.8 mmol/L), together with low levels of renin and aldosterone. However, 2 of the other 3 women not bearing the variant were also hypertensive, and the confounding effect of the antihypertensive therapy made it difficult to evaluate any tendency to hypokalemia. Among the 2 untreated sisters (II-2 and II-3), the one heterozygous G589S sister was clearly hypertensive (average of 164/93 mm Hg on a 30-minute oscillometric blood pressure reading), whereas the sister with a wild-type genotype had normal blood pressure values (116/69 mm Hg in the same conditions). Both sisters had low potassium levels, as well as low plasma renin (II-2, 7 pg/mL; II-3, 10 pg/mL) and aldosterone levels (II-2, 51 pg/mL; II-3, 51 pg/mL), but a marked weight difference (see Figure 3⇓). Thus, in this family, it was difficult to distinguish between the effect of the βENaC genetic variant on blood pressure and other factors such as obesity, familial dyslipidemia, and non–insulin-dependent diabetes mellitus.
The critical role of ENaC in sodium reabsorption and the existence of mutations of βENaC at the origin of Liddle’s syndrome, a rare and severe form of salt-sensitive hypertension, led to the hypothesis that more subtle variants of this channel subunit could account for a proportion of essential forms of hypertension, especially those characterized with low plasma renin level and those occurring in populations of African descent. We report here the results obtained by a systematic screening of the whole coding sequence of βENaC in a large set of white hypertensive subjects with family history of hypertension, as well as in 50 hypertensive patients of African origin. Because only a preliminary and negative study limited to the carboxy-terminal end of βENaC (amino acids 556 to 627) in 90 unselected Japanese hypertensive patients20 has been reported so far, this study is the first to look extensively for mutations in all the coding sequences of βENaC in a very large set of hypertensive subjects.
Seven amino acid changes were found. Five are located within the last exon, which codes for the second transmembrane domain and the intracytoplasmic carboxy terminus already involved in Liddle’s syndrome. Two others were found in exon 8, which codes for a segment of the extracellular loop. Among the 5 variants affecting the last exon in our population, 3 (G589S, R597H, and T594M) were identified in the region F588 GFQPDTAPRSPNTGPYPSEQALP611, corresponding to a stretch of amino acids located near the PY motif.14 This region is poorly conserved between humans, rats, and Xenopus. Variant R624C removes a putative phosphorylation site for protein kinase C, which is found in β and γ subunits from humans, rats, and Xenopus. The two variants detected in exon 8 (V434M and G442V) are located in the extracellular loop, outside the highly conserved pre-M2 segment, which is involved in the formation of the ionic pore.1 21
The frequency of the genetic variants of βENaC in patients with essential hypertension was approximately 1% (5 of 475) in the white population but reached 44% (22 of 50) in patients of African origin (Table 6⇑). The major part of this high prevalence in the latter group was due to 2 frequent polymorphisms: T594M (6%) and G442V (36%), located in exon 12 and exon 8, respectively, but not in linkage disequilibrium. We had no access to an unbiased control population from the West Indies to test the potential association of these genetic variants with hypertension. Similar T594M frequencies were reported by Su et al22 in normotensive (6.7%) and hypertensive (5.6%) African Americans. Using two different assays, we could not detect any effect of the T594M and G442V variants when expressed into Xenopus oocytes. Thus, available data indicate that these polymorphisms are not likely to be responsible for the high prevalence of salt sensitivity and hypertension observed in the populations of African ancestry.23
The possible functionality of the rare genetic variants (G589S, R597H, R624C, E632G, and V434M) can be appreciated from in vivo and in vitro experiments. In vivo, it was difficult to evaluate from the clinical and biological profile of the affected individuals. Even though some patients had a tendency to hypokalemia, a low-renin profile, and an early onset of hypertension, their characteristics did not suggest a typical syndrome of volume expansion due to excessive Na+ reabsorption. However, one must keep in mind the heterogeneity of the clinical and biological presentation of individuals affected with more stringent mutations leading to Liddle’s syndrome.24 Another classic methodology to analyze the in vivo effect of rare genetic variants is to perform linkage analysis in large pedigrees of affected probands. For most of these variants, we were unable to use this strategy because most of the first-degree relatives of the affected individuals were living in the West Indies or were currently treated for hypertension, which makes the evaluation of blood pressure level or of any biochemical marker difficult. The clinical and biological analysis of the white pedigree bearing the G589S variant, which exhibited the highest ENaC activity, shows the difficulty to demonstrate a possible mild genetic effect in a complex multifactorial trait such as hypertension.
In vitro, expression in Xenopus oocytes followed by measurement of the amiloride-sensitive Na+ current has been the gold standard technique used to evaluate the functionality of mutations of either ENaC subunit in Liddle’s syndrome. In this study, the Na+ changes of current obtained after expression of the βENaC genetic variants were not statistically significant in most cases (Figure 2⇑). These data were compared with the measurement of 22Na+ flux in a large number of eggs injected with wild-type or mutant cRNAs. Only those containing the Liddle’s mutation displayed consistent positive results in both types of measurements. Our results are in agreement with the absence of significant Na+ current increase following expression of variants obtained by systematic alanine replacement of the amino acids of the carboxy-terminal part of the βENaC, with the exception of those affecting the PY motif.25 In particular, the lack of significant increase after expression of mutation R624C is consistent with the negative results obtained after expression of R624A26 and the absence of in vitro phosphorylation of the carboxy-terminal end of the βENaC by protein kinase C.1
However, the lack of significant increase in Na+ current observed after transient expression of these variants in Xenopus oocytes does not completely rule out any functional impact. Indeed, in vivo, even a single additional mEq of Na+ per day would result in a yearly accumulation of 365 mEq Na+, probably leading to plasma volume expansion and low-renin hypertension. In addition, it is not known whether amphibian cells such as Xenopus oocytes possess the whole cellular machinery involved in the complex regulation of the ENaC in mammalian cells. For instance, cAMP stimulates channel activity in renal cortical collecting tubules, in distal airways, or after heterologous expression of the 3 ENaC subunits in MDCK or Vero cells but has no effect on the channel expressed in Xenopus oocytes.1 One might speculate that some key element of the complex cellular machinery involved in the regulation of ENaC is missing in Xenopus oocytes. Other experimental systems, such as immortalized human lymphocytes, might represent alternative systems of expression.27 28 This possibility is suggested by a recent report by Cui et al, 29 who have described an increased effect of cAMP and a loss of protein kinase C inhibition in lymphocytes of subjects homozygous for T594M.
In conclusion, a systematic screening of the coding sequences of βENaC in a large set of hypertensive patients detected some rare genetic variants and two polymorphisms present almost exclusively in subjects of African ancestry. For most of them, no significant change was observed after expression into Xenopus oocytes. Although we cannot exclude the presence of mutations or polymorphisms in the 5′ or 3′ region of the gene that may dictate its expression or regulation, these results are not in favor of a substantial contribution of the βENaC to the genetics of essential hypertension.
Selected Abbreviations and Acronyms
|ENaC||=||epithelial Na+ channel|
|PCR||=||polymerase chain reaction|
|SSCP||=||single-strand conformational polymorphism|
This project was partly supported by the Société Française d’Hypertension Artérielle. Michel Lazdunski and Pascal Barbry’s investigations were supported by grants from the Centre National de la Recherche Scientifique and the Association Française de Lutte contre la Mucoviscidose. We gratefully thank Cécile Dumont for her technical assistance and Albert Vuagnat and Gilles Chatellier for their statistical advice. The expert technical assistance of Valérie Friend, Martine Jodar, and Dahvya Doume is greatly acknowledged. Some initial in vitro experiments were performed by Dr Valérie Urbach. We thank Emma Baker (London, UK) and Anil Menon (Cincinnati, Ohio) for providing unpublished information.
- Received December 10, 1997.
- Revision received January 2, 1998.
- Accepted February 6, 1998.
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