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Advances in molecular research techniques have enabled a new frontier in discerning the mechanisms responsible for monogenic diseases. In this review, we discuss the current research on the molecular pathways governing blood pressure disorders with a Mendelian inheritance pattern, each presenting with a unique pathophysiology. Glucocorticoid Remediable Aldosteronism (GRA) and Apparent Mineralocorticoid Excess (AME) are caused by mutations in regulatory enzymes that induce increased production of mineralocorticoids or inhibit degradation of glucocorticoids, respectively. Geller syndrome is due to a point mutation in the hormone responsive element of the promotor for the mineralocorticoid receptor, rendering the receptor susceptible to activation by progesterone, leading to hypertension during pregnancy. Pseudohypoaldosteronism type II (PHA-II), also known as Gordon’s syndrome or familial hyperkalemic hypertension, is a more variable disorder typically characterized by hypertension, high plasma potassium and metabolic acidosis. Mutations in a variety of intracellular enzymes that lead to enhanced sodium reabsorption have been identified. In contrast, hypertension in Liddle’s syndrome, which results from mutations in the Epithelial sodium Channel (ENaC), is associated with low plasma potassium and metabolic alkalosis. In Liddle’s syndrome, truncation of one the ENaC protein subunits removes a binding site necessary protein for ubiquitination and degradation, thereby promoting accumulation along the apical membrane and enhanced sodium reabsorption. The myriad effects due to mutation in phosphodiesterase 3A (PDE3A) lead to severe hypertension underlying sodium-independent autosomal dominant hypertension with brachydactyly. How mutations in PDE3A result in the phenotypic features of this disorder are discussed. Understanding the pathologies of these monogenic hypertensive disorders may provide insight into the causes of the more prevalent essential hypertension and new avenues to unravel the complexities of blood pressure regulation.

Liddle's syndrome, an autosomal dominant form of monogenic hypertension, is characterized by salt-sensitive hypertension with early penetrance, hypokalemia, metabolic alkalosis, suppression of plasma rennin activity and aldosterone secretion, and a clear-cut response to epithelial sodium channel blockers but not spironolactone therapy. Here, we describe the case of a 16-year-old boy patient with resistant hypertension (maintain 170-180/100-110 mm Hg after administration four kinds of antiypertensive drugs) and severe hypokalemia. After a series of checks, we exclude primary aldosteronism and renal artery stenosis and other diseases. Finally, the Liddle syndrome was diagnosed because of the DNA sequencing found that the proband's mother and himself had mutations P616L (c.1847 C>T) in the SCNN1B gene. Liddle syndrome should be considered as a cause of hypertension in children or adolescents particularly with suppressed renin activity. Early diagnosis and appropriately tailored treatment avoid complications of long-term unrecognized or inappropriately managed hypertension. Genetic testing has made it possible to make accurate diagnoses and develop tailored therapies for mutation carriers. The role of genetic testing and genetic counseling in establishing the early diagnosis of Liddle's syndrome is important.

Summary Resistant hypertension is a common clinical problem in South Africa and is frequently associated with low renin and aldosterone levels, especially in black Africans. In South Africa, novel variants in the epithelial sodium channel (ENaC) have been described to be associated with varying degrees of hypokalaemia and hypertension due to primary sodium retention. We report here a case of Liddle’s syndrome due to a novel c.1709del11 (p.Ser570Tyrfs*20) deletion in the beta-subunit of the ENaC in a young black African male. We discuss the likely pathogenesis of hypertension in this setting as well as the treatment options available in South Africa aimed at the ENaC. This case highlights the need for vigilance in detecting and appropriately treating low-renin and low-aldosterone hypertension in view of the frequency of the described variants of the ENaC channel in our cuntry. Specific therapy such as amiloride should be made more widely available.

Advances in genetic mapping and sequencing techniques have led to substantial progress in the study of rare monogenic (Mendelian) forms of abnormal blood pressure. Many disease-defining pathways for hypertension have been identified in the past two decades. Perturbations in renal salt handling appear to be a common mechanism underlying these rare syndromes of hypertension. Excess activation at various points in the mineralocorticoid signaling pathway and malfunctioning of the autonomic (specifically sympathetic) nervous system have both been implicated in inducing hypertension, while complementary studies examining low blood pressure phenotypes have identified novel pathways exclusively linked to renal salt wasting in either the thick ascending limb or the distal nephron. The genetic defects and the physiological and cellular pathways affected in these various disorders are reviewed here. Importantly, studies have suggested that genetic variation affecting these same genes and pathways may play an important role in explaining the variation of blood pressure levels in the general population. The investigation of rare syndromes of human blood pressure variation has important implications for improving the diagnosis and treatment of hypertension.

BACKGROUND: Liddle's syndrome (LS) is a monogenic form of hypertension simulating a mineralocorticoid excess, and is currently suspected in young hypokalemic hypertensives. The aims of the study were: (i) to evaluate the clinical phenotype of LS in a newly identified Italian family of Sicilian origin carrying a gain-of-function mutation of the β subunit of the epithelial sodium channel (ENaC) (P617L) previously reported by our group in an apparently unrelated Sicilian patient presenting the typical phenotype of LS including hypokalemia; (ii) to determine whether an unknown biological relationship exists between the newly identified family and the family of the proband previously reported. METHODS: Genetic analysis was performed in the present family, in the individual in which the βP617L mutation was first observed, and in his relatives. RESULTS: βP617L mutation was identified in the proband and in three maternal relatives. None of them showed hypokalemia. Mild to severe early onset hypertension and left ventricular hypertrophy were present in all of them. Analysis of mitochondrial DNA (mtDNA) and Y chromosome profiles in the present family and in the proband's family previously reported showed the absence of a relationship between them. The availability of only one carrier of the mutation in one of the two families meant that a genetic analysis able to assess a founder effect was not feasible. CONCLUSIONS: LS should be considered in all cases of early onset hypertension, independently of the plasma potassium concentration. The incidence of LS may be greater than is currently thought, because hypokalemia is not invariably present.

Background A high prevalence of the R563Q mutation of the epithelial sodium channel β-subunit has been reported in South African hypertensives compared with unrelated normotensive controls. To delineate the effects of this mutation against a more uniform genetic background, this study investigated the association of the mutation with hypertension within affected kindreds. Methods Forty-five index patients and members of their kindreds were studied. Blood pressure, serum potassium and the presence of the R563Q mutation were determined. Results Of the 136 individuals studied, 89 were heterozygous for the R563Q mutation and 47 homozygous RR. The mean arterial pressure was significantly higher in the R563Q heterozygous group (p = 0.005) after adjusting for gender, race, age and kindred membership. Of the R563Q heterozygous subjects, 71 (80%) had hypertension, while 17 (36%) of the R563Q homozygous RR subjects were hypertensive. Six R563Q heterozygous subjects had hypokalaemia and one R563Q homozygous RR subject had hypokalaemia, but the difference was not statistically significant. Two heterozygous patients had Liddle’s syndrome, both occurring during pregnancy. Conclusion The R563Q mutation of β-ENaC is associated with hypertension within affected kindreds, but does not usually cause the full Liddle’s syndrome phenotype.

The knowledge of the genetic bases of hypertension has improved over the last decade; this area of research has high priority due to the high incidence of hypertension and its impact on public health. Monogenetic mineralocorticoid hypertension syndromes are associated with suppressed plasma renin activity due to excessive activation of the mineralocorticoid pathway. We review the pathophysiology, phenotype, and method of diagnosis for familial hyperaldosteronism type I and type II, hypertensive forms of congenital adrenal hyperplasia, 11β-hydroxysteroid dehydrogenase type 2 deficiency, Liddle’s syndrome, an activating mutation of the MR, and glucocorticoid resistance. We also review some genes that could contribute to essential hypertension.

Hypokalemia is a common clinical problem. The kidney is responsible for long term potassium homoeostasis, as well as the serum potassium concentration. The main nephron site where K secretion is regulated is the cortical collecting duct, mainly via the effects of aldosterone. Aldosterone interacts with the mineralocorticoid receptor to increase sodium reabsorption and potassium secretion; the removal of cationic sodium makes the lumen relatively electronegative, thereby promoting passive potassium secretion from the tubular cell into the lumen through apical potassium channels. As a result, any condition that decreases the activity of renal potassium channels results in hyperkalemia (for example, amiloride intake or aldosterone deficiency) whereas their increased activity results in hypokalemia (for example, primary aldosteronism or Liddle's syndrome). The cause of hypokalemia can usually be determined from the history. If there is no apparent cause, the initial step is to see if hypokalemia is in associated with systemic hypertension or not. In the former group hypokalaemia is associated with a high mineralocorticoid effect or hyperactive sodium channel as in Liddle's syndrome. In hypertensive hypokalemic patients, measurement of the renin, aldosterone, and cortisol concentrations would be of help in differential diagnosis.

Liddle’s syndrome, an inherited form of hypertension, is caused by mutations that delete or disrupt a C-terminal PY motif in the epithelial Na + channel (ENaC). Previous work indicates that these mutations increase expression of ENaC at the cell surface by disrupting its binding to Nedd4-2, an E3 ubiquitin–protein ligase that targets ENaC for degradation. However, it remains uncertain whether this mechanism alone is responsible; increased activity of ENaC channels could also contribute to excessive Na + transport in Liddle’s syndrome. ENaC activity is controlled in part by its cleavage state; proteolytic cleavage produces channels with a high open-state probability, whereas uncleaved channels are inactive. Here, we found that Liddle’s syndrome mutations have two distinct effects of ENaC surface expression, both of which contribute to increased Na + transport. First, these mutations increased ENaC expression at the cell surface; second, they increased the fraction of ENaC at the cell surface that was cleaved (active). This disproportionate increase in cleavage was reproduced by expression of a dominant-negative Nedd4-2 or mutation of ENaC ubiquitination sites, interventions that disrupt ENaC endocytosis and lysosomal degradation. Conversely, overexpression of Nedd4-2 had the opposite effect, decreasing the fraction of cleaved ENaC at the cell surface. Thus, the data not only suggest that Nedd4-2 regulates epithelial Na + transport in part by controlling the relative expression of cleaved and uncleaved ENaC at the cell surface but also provide a mechanism by which Liddle’s syndrome mutations alter ENaC activity.

Liddle's syndrome has been known as a disorder associated with abnormal sodium reabsorption in the distal tubule and transmitted as a rare autosomal dominant trait. It is caused by mutations in the SCNN1B or SCNN1C gene, which truncate the cytoplasmic carboxyl terminus of the beta and gamma subunit of the epithelial sodium channel (ENaC). Genetic analysis of ENaC in a Chinese family with Liddle's syndrome revealed P616H of SCNN1B coaggregated with the phenotype, while this variant was not detected in 100 unrelated subjects. No mutation at gamma ENaC could be detected in all members of the family. P616H is located in the conserved proline-rich PY motif of the betaENaC. The PY motif can interact with the WW domain in Nedd4 and affect the activity of ENaC. Structural bioinformatics analysis confirmed that the functional interaction between Nedd4 and ENaC reduces in Liddle-ENaC (P616H) when compared with wild-type ENaC. In summary, P616H may be an underlying mechanism for the signs and symptoms of this family.

Currents through epithelial Na channels (ENaCs) were measured in the cortical collecting tubule (CCT) of mice expressing truncated β-subunits of ENaC, reproducing one of the mutations found in human patients with Liddle's syndrome. Tubules were isolated from mice homozygous for the Liddle mutation (L/L) and from wild-type (WT) littermates. Amiloride-sensitive currents ( INa) from single cells were recorded under whole cell clamp conditions. CCTs from mice kept under control conditions and fed a diet with normal levels of Na had very small INas (WT: 18 ± 13 pA; L/L: 22 ± 8 pA at Vm= –100 mV) that were not different in WT and L/L animals. However, the L/L mice had much larger currents when the animals were fed a low-Na diet (WT: 256 ± 127 pA; L/L: 1,820 ± 330 pA) or infused with aldosterone (WT: 285 ± 63 pA; L/L: 1,600 ± 280 pA). Currents from L/L mice were also larger when animals were pretreated with a high-K diet but not when the CCTs were stimulated in vitro with 8-CTP-cAMP. Noise analysis of amiloride-induced fluctuations in INashowed that single-channel currents at Vm= 0 mV were slightly smaller in L/L mice (WT: 0.33 pA; L/L: 0.24 pA). This difference could be attributed to a decrease in driving force since current-voltage analysis indicated that intracellular Na was increased in the L/L animals. Analysis of spontaneous channel noise indicated that the open probability was similar in the two genotypes(WT: 0.77; L/L: 0.80). Thus the increase in whole cell current is attributed to a difference in the density of conducting channels.

Serine proteases, both soluble and cell-attached, can activate the epithelial sodium channel (ENaC) proteolytically through release of a putative 43-mer inhibitory tract from the ectodomain of the γ-subunit. ENaC controls renal Na(+) excretion and loss-of-function mutations lead to low blood pressure, while gain-of-function mutations lead to impaired Na(+) excretion, hypertension, and hypokalemia. We review an emerging pathophysiological concept that aberrant glomerular filtration of plasma proteases, e.g., plasmin, prostasin, and kallikrein, contributes to proteolytic activation of ENaC, both in acute conditions with proteinuria, like nephrotic syndrome and preeclampsia, and in chronic diseases, such as diabetes with microalbuminuria. A vast literature on renin-angiotensin-aldosterone system and volume homeostasis from the last four decades show a number of common characteristics for conditions with albuminuria compatible with impaired renal Na(+) excretion: hypertension and volume retention is secondary to proteinuria in, e.g., preeclampsia and nephrotic syndrome; plasma concentrations of renin, angiotensin II, and aldosterone are frequently suppressed in proteinuric conditions, e.g., preeclampsia and diabetic nephropathy; blood pressure is salt-sensitive in conditions with microalbuminuria/proteinuria; and extracellular volume is expanded, plasma atrial natriuretic peptide (ANP) concentration is increased, and diuretics, like amiloride and spironolactone, are effective blood pressure-reducing add-ons. Active plasmin in urine has been demonstrated in diabetes, preeclampsia, and nephrosis. Urine from these patients activates, plasmin-dependently, amiloride-sensitive inward current in vitro. The concept predicts that patients with albuminuria may benefit particularly from reduced salt intake with RAS blockers; that distally acting diuretics, in particular amiloride, are warranted in low-renin/albuminuric conditions; and that urine serine proteases and their activators may be pharmacological targets.

Liddle's syndrome: A novel mouse Nedd4 isoform regulates the activity of the epithelial Na + channel . The epithelial Na + channel (ENaC), which plays an essential role in renal Na + handling, is composed of three subunits (αβγ), each containing a conserved PY motif at the C terminus. In Liddle's syndrome, an inherited form of salt-sensitive hypertension, the PY motifs of either β or γENaC are deleted or modified. We have recently shown that a ubiquitin-protein ligase Nedd4 binds via its WW domains to these PY motifs on ENaC, that ENaC is regulated by ubiquitination, and that Xenopus laevis Nedd4 (xNedd4) controls the cell surface pool of ENaC when coexpressed in Xenopus oocytes. Interestingly, Na + transporting cells, derived from mouse cortical collecting duct, express two different Nedd4 isoforms, which we have termed mNedd4-1 and mNedd4-2. Only mNedd4-2, which is orthologous to xNedd4, but not mNedd4-1, is able to regulate ENaC activity, and this property correlates with the capability to bind to the ENaC complex. Hence, Nedd4-2 may be encoded by a novel susceptibility gene for arterial hypertension.

The kidneys play a pivotal role in causing some forms of hypertension and probably a permissive role in most, if not all, forms of hypertension. This concept of the critical role of the kidneys has been postulated for many years but has been solidified by the molecular unraveling of several monogenic forms of hypertension such as Liddle's syndrome, apparent mineralocorticoid excess and glucocorticoid-remedial aldosteronism. These and other hypertensive disorders cause sodium retention through excess Na reabsorption in the distal nephron. Some disorders of salt wasting and relative hypotension such as Bartter's syndrome, Gitelman's syndrome and pseudohypoaldosteronism also localize to Na transport abnormalities in the distal nephron. Hypertensive in the general population may also result from subtle abnormalities in sodium balance resulting from alterations in the distal nephron.

The epithelial Na+channel (ENaC) is comprised of three homologous subunits: α, β, and γ, all of which are required for formation of the fully functional channel. This channel is responsible for salt reabsorption in the kidney, the airway, and the large bowel. Mutations in ENaC can cause human disease by increasing channel function in Liddle’s syndrome, a form of hereditary hypertension, or by decreasing channel function in pseudohypoaldosteronism type I, a salt-wasting disease of infancy. We previously showed that ENaC is expressed on the cell surface as a minimally glycosylated, Triton-insoluble protein. In the present study we found that ENaC existed initially as a Triton-soluble protein that contained high-mannose glycosylation, presumably in the endoplasmic reticulum. This form of the protein disappeared as the Triton-insoluble, minimally glycosylated form became the more prevalent species. In pulse-chase studies of individually expressed subunits, we found that the Triton-soluble form of β-ENaC accumulated initially, whereas the Triton-soluble form of α-ENaC decreased throughout the time course. However, when all three subunits were coexpressed, the α- and β-subunits showed a similar pattern. The complex became Triton insoluble at some point after the endoplasmic reticulum, as incubation at 15°C blocked the conversion to the insoluble form. Deletion of the carboxy-terminal tail of β-ENaC causes Liddle’s syndrome. This mutation increased the amount of newly synthesized Triton-insoluble ENaC heteromultimers but did not affect the half-life of insoluble protein. Therefore, subunit composition and mutations in individual subunits can influence biosynthesis of the ENaC complex.

Epithelial Na+ channels (ENaC) are inhibited by the cystic fibrosis transmembrane conductance regulator (CFTR) upon activation by protein kinase A. It is, however, still unclear how CFTR regulates the activity of ENaC. In the present study we examined whether CFTR interacts with ENaC by interfering with the Nedd4- and ubiquitin-mediated endocytosis of ENaC. Various C-terminal mutations were introduced into the three alpha-, beta-, and gamma-subunits of the rat epithelial Na+ channel, thereby eliminating PY motifs, which are important binding domains for the ubiquitin ligase Nedd4. When expressed in Xenopus oocytes, most of the ENaC stop (alpha-H647X, beta-P565X, gamma-S608X) or point (alpha-P671A, beta-Y618A, gamma-P(624-626)A) mutations induced enhanced Na+ currents when compared with wild type alpha,beta,gamma-rENaC. However, ENaC currents formed by either of the mutant alpha-, beta-, or gamma-subunits were inhibited during activation of CFTR by forskolin (10 micromol/l) and 3-isobutyl-1-methylxanthine (1 mmol/l). Antibodies to dynamin or ubiquitin enhanced alpha,beta,gamma-rENaC whole cell Na+ conductance but did not interfere with inhibition of ENaC by CFTR. Another mutant, beta-T592M,T593A-ENaC, also showed enhanced Na+ currents, which were down-regulated by CFTR. Moreover, activation of ENaC by extracellular proteases and xCAP1 does not disturb CFTR-dependent inhibition of ENaC. We conclude that regulation of ENaC by CFTR is distal to other regulatory limbs and does not involve Nedd4-dependent ubiquitination.

Liddle's syndrome is an inherited form of hypertension linked to mutations in the epithelial Na+ channel (ENaC). ENaC is composed of three subunits (α, β, γ), each containing a COOH-terminal PY motif (xPPxY). Mutations causing Liddle's syndrome alter or delete the PY motifs of β- or γ-ENaC. We recently demonstrated that the ubiquitin–protein ligase Nedd4 binds these PY motifs and that ENaC is regulated by ubiquitination. Here, we investigate, using the Xenopus oocyte system, whether Nedd4 affects ENaC function. Overexpression of wild-type Nedd4, together with ENaC, inhibited channel activity, whereas a catalytically inactive Nedd4 stimulated it, likely by acting as a competitive antagonist to endogenous Nedd4. These effects were dependant on the PY motifs, because no Nedd4-mediated changes in channel activity were observed in ENaC lacking them. The effect of Nedd4 on ENaC missing only one PY motif (of β-ENaC), as originally described in patients with Liddle's syndrome, was intermediate. Changes were due entirely to alterations in ENaC numbers at the plasma membrane, as determined by surface binding and immunofluorescence. Our results demonstrate that Nedd4 is a negative regulator of ENaC and suggest that the loss of Nedd4 binding sites in ENaC observed in Liddle's syndrome may explain the increase in channel number at the cell surface, increased Na+ reabsorption by the distal nephron, and hence the hypertension. J. Clin. Invest. 103:667–673 (1999)

In Liddle's syndrome, a rare inherited form of hypertension, epithelial sodium channel mutations appear to cause high blood pressure by increasing sodium reabsorption through sodium channels in the renal distal tubule. This increase in channel activity has not been confirmed previously by in vivo measurement. We have made transnasal potential difference measurements (effective in detection of increased sodium channel activity in cystic fibrosis) in three brothers with genetically proven Liddle's syndrome, their unaffected sister, and 40 normotensive controls. Maximum potential difference after 2 wk off treatment in the affected brothers was -30.4+/-1.2 mV (values mean+/-SD, lumen-negative with respect to submucosa) and was significantly more lumen-negative than that of the control group (-18.6+/-6.8 mV, P = 0.0228) or the unaffected sister (-18.25 mV, P < 0.01). The change in potential difference after topical application of 10(-)4 M amiloride was greater in the Liddle's patients, 14.0+/-2.1 mV, than in controls (7.9+/-3.9 mV, P = 0.0126) or the unaffected sister (5.5 mV, P < 0.05). This is the first in vivo demonstration of increased sodium channel activity in Liddle's syndrome. If these results are confirmed in other kindreds with this condition, then nasal potential difference measurements could provide a simple clinical test for Liddle's syndrome.

Liddle's syndrome is an autosomal dominant form of salt sensitive hypertension caused by mutations in the beta or gamma subunit of the epithelial sodium channel. Systematic mutagenesis studies revealed that a conserved PPPXY sequence (PY motif) of the C-terminus of the alpha, beta, or gamma subunits might be involved in the regulation of the channel activity. However, only two missense mutations in the PY motif of the beta subunit have been reported to cause Liddle's syndrome. We sequenced the C-termini of the beta and gamma subunits of the epithelial sodium channel in a Japanese family clinically diagnosed as having Liddle's syndrome and found a new missense mutation in the PY motif of the beta subunit, P615S. Expression studies with P615S mutant in Xenopus oocytes resulted in an about 3-fold increase in the amiloride-sensitive sodium current compared to the wild type (p = 0.001). These findings provide further clinical evidence for the hypothesis that a conserved PY motif may be critically important for the regulation of the epithelial sodium channel.

Abstract —Liddle’s syndrome is a rare monogenic form of hypertension caused by truncating or missense mutations in the C termini of the epithelial sodium channel β- or γ-subunits. These mutations delete or alter a conserved proline-rich amino acid sequence referred to as the PY-motif. We report here a Liddle’s syndrome family with a βArg564X mutation with a premature stop codon deleting the PY-motif of the β-subunit. This family shows marked phenotypic variation in blood pressure, serum potassium levels, and age of onset of hypertension. Given the similarity with primary hypertension, changes in the C termini of the β- or γ-subunits may contribute to the development of primary hypertension or to hypertension associated with diabetic nephropathy. Accordingly, the coding sequences for the cytoplasmic C termini of the β- and γ-subunits were screened for mutations with the use of polymerase chain reaction, single-strand conformation polymorphism, and direct DNA sequencing in 105 subjects with primary hypertension and 70 subjects with diabetic nephropathy. One frequent polymorphism was identified, but its frequency did not differ among subjects with primary hypertension, subjects with diabetic nephropathy, or control subjects. Two of the 175 subjects with primary hypertension or diabetic nephropathy showed variants that were not present in 186 control subjects. None of the variants changed the PY-motif sequence. In conclusion, a βArg564X mutation is the likely cause of Liddle’s syndrome in this Swedish family, but it is unlikely that mutations in the β- and γ-subunit genes of the epithelial sodium channel play a significant role in the pathogenesis of primary hypertension or diabetic nephropathy.

Activity of the epithelial sodium channel (ENaC) is a key determinant of sodium homeostasis and blood pressure. Liddle's syndrome, an inherited form of hypertension, is caused by mutations that delete or alter PY domains in the carboxyl termini of beta or gamma ENaC subunits, leading to increased channel activity. In this study we investigated the mechanism of this effect by analysis of wild-type and mutant ENaC activity in Xenopus oocytes. By inhibiting insertion of new channels into the plasma membrane with brefeldin A, we demonstrate that the half-life of the activity of channels containing Liddle's mutations is markedly prolonged compared with wild-type channels (t1/2 of 30 h in mutant versus 3.6 in wild-type, p < 0.001). We investigated the involvement of clathrin-coated pit-mediated endocytosis by co-expressing a dominant-negative dynamin mutant with wild-type ENaC in oocytes. Expression of this specific inhibitor of endocytosis leads to a large increase in the activity of wild-type channels, demonstrating that normal turnover of this channel is through the clathrin-coated pit pathway. In contrast, co-expression of Liddle's mutations and dynamin mutants leads to no further increase in channel activity, consistent with one of the effects of Liddle's mutations being the loss of endocytosis of these channels. These findings demonstrate the normal mechanism of turnover of ENaC from the cell surface and demonstrate a mechanism that can account for the increased number of channels in the plasma membrane seen in Liddle's syndrome.

Liddle’s syndrome is an autosomal dominant form of hypertension that resembles primary hyperaldosteronism, is characterized by the early onset of hypertension with hypokalemia and suppression of both PRA and aldosterone, and is caused by mutations in the carboxyl-terminus of theβ - or γ-subunits of the renal epithelial sodium channel. We describe a kindred (K176) whose distinguishing clinical features were mild hypertension and decreased aldosterone secretion. The index case was a 16-yr-old girl with intermittent mild hypertension and hypokalemia and subnormal PRA, aldosterone, 18-hydroxycorticosterone, and deoxycortisol levels, but normal cortisol/cortisone metabolite ratio and cortisol half-life. A frameshift mutation in the carboxyl-terminus of the β-subunit of the epithelial sodium channel was identified in the index case, establishing the diagnosis of Liddle’s syndrome. Sixteen at-risk relatives of the index case were tested. Seven new subjects were heterozygous for the mutation found in the index case, and two deceased obligate carriers were identified. All genetically affected adult subjects had a history of mild hypertension, and four had a history of hypokalemia. Basal and postcosyntropin plasma aldosterone and urinary aldosterone levels were significantly suppressed in those positive for the mutation. The family demonstrates variability in the severity of hypertension and hypokalemia in this disease, raising the possibility that this disease may be underdiagnosed among patients with essential hypertension.

Blood pressure responses to increases and decreases in dietary salt intake are heterogeneous. In some hypertensive individuals, decreases in blood pressure with salt restriction are clinically significant and approach that achieved with medication. In others, little or no change in blood pressure occurs, whereas in still others, blood pressure may actually increase with salt restriction. The heterogeneous responses are partly acquired and involve the influences of age, the intake of other electrolytes, and the influence of certain medications. Genetic predisposition may also play a substantial role because salt sensitivity is increased in black individuals and in persons with non-insulin-dependent diabetes mellitus. Some uncommon but readily diagnosed salt-sensitive genetic syndromes, such as glucocorticoid-remediable aldosteronism and Liddle syndrome, have been identified. Short-term volume expansion and contraction and longer-term dietary interventions appear to be reproducible and may be used to identify salt-sensitive and salt-resistant individuals; however, these maneuvers are cumbersome and cannot be used on a large scale. Molecular genetic techniques for identifying individuals with salt-sensitive and salt-resistant essential hypertension are not yet available, but if the putative gene polymorphisms are identified, such techniques may replace the current trial-and-error methods.

The 14-year follow-up of a female patient with Liddle's syndrome (LS), a rare disease characterized by hypertension, hypokalemic alkalosis, and negligible aldosterone secretion due to renin suppression, is described. The disease was diagnosed at the age of 10 months (youngest identification). The patient was repeatedly investigated during follow-up for plasma renin activity (PRA), plasma aldosterone concentration (PA), serum sodium and potassium (K) concentration, blood pressure (BP), somatic anthropometry, and mental development. Noteworthy results included: persistent low circulating K, PRA, and PA and high BP, coinciding with unauthorized withdrawal of the triamterene therapy. These findings are in keeping with the hypothesis that LS results from a pathogenetic disorder which is not correctable with age. The triamterene therapy was effective in correcting the endocrine and metabolic disorders as well as arterial hypertension, but did not prevent a deficit in mental and physical development. However, the information derived from this study allows further clarification of the clinical picture of the disease.

The epithelial amiloride-sensitive sodium channel (ENaC) controls transepithelial Na + movement in Na + -transporting epithelia and is associated with Liddle syndrome, an autosomal dominant form of salt-sensitive hypertension. Detailed analysis of ENaC channel properties and the functional consequences of mutations causing Liddle syndrome has been, so far, limited by lack of a method allowing specific and quantitative detection of cell-surface-expressed ENaC. We have developed a quantitative assay based on the binding of 125 I-labeled M 2 anti-FLAG monoclonal antibody (M 2 Ab*) directed against a FLAG reporter epitope introduced in the extracellular loop of each of the α, β, and γ ENaC subunits. Insertion of the FLAG epitope into ENaC sequences did not change its functional and pharmacological properties. The binding specificity and affinity ( K d = 3 nM) allowed us to correlate in individual Xenopus oocytes the macroscopic amiloride-sensitive sodium current (I Na ) with the number of ENaC wild-type and mutant subunits expressed at the cell surface. These experiments demonstrate that: ( i ) only heteromultimeric channels made of α, β, and γ ENaC subunits are maximally and efficiently expressed at the cell surface; ( ii ) the overall ENaC open probability is one order of magnitude lower than previously observed in single-channel recordings; ( iii ) the mutation causing Liddle syndrome (β R564stop) enhances channel activity by two mechanisms, i.e., by increasing ENaC cell surface expression and by changing channel open probability. This quantitative approach provides new insights on the molecular mechanisms underlying one form of salt-sensitive hypertension.

Mutations in beta or gamma subunit of the epithelial sodium channel (ENaC) have been found to cause a hereditary form of human hypertension, Liddle syndrome. Most of the mutations reported are either nonsense mutations or frame shift mutations which would truncate the cytoplasmic carboxyl terminus of the beta or gamma subunits of the channel, suggesting that these domains are important for the normal regulation of this channel. We sequenced ENaC in a family with Liddle syndrome and found a missense mutation in beta subunit which predicts substitution of Tyr by His at codon 618, 2 bp downstream from a missense mutation (P616L) that has been reported recently. Presence of this mutation correlates with the clinical manifestations (hypertension, hypokalemia, suppressed aldosterone secretion) in this kindred. Functional expression studies in the Xenopus oocytes revealed constitutive activation of the Y618H mutant indistinguishable from that observed for the deletion mutant (R564stop) identified in the original pedigree of Liddle. Our data suggest that the region between Pro616 and Tyr618 is critically important for regulation of ENaC activity.

Liddle syndrome is a mendelian form of hypertension characterized by constitutively elevated renal Na reabsorption that can result from activating mutations in the beta or gamma subunit of the epithelial Na channel. All reported mutations have deleted the last 45-76 normal amino acids from the cytoplasmic C terminus of one of these channel subunits. While these findings implicate these terminal segments in the normal negative regulation of channel activity, they do not identify the amino acid residues that are critical targets for these mutations. Potential targets include the short highly conserved Pro-rich segments present in the C terminus of beta and gamma subunits; these segments are similar to SH3-binding domains that mediate protein-protein interaction. We now report a kindred with Liddle syndrome in which affected patients have a mutation in codon 616 of the beta subunit resulting in substitution of a Leu for one of these highly conserved Pro residues. The functional significance of this mutation is demonstrated both by the finding that this is a de novo mutation appearing concordantly with the appearance of Liddle syndrome in the kindred and also by the marked activation of amiloride-sensitive Na channel activity seen in Xenopus oocytes expressing channels containing this mutant subunit (8.8-fold increase compared with control oocytes expressing normal channel subunits; P = 0.003). These findings demonstrate a de novo missense mutation causing Liddle syndrome and identify a critical channel residue important for the normal regulation of Na reabsorption in humans.