PHYSIOLOGY IN MEDICINE
In collaboration with
The American Physiological Society, Thomas E. Andreoli, MD, Editor
Liddle’s Syndrome
Biff F. Palmer, MD, Robert J. Alpern, MD
L
iddle’s syndrome is characterized by hypertension
in the setting of hypokalemic metabolic alkalosis.
Clinically these patients resemble those with pri-
mary hyperaldosteronism. However, the hallmark of this
disorder is the finding of markedly suppressed serum al-
dosterone levels and the lack of response to administra-
tion of the mineralocorticoid receptor blocker spirono-
lactone. In the original classic report of this disorder in
1963, Liddle et al (1) concluded that the pathogenesis of
this disorder was due to a tendency of the kidney to con-
serve Na1 and excrete K1 despite the virtual absence of
mineralocorticoids. In the last several years the specific
mechanism underlying this tendency has been eluci-
dated. The disorder has been localized to a specific muta-
tion in the epithelial Na1 channel located in the collect-
ing duct of the kidney. This mutation results in constitu-
tive overactivity of the channel, resulting in a clinical
picture virtually identical to that of hyperaldosteronism.
The sequence of events that started with the original re-
port of this disorder and continued to the present day
understanding of the precise molecular defect is illustra-
tive of how keen clinical deductive reasoning and tech-
niques of molecular biology can complement each other
in enhancing our understanding of a clinical disorder.
CLINICAL CHARACTERISTICS OF THE
INDEX CASE
The original report of a patient with Liddle’s syndrome
involved a 16-year-old girl who was being evaluated for
persistent hypertension in the setting of hypokalemic
metabolic alkalosis (1). Despite the similarity to primary
hyperaldosteronism, a number of observations suggested
an alternative diagnosis. First, basal aldosterone secretory
rates were suppressed and failed to increase in response to
a low NaCl diet or correction of the serum K1 concentra-
tion. In addition, excess secretion of an alternative com-
Am J Med. 1998;104:301–309.
From the Department of Internal Medicine, University of Texas South-
western Medical Center, Dallas, Texas.
Requests for reprints should be addressed to Biff F. Palmer, MD,
Associate Professor of Internal Medicine, Department of Internal Med-
icine, University of Texas Southwestern Medical Center, 5323 Harry
Hines Blvd., Dallas, Texas 75235.
q1998 by Excerpta Medica, Inc.
All rights reserved.
pound with mineralocorticoid activity was excluded by
the finding of a high salivary Na1/K1 ratio and by the
failure to modify urine electrolyte excretion after admin-
istration of a mineralocorticoid receptor blocker. Fur-
thermore, measurements of mineralocorticoid metabo-
lites in the urine were all within normal limits.
To further characterize renal Na1 handling in this pa-
tient, the urinary response to a low NaCl diet and admin-
istration of exogenous aldosterone was examined. After
placing the patient on a 9 mEq/day Na1 diet, urinary Na1
excretion fell, but not to the extent that would be ob-
served in an otherwise normal subject. To differentiate
whether this residual renal Na1 wasting was secondary to
inadequate mineralocorticoid activity or an end organ
resistance to mineralocorticoids, the patient was treated
with exogenous aldosterone while on the low NaCl diet.
Following administration of aldosterone, urinary Na1
excretion fell maximally, suggesting the inability to max-
imally conserve Na1 was the result of inadequate circu-
lating levels of aldosterone and not due to an intrinsic
renal defect in Na1 reabsorption. Urinary Na1 excretion
was then examined and compared with that of a group of
patients with known Addison’s disease who were treated
with glucocorticoids and an amount of aldosterone that
would produce serum levels comparable with that found
in the patient with Liddle’s syndrome. In patients with
Addison’s disease urinary Na1 excretion remained much
higher than in the patient with Liddle’s syndrome, and
ultimately the Addison’s patients developed volume de-
pletion and hypotension. By comparison, urinary Na1
excretion was much lower in the Liddle’s patient, suggest-
ing that the kidney was reabsorbing a greater amount of
Na1 by a mechanism independent of mineralocorticoid
activity.
Maneuvers designed to decrease aldosterone synthesis
or block its peripheral activity also suggested a mecha-
nism intrinsic to the kidney. Administration of an inhib-
itor of aldosterone secretion caused the low basal levels of
aldosterone to fall even further. Despite these lower lev-
els, urinary electrolyte excretion was unaffected. Simi-
larly, administration of the aldosterone receptor blocker
spironolactone did not affect urinary electrolyte excre-
tion or correct the hypokalemia.
The patient, however, did respond to triamterene, an
agent that directly inhibits the apical membrane Na1
0002-9343/98/$19.00 301
PII S0002-9343(98)00018-7
 Liddle’s Syndrome/Palmer and Alpern
channel in the collecting duct. Following the administra-
tion of this drug there was a marked increase in urinary
Na1 excretion and decrease in urinary K1 excretion.
When triamterene was combined with a low NaCl diet,
the patient’s hypertension was successfully corrected.
These initial observations suggested that Liddle’s syn-
drome was the result of persistent renal salt retention
secondary to a mechanism that was intrinsic to the kid-
ney. The resultant volume expansion could account for
the development of hypertension and the finding of sup-
pressed aldosterone secretory rates. The presence of hy-
pokalemia and metabolic alkalosis as well as the beneficial
response to triamterene suggested that the site of renal
Na1 retention was in the distal nephron.
The next advance in the understanding of Liddle’s syn-
drome came from studies performed in the same patient,
but more than 30 years later (2). During this time, the
patient had developed renal failure from unknown causes
and subsequently underwent a successful cadaveric renal
transplant. At the time of repeat evaluation, the patient
was noted to have only mild hypertension and no evi-
dence of hypokalemia or metabolic alkalosis. Urinary
electrolytes showed no evidence of K1 wasting. When the
patient was placed on a low NaCl diet, plasma renin ac-
tivity and the plasma aldosterone concentration in-
creased normally and to a magnitude similar to control
transplant recipients. The fact that the clinical manifesta-
tions and abnormal physiologic studies had normalized
in this patient after a successful renal transplant con-
firmed that the defect in Liddle’s syndrome was intrinsic
to the kidney.
LIDDLE’S SYNDROME IS AN
INHERITED DISORDER
Investigation of the patient’s family showed other
members to have similar clinical abnormalities. The
pattern of expression of these abnormalities were
found to best fit an autosomal dominant pattern of
inheritance. Subsequent description of other kindreds
have confirmed that Liddle’s syndrome is an autoso-
mal dominant disorder (3–5).
The classical phenotype of hypertension, hypokalemia,
and metabolic alkalosis is only variably expressed in fam-
ily members with Liddle’s syndrome, suggesting variable
penetrance of the gene involved with the disorder. This
variability is particularly evident with regard to the serum
K1 concentration (1,2). Although hypertensive members
of the original family had lower serum K1 concentrations
as a group when compared with unaffected family mem-
bers, there were affected individuals defined by the pres-
ence of hypertension who were normokalemic. In addi-
tion, 1 or 2 patients in the original report were noted to be
hypokalemic but have no evidence of hypertension. Some
302 March 1998 THE AMERICAN JOURNAL OF MEDICINEt Volume 104
patients were found to be normotensive and normokale-
mic but have low aldosterone excretion rates and low
ratios of aldosterone to K1.
A similar variability is seen with regard to the plasma
HCO2 3 concentration. In some patients there is no evi-
dence of metabolic alkalosis whereas in others the serum
HCO2 3 concentration is clearly elevated. In general, the
serum HCO2 3 concentration is highest in those patients
with the greatest degree of hypokalemia. The severity and
age of onset of hypertension also varies among affected
family members. In general, hypertension is present in
the teenage years but occasionally occurs at an even ear-
lier age.
Another prominent feature noted in the original ped-
igree was an increased risk of premature death due to
stroke or heart failure. Several of the affected family
members had poorly controlled hypertension and died at
an early age. As described earlier, the index patient devel-
oped renal failure and required hemodialysis. Many years
prior to the need for dialysis, this patient had undergone
a renal biopsy that only showed a nonspecific increase in
the cellularity of several glomeruli with occasional adhe-
sions. In a more recently described pedigree, another pa-
tient and possibly a second developed renal failure that
required dialysis treatment (5). The cause of the renal
failure in these patients was said to be secondary to neph-
rosclerosis. Whether these examples of end organ damage
are simply the result of long-standing poorly controlled
hypertension or represent an intrinsic feature of the ge-
netic disorder is not known.
PATHOGENESIS OF THE CLINICAL
FEATURES
Based on observations that the clinical manifestations of
Liddle’s syndrome are reversed by treatment with the
Na1 channel inhibitor triamterene, but not with miner-
alocorticoid receptor blockers, it was concluded that the
defect is secondary to a primary increase in electrogenic
Na1 absorption in the distal nephron. Subsequent stud-
ies, described below, have shown that Liddle’s syndrome
is due to an activating mutation in the distal nephron
apical membrane Na1 channel. The observation that
such a primary increase in Na1 transport can lead to hy-
pokalemia and alkalosis has taught us much about the
interrelationships between the processes of Na1, K1, and
H1 transport in the distal nephron.
Hypokalemia
Hypokalemia is variably present in patients with Liddle’s
syndrome. To address the mechanisms responsible, we
first briefly review the determinants of renal K1 excre-
tion. Potassium is freely filtered by the glomerulus. The
bulk of filtered K1 is reabsorbed in the proximal tubule
 Liddle’s Syndrome/Palmer and Alpern

Figure 1. Cell model for Na1, K1, and H1 transport in the principal cell and the intercalated cell under normal conditions and in
Liddle’s syndrome. Constitutive activation of the Na1 channel in Liddle’s syndrome increases the luminal electronegativity. The
increase in cell Na1 causes an increase in the activity of the Na1/K1 ATPase, which in turn results in an increase in cell K1 in principal
cells. K1 secretion is enhanced secondary to the favorable diffusion and electrical gradient across the apical membrane. The increase
in the transepithelial electrical gradient enhances H1 secretion by the intercalated cell. See text for detailed discussion.

and loop of Henle so that under most physiologic and
pathologic conditions, K1 delivery to the distal nephron
remains small and is fairly constant. Secretion of K1 oc-
curs in the distal nephron primarily in the initial collect-
ing tubule and the cortical collecting duct. Here, the rate
of K1 secretion is regulated and varies according to phys-
iologic needs. Potassium secretion in the distal nephron is
generally responsible for most of urinary K1 excretion.
The cell that is responsible for K1 secretion in the ini-
tial collecting tubule and the cortical collecting duct is the
principal cell. This cell, shown in Figure 1, possesses a
basolateral membrane Na1/K1 adenosine triphos-
phatase (ATPase), which is responsible for the active
transport of Na1 out of and K1 into the cell. The result-
ant low cell Na1 concentration and high cell K1 concen-
tration provide favorable diffusion gradients for the
movements of Na1 from lumen to cell and K1 from cell
to lumen across the apical membrane. Both the move-
ments of Na1 and K1 across the apical membrane occur
via well-defined Na1 and K1 channels.
The rate of K1 movement across the apical membrane
K channel is determined by the cell K1 concentration,
1 tration. Although increased distal delivery of Na1 and
luminal K1 concentration, the potential (voltage) differ-
ence across the apical membrane, and the permeability of
the apical membrane to K1. Two of the most important
physiologic regulators of K1 excretion are mineralocor-
ticoid activity and distal delivery of Na1 and water. Min-
eralocorticoids stimulate K1 secretion by affecting sev-
eral of the determinants of K1 channel permeation dis-
cussed above. First, mineralocorticoids stimulate Na1
reabsorption across the apical membrane, which depo-
larizes the cell relative to the lumen, thereby increasing
the electrical gradient favoring K1 secretion. In addition,
increases in apical Na1 entry increase cell Na1 concen-
tration, causing the Na1/K1 ATPase to run more rapidly,
and secondarily increasing cell K1 concentration. Sec-
ond, mineralocorticoids increase intracellular K1 con-
centration by directly stimulating the activity of the baso-
lateral membrane Na1/K1 ATPase. Third, mineralocor-
ticoids directly increase the permeability of the apical
membrane to K1.
A second important factor that affects K1 secretion is
the distal delivery of Na1 and water. In general, distal
delivery of Na1 and water change in parallel under most
physiologic and pathologic conditions. Increased distal
delivery of Na1 stimulates distal Na1 absorption, which
again depolarizes the cell and drives the Na1/K1 ATPase
to run faster. Increased flow rates also increase K1 secre-
tion by diluting and thus lowering luminal K1 concen-
water and increased mineralocorticoid activity can each
stimulate renal K1 secretion, under normal physiologic
conditions these two determinants are inversely regu-
lated by effective arterial volume. Decreases in effective
arterial volume increase aldosterone secretion, but lower
distal delivery of Na1 and water secondary to enhance-
ment of reabsorption in the proximal nephron. It is for

March 1998 THE AMERICAN JOURNAL OF MEDICINEt Volume 104 303

 Liddle’s Syndrome/Palmer and Alpern
this reason that renal K1 excretion is relatively indepen-
dent of volume status.
Primary increases in mineralocorticoid levels are well
known to cause hypokalemia by stimulating the apical
membrane Na1 channel as well as by directly stimulating
pathways of K1 secretion. The finding of hypokalemia in
Liddle’s syndrome where there is not direct involvement
of K1 secretory pathways provides the best evidence that
isolated increases in apical membrane Na1 entry can
stimulate K1 secretion and result in hypokalemia (Figure
1). Increased apical membrane Na1 permeability leads to
increased Na1 flux into the cell, which depolarizes the
apical membrane and increases the rate of the Na1/K1
ATPase. The resulting increase in cell K1 concentration
along with the voltage changes enhance K1 secretion. In-
creased Na1 reabsorption leads to volume expansion,
which decreases proximal Na1 and water reabsorption,
and as a result ensures adequate distal delivery of Na1
and water. Increased distal delivery of Na1 coupled to
persistent overactivity of the Na1 channel provides for
sustained renal K1 wasting.
Although hypokalemia is common in patients with
Liddle’s syndrome, it is not invariably present. In fact,
approximately 50% of the patients described in one kin-
dred were found to be hypertensive but normokalemic.
One factor limiting the development of hypokalemia in
affected family members is suppressed circulating aldo-
sterone levels. Decreased mineralocorticoid activity re-
moves two driving forces that tend to augment K1 secre-
tion in the collecting tubule, namely, direct activation of
the basolateral membrane Na1/K1 ATPase and increased
permeability of the apical K1 channel. The lack of these
mineralocorticoid-mediated effects may be one reason
why some patients have only minimal or no hypokalemia
at all. Another factor that may limit the development of
hypokalemia in some patients is variability in dietary salt
intake. If dietary salt is restricted, distal delivery of Na1
will decrease. As a result, the magnitudes of cell depolar-
ization and increased Na1/K1 ATPase flux will decline
and result in a decreased driving force for K1 secretion.
The absence of hypokalemia in patients with Liddle’s
syndrome is not without precedence, since a normal se-
rum K1 concentration has been described in patients
with primary mineralocorticoid excess of varying etiolo-
gies (6,7).
Metabolic Alkalosis
Metabolic alkalosis was a prominent clinical feature in
the index case. However, like hypokalemia, there is a great
deal of variability in the degree to which this abnormality
is expressed. The generation and maintenance of meta-
bolic alkalosis in Liddle’s syndrome is secondary to an
increase in H1 secretion in the initial cortical collecting
tubule and cortical collecting duct. Acidification in this
nephron segment is mediated by a H1 translocating
304 March 1998 THE AMERICAN JOURNAL OF MEDICINEt Volume 104
ATPase located on the apical membrane of a intercalated
cells. The HCO2 3 generated intracellularly exits the baso-
lateral membrane by way of a Cl2/HCO2 3 exchanger; the
Cl2 that enters the cell on the exchanger exits on a baso-
lateral membrane Cl2 channel. The H1 ATPase is not
coupled to Na1 directly, but its rate is secondarily af-
fected by Na1 transport owing to changes in transepithe-
lial voltage. Increases in the rate of Na1 reabsorption in
the principal cell lead to the generation of a lumen nega-
tive transepithelial potential. Because of the nature of the
membrane resistances in the a intercalated cell, a lumen
negative transepithelial potential change leads to voltage
changes in which the cell is slightly more negative with
respect to the interstitium, and the lumen is significantly
more negative relative to the cell (Figure 1). This leads to
parallel increases in the rate of the apical membrane H1
ATPase and the basolateral membrane Cl2 channel. In-
creases in Cl2/HCO2 3 exchange are likely driven by a sec-
ondary increase in cell pH.
As with K1 secretion, mineralocorticoids stimulate
H secretion by increasing Na1 reabsorption in the prin-
1
cipal cell, and by direct effects on H1 secretion in the a
intercalated cell. Once again, the finding that metabolic
alkalosis is associated with Liddle’s syndrome, where the
only direct effect is on Na1 absorption in the principal
cell, provides the best evidence that increases in Na1
transport alone can stimulate distal H1 secretion and re-
sult in alkalosis.
In Liddle’s syndrome aldosterone levels are suppressed
but the activity of the Na1 channel is constitutively in-
creased. In addition, distal Na1 delivery is plentiful as a
result of the volume-expanded state. As with the develop-
ment of hypokalemia, this combination of increased dis-
tal Na1 delivery and increased activity of the Na1 chan-
nel leads to increased luminal electronegativity, thus aug-
menting H1 secretion (Figure 1). As long as dietary NaCl
is adequate, distal H1 secretion will remain stimulated.
However, restriction of dietary NaCl would decrease dis-
tal Na1 delivery and cause the lumen potential to become
less negative. As a result, H1 secretion would decrease,
thereby limiting the development of metabolic alkalosis.
Along these lines, variations in dietary salt intake may be
one factor explaining the variable presence of metabolic
alkalosis in this patient population.
An additional factor that can contribute to the variable
presence of metabolic alkalosis in Liddle’s syndrome is the
varying degree of hypokalemia. Potassium depletion has
several effects on renal acidification mechanisms that con-
tribute to the maintenance and generation of metabolic al-
kalosis. Potassium depletion can lead to a decrease in glo-
merular filtration rate, which would lower the filtered load
of HCO2 3 and secondarily help to maintain metabolic alka-
losis. In addition, K1 depletion has been demonstrated to
stimulate rates of proximal and distal tubular H1 secretion.
The latter may be related to activation of an H1/K1 ATPase
 Liddle’s Syndrome/Palmer and Alpern
in the collecting duct. Lastly, K1 depletion leads to an adap-
tive increase in the enzymes that synthesize ammonia, caus-
ing increased rates of ammoniagenesis in the proximal tu-
bule. While all of these effects contribute to the generation
and maintenance of metabolic alkalosis, K1 deficiency also
inhibits aldosterone secretion, an effect that inhibits Na1
absorption and acidification. The net result is that K1 defi-
ciency alone does not cause significant metabolic alkalosis
unless severe.
By contrast, if K1 depletion occurs in a setting where
mineralocorticoid secretion is nonsuppressible, then the
stimulatory effects on renal acidification dominate and
metabolic alkalosis will develop. Indeed, in patients with
primary hyperaldosteronism, patients with the most se-
vere K1 depletion have the most marked metabolic alka-
losis (8). While aldosterone levels are suppressed in Lid-
dle’s syndrome, persistent overactivity of the Na1 chan-
nel mimics a nonsuppressible mineralocorticoid state. As
a result, one would expect metabolic alkalosis to be more
prominent in patients who were also hypokalemic.
Hypertension with Suppressed Plasma Renin
Activity and Serum Aldosterone Levels
Overactivity of the Na1 channel is also responsible for the
development of hypertension in these patients. The hy-
pertension is accompanied by absolute suppression of al-
dosterone and renin secretion secondary to the chroni-
cally volume expanded state. The chronicity of this sup-
pression is reflected in renal biopsy material
demonstrating atrophy of the juxtaglomerular apparatus
(9). The failure of a low salt diet to increase plasma renin
activity and serum aldosterone levels in the index case
prior to her renal transplant was most likely a reflection of
this atrophy.
Despite unrelenting Na1 reabsorption in the collecting
duct, patients with Liddle’s syndrome have not been
noted to develop edema. This lack of edema formation is
presumably based on a similar physiologic mechanism as
explains the absence of edema formation in patients with
primary hyperaldosteronism. If aldosterone is given to a
normal subject on an adequate salt intake, NaCl and wa-
ter retention and K1 loss are seen initially, leading to a
rise in blood pressure, weight gain, and K1 depletion.
After a weight gain of approximately 3 kg, however, a
spontaneous natriuresis ensues, returning the urinary
Na1 excretion to normal. This phenomenon is referred
to as aldosterone escape and is likely due to suppressed
Na1 reabsorption in other nephron segments (10,11).
CHARACTERIZATION OF THE
MOLECULAR DEFECT IN LIDDLE’S
SYNDROME
The clinical data in patients with Liddle’s syndrome point
to an abnormality in the epithelial Na1 channel that re-
March 1998
sults in constitutive overactivity of the channel. Based on
the responsiveness of patients to triamterene and the un-
responsiveness to spironolactone, the precise location of
this abnormality was predicted to lie somewhere in the
pathway between the mineralocorticoid receptor and the
channel. Subsequent studies localized the defect to the
epithelial Na1 channel.
The basis for understanding the defect in Liddle’s syn-
drome came from studies that defined the molecular
structure of the epithelial Na1 channel utilizing expres-
sion cloning. With this technique, a cDNA library is gen-
erated from tissue known to express the protein of inter-
est, in this case, the colon. mRNA synthesized from
clones is injected into Xenopus oocytes and screened for
its ability to induce an amiloride sensitive Na1 current.
Utilizing this technique Canessa et al (12) and Lingueglia
et al (13) independently cloned a cDNA derived from rat
colon that induced amiloride-sensitive Na1 conductance
when expressed in Xenopus oocytes. This first Na1 chan-
nel clone, termed arENaC (a subunit of rat epithelial Na
channel), encoded a protein that was qualitatively similar
to that found in epithelia in that it was highly sensitive to
amiloride and was highly selective for Na1. Quantita-
tively, however, the conductance was relatively small,
suggesting that there were other subunits required for full
channel conductance. This prediction was later proved
when Canessa et al (14) identified two additional sub-
units, termed b and g rENaC. Expression of the b and g
subunits alone in oocytes failed to elicit a significant
amiloride-sensitive channel current. By contrast, when
all three of the subunits were expressed, maximal channel
conductance was obtained. It was concluded that
arENaC was sufficient to induce channel activity but that
b and g rENaC were required for maximal expression of
channel activity.
The three subunits of the channel share significant se-
quence homologies and appear to have the same mem-
brane topology. Each subunit consists of two transmem-
brane domains, a large extracellular domain, and two
short cytoplasmic tails. The extracellular domain con-
tains four potential N-glycosylation sites. The roles that
each subunit plays in channel formation and the exact
stoichiometry of a, b, and g in the intact channel are
unknown. Studies examining the kinetic behavior of the
Na1 channel in lipid bilayers suggest that the functional
channel is comprised of a minimum of three conductive
elements arranged in what has been described as a triple-
barrel configuration (15,16).
Once the subunits of the Na1 channel were cloned, the
stage was set to define the exact molecular defect in Lid-
dle’s syndrome. Shimkets et al (3) examined the original
kindred described by Liddle and found complete linkage
of the gene encoding the b subunit with affected family
members. Direct sequence analysis of the gene revealed a
single base substitution involving a C for G at the first
THE AMERICAN JOURNAL OF MEDICINEt Volume 104 305
 Liddle’s Syndrome/Palmer and Alpern
nucleotide of codon Arg-564. This substitution intro-
duced a premature stop codon that left the second trans-
membrane domain intact but removed virtually the en-
tire cytoplasmic carboxyl tail of the protein. No unaf-
fected members of the kindred demonstrated this
mutation. Analysis of four other kindreds by the same
group revealed either premature termination or frame
shift mutations in this same carboxyl-terminal domain.
The clustering of these mutations within a small region of
the carboxyl terminus of the b subunit in all five kindreds
suggested that this region of the protein was an important
structural determinant regulating channel activity.
With knowledge of the specific mutation in the origi-
nal kindred, Schild et al (17) reported on the functional
consequences of this mutation in the rat epithelial Na
channel (rENaC). The rat and human homologues of
ENaC share a high degree of sequence homology, includ-
ing 81% identity in the carboxyl-terminal segment of the
b subunit. After introducing a premature stop codon at
the Arg-564 codon in the b chain, channel activity was
studied in the Xenopus laevis oocyte expression system.
This mutation resulted in a significant increase in channel
activity but preserved other features of the channel, in-
cluding Na1 selectivity and affinity for amiloride. As in
the original Liddle’s kindred, this mutation deleted the
major portion of the cytoplasmic tail of the b subunit.
The fact that the biophysical and pharmacologic proper-
ties of the channel remained intact suggested that this
region was not critically involved in the pore-forming
region of the channel, but rather played a role in the reg-
ulation of channel activity. This conclusion was strength-
ened when it was found that the corresponding deletion
introduced into the a subunit, the subunit that primarily
constitutes the pore, only caused a slight increase in chan-
nel activity. By contrast, deletion of the carboxyl-terminal
portion of the g subunit increased channel activity quan-
titatively to an equivalent degree as was observed with the
mutant b subunit. This observation implied that the g
subunit was also important in regulating channel activity
and that mutations in this subunit might also give rise to
Liddle’s syndrome.
As was predicted form studies in Xenopus oocytes, a
kindred with Liddle’s syndrome was subsequently de-
scribed in which a mutation was localized to the g subunit
(4). In this kindred a single base substitution was found to
generate a premature stop codon at codon Trp-574. The
protein encoded by the mutant gene was characterized by
a deletion of the last 76 amino acids in the cytoplasmic
carboxyl terminus. Introduction of this termination
codon into the corresponding region of the rat gENaC
resulted in a significant increase in amiloride-sensitive
Na1 current as measured in Xenopus oocytes. This in-
crease in channel activity was indistinguishable from the
increase in channel activity seen in activated oocytes ex-
pressing the truncated b subunit.
306 March 1998 THE AMERICAN JOURNAL OF MEDICINEt Volume 104
To date, all mutations described in kindreds with Lid-
dle’s syndrome have been confined to the cytoplasmic tail
of the carboxyl terminus of either the b or g subunit. The
clustering of mutations to this specific region of the re-
spective subunits indicates that the cytoplasmic carboxyl
terminus of both the b and g subunits is required for
normal negative regulation of channel activity. The pre-
cise mechanism by which these mutations result in con-
stitutive activation of the Na1 channel is not entirely
clear. One possible mechanism is that the cytoplasmic
tails of the b and g subunits normally act as blocking
moieties, accounting for the gated behavior of the Na1
channel. In this regard, the mutations in the kindreds
described by Shimkets et al (3) resulted in the loss of 45 to
70 amino acids of the terminal portion of the b subunit,
whereas the mutation in the kindred described by Hans-
son et al (4) caused the loss of the last 76 amino acids of
the g subunit. Since these truncating mutations lead to
the removal of large portions of the cytoplasmic tail of the
respective subunits, any potential blocking activity would
likely be lost resulting in a non-gated channel free to con-
duct Na in an unrestricted manner.
Evidence to support this possibility has come from
studies examining the Na1 channel in lymphocytes taken
from members of the original Liddle’s kindred. Human
lymphocytes are known to express amiloride-sensitive
Na1 channels that are functionally similar to those ex-
pressed in principal cells of the renal cortical collecting
duct (18). Utilizing the technique of whole cell patch
clamp, Bubien et al (19) found that the amiloride sensi-
tive Na1 channel in lymphocytes from affected family
members was constitutively activated. Addition of a pep-
tide identical to the terminal 10 amino acids of the trun-
cated b subunit to the cytoplasm of the cell resulted in a
reversal of the constitutive basal activation in lympho-
cytes from affected patients, while it had no effect on
channel activity in lymphocytes from unaffected family
members. In a subsequent paper, this same group exam-
ined the lymphocyte amiloride-sensitive Na1 channel
from patients affected with Liddle’s syndrome after first
reconstituting the channel in planar lipid bilayers (20).
These channels demonstrated constitutive activation fol-
lowing reconstitution. Exposing the cytoplasmic face of
the bilayer to increasing concentrations of this same 10
amino acid peptide resulted in a dose-dependent reversal
of basal constitutive activation of the Na1 channel. This
inhibitory affect of the peptide on channel activity was
dependent on the presence of GTP. Similarly, application
of the carboxyl-terminal 10 amino acid peptide from the
g subunit also inhibited channel activity, but to a lesser
extent. By contrast, the carboxyl-terminal 10 amino acid
peptide from the a subunit was without effect. These re-
sults are consistent with the possibility that the terminal
portions of the b and g subunit act as blocking peptides,
 Liddle’s Syndrome/Palmer and Alpern
thereby providing a gating mechanism for the regulation
of Na1 movement through the channel.
Although the removal of a gating function is an attrac-
tive explanation as to how these truncating mutations
give rise to constitutive Na1 channel activity, it does not
readily explain increased channel activity in nontruncat-
ing mutations. Hannson et al (21) has described a kin-
dred with all the features typical of Liddle’s syndrome in
which a single nucleotide substitution changes proline
616 to leucine in the carboxyl terminus of the b subunit.
Introduction of this mutation into the corresponding po-
sition of the rat bENaC results in a marked increase in
Na1 channel activity when expressed in Xenopus oocytes.
Tamura et al (5) has also identified a missense mutation
in the b subunit only two base pairs downstream from the
mutation described by Hannson et al in a kindred with
classic Liddle’s syndrome. As in the previous report, the
introduction of this mutation into the corresponding po-
sition of the rat bENaC caused increased Na1 channel
activity measured in Xenopus oocytes.
The localization of these two nontruncating missense
mutations identified a specific region in the b subunit
that plays a key role in inhibition of the Na1 channel. The
amino acid sequence of the a, b, and g subunits share
approximately 35% total homology. In examining the cy-
toplasmic tail of these subunits, there is only a low level of
identity among them. However, within the cytoplasmic
tail is a proline-rich region (termed P2) that is highly
conserved (22). This region contains a PPPXY sequence
that is called the PY motif. Thus far all reported kindreds
have mutations that either delete (truncating mutations)
or directly alter (missense mutations) this motif. The im-
portance of this motif in channel regulation is supported
by studies that have examined channel activity in Xenopus
oocytes after inducing sequential mutations or amino
acid substitutions along the length of the carboxyl termi-
nus in the a, b, and g subunits (22). In these studies a
stimulatory effect on channel activity is only seen when
the mutation affects the PY motif. Of interest, this stim-
ulatory effect on channel activity is observed not only
when the mutation is targeted to the PY motif on the b or
g subunits but also when the mutation is restricted to the
PY motif on the a subunit.
Proline-rich regions are known to be often involved in
protein-protein interactions. This fact led investigators to
consider the possibility that a protein may exist whose
function is to regulate channel activity via interaction
with the PY motif on each of the three subunits. Bork and
Sudol (23) and Andre and Springael (24) have recently
identified a novel domain called the WW domain in the
yes-associated protein YAP65, as well as several other un-
related proteins, including Nedd4. The WW domain is a
38 amino acid module that contains two highly conserved
tryptophans and two conserved prolines. Based on mu-
tational analysis, Chen and Sudol (25) recently proposed
March 1998
that the WW domain of YAP65 binds to a consensus se-
quence of XPPXY called the PY motif. This PY motif
matches the PY motif located in the proline rich P2 region
of the b and g subunits. These observations suggest that a
WW domain-containing protein may exist whose func-
tion is to bind to the PY motif of the b and g subunits of
the epithelial Na1 channel, inhibiting the channel.
Recent evidence suggests that Nedd4 is such a protein.
Nedd4 was originally isolated from a mouse brain library
but is known to be expressed in Xenopus oocytes as well as
the kidney (26,27). Of particular interest, preliminary ev-
idence indicates that the protein is primarily expressed in
the cortical collecting duct, the same location as the Na1
channel (27). The rat homologue of Nedd4 contains three
WW domains. Staub et al (27) have recently shown that
rNedd4 can bind to the b and g subunits of the Na1
channel and that this binding is mediated by the WW
domain. The WW domain binding was specific for the
proline rich P2 region of the 2 subunits. In addition, two
single-point mutations in the PY motif of the b subunit
corresponding to the missense mutations in kindreds de-
scribed by Hansson (21) and Tamarka (5) abolished
rNedd4-WW binding. Staub (27) also showed that the
rNedd4 WW domain could bind to the P2 region of the a
subunit, although this interaction was weaker when com-
pared with that with the b and g subunits. Together these
observations suggest that Nedd4 binds to the proline rich
P2 region of each of the subunits of the Na1 channel via a
WW domain-PY motif interaction. Interference in this
binding by either mutations that delete the P2 regions of
the b and g subunits or alter residues in the PY motifs of
these subunits results in activation of the channel.
The mechanism by which Nedd4 regulates the Na1
channel is currently unknown. In addition to the three
WW domains, Nedd4 contains a CaLB/C2 domain and a
ubiquitin ligase homology (Hect) domain. The CaLB/C2
domain has been shown to bind to lipid in a Ca21-depen-
dent manner (28). Such binding may allow this domain
to associate with the plasma membrane and participate in
endocytosis and/or exocytosis of membrane compo-
nents. The precise function of the CaLB/C2 domain in
Nedd4 is unknown.
The ubiquitin ligase homology (Hect) domain shares
sequence homology with several proteins including
E6-AP (29,30). ‘‘Hect’’ refers to homology to the E6-AP
carboxyl terminus. Ubiquination regulates protein
breakdown by tagging proteins destined for degradation
(31). The ubiquitin group is ligated by ubiquitin ligase,
and the tagged protein is broken down in proteosomes.
Given this makeup of Nedd4, the following model has
been proposed for the Nedd4-Na1 channel interaction
(27). The WW domain of Nedd4 would bind to the PY
motif contained within the P2 region of all three subunits
of the Na channel. This binding would bring the CaLB/C2
domain and the ubiquitin ligase domain in close proxim-
THE AMERICAN JOURNAL OF MEDICINEt Volume 104 307
 Liddle’s Syndrome/Palmer and Alpern

Figure 2. Under normal conditions the removal and degradation of Na1 channels from the apical membrane of the collecting duct
is initiated by Nedd4 binding to a specific PY motif located on the carboxyl terminal cytoplasmic tails of the a, b, and g subunits. A
mutation in which the PY motif of one of the subunits is either deleted or altered leads to a failure of Nedd4 binding and results in an
increase in channel density. Increased channel density can account for constitutive Na1 reabsorption. To date, all mutations in
Liddle’s syndrome have been localized to either the b or g subunits of the Na1 channel. Based on experimental studies a mutation in
the PY motif of the a subunit may also give rise to Liddle’s syndrome. See text for detailed discussion.

ity to the channel. The CaLB/C2 domain could then as-
sociate with the cell membrane and initiate endocytosis of
the Na1 channel. The ubiquitin ligase homology (Hect)
domain would act to tag the channel for eventual degra-
dation. The end result would be a decrease in the number
of channels. Interference in the binding of Nedd4 to the
subunits would prevent channel degradation and in-
crease total channel density resulting in excess channel
activity (Figure 2). The model assumes that Nedd4 has to
bind to all three subunits in order to initiate the degrada-
tive process, since deletion or mutation of the PY motif in
a single subunit can give rise to increased channel activity.
Although mutations in aENaC causing Liddle’s syn-
drome have not yet been reported, the model suggests
that the a subunit may be a potential target gene.
According to this model, Liddle’s syndrome would
be associated with an increase in channel density. In
support of this possibility, patch clamp studies have
shown that deletions of the b subunit causing Liddle’s
syndrome result in an increase in overall channel ac-
tivity without altering the single channel conductance,
open state probability, selectivity, or pharmacologic
properties (17,32). Studies by Snyder et al (32) have
provided additional evidence indicating that increased
Na1 channel activity is the result of increased channel
density in Liddle’s syndrome. These investigators ex-
pressed both the wild type Na1 channel and a Na1
channel containing the Liddle’s associated truncated b
subunit in MDCK epithelia. Both channels were found
to be localized to the apical membrane of the epithelia,
indicating that the Liddle’s mutation did not disrupt
normal membrane targeting. However, using tech-
niques of immunostaining revealed a significant in-
crease in the cell-surface expression of the channel
containing the Liddle’s mutation when compared with
the normal channel. A similar increase in surface ex-
pression was found in studies using a channel in which
the b subunit contained a missense mutation within
the proline rich P2 region.
In summary, the available data suggest that Liddle’s
syndrome is the result of specific mutations that pre-
vent the binding of a regulatory protein to a specific
proline-rich region located in the carboxyl terminal of
the three subunits that comprise the Na1 channel. The
failure of this binding prevents the normal degradation
of the Na1 channel so that the total number of chan-
nels is increased in the cell membrane. This increase in
channel density gives rise to constitutive activation of
the channel, ultimately accounting for the develop-
ment of hypertension, hypokalemia, and metabolic al-
kalosis. Although Liddle’s syndrome is a rare disease,
insights gained from the study of this disorder may
eventually prove useful in elucidating the pathogenesis
of other, more common forms of hypertension. It is
possible that some forms of salt-sensitive hypertension
may prove to be the result of more subtle mutations in
the subunits of the Na1 channel or in the proteins that
regulate channel activity and number.

308 March 1998 THE AMERICAN JOURNAL OF MEDICINEt Volume 104

 Liddle’s Syndrome/Palmer and Alpern
REFERENCES
1. Liddle GW, Bledsoe T, Coppage WS Jr. A familial renal disorder
simulating primary aldosteronism but with negligible aldosterone
secretion. Trans Assoc Am Physicians. 1963;76:199 –213.
2. Botero-Velez M, Curtis JJ, Warnock DG. Brief report: Liddle’s syn-
drome revisited. A disorder of sodium reabsorption in the distal
tubule. NEJM. 1994;330:178 –181.
3. Shimkets RA, Warnock DG, Bositis CM, et al. Liddle’s syndrome:
heritable human hypertension caused by mutations in the b sub-
unit of the epithelial sodium channel. Cell. 1994;79:407– 414.
4. Hansson JH, Nelson-Williams C, Suzuki H, et al. Hypertension
caused by a truncated epithelial sodium channel g subunit: genetic
heterogeneity of Liddle syndrome. Nature Genetics. 1995;11:76 – 82.
5. Tamura H, Schild L, Enomoto N, et al. Liddle disease caused by a
missense mutation of b subunit of the epithelial sodium channel
gene. J Clin Invest. 1996;97:1780 –1784.
6. Young WF, Hogan MJ, Klee GG, et al. Primary aldosteronism: di-
agnosis and treatment. Mayo Clin Proc. 1990;65:96 –110.
7. Rich GM, Ulick S, Cook S, et al. Glucocorticoid-remediable aldo-
steronism in a large kindred: clinical spectrum and diagnosis using
a characteristic biochemical phenotype. Ann Intern Med. 1992;116:
813– 820.
8. Kassirer JP, London AM, Goldman DM, Schwartz WB. On the
pathogenesis of metabolic alkalosis in hyperaldosteronism. Am J
Med. 1970;49:306 –315.
9. Nakada T, Koike H, Akiya T, et al. Liddle’s syndrome, an uncom-
mon form of hyporeninemic hypoaldosteronism: functional and
histopathological studies. J Urology. 1987;137:636 – 640.
10. Gonzalez-Campoy J, Romero J, Knox FG. Escape from the sodium-
retaining effects of mineralcorticoids: role of ANF and intrarenal
hormone systems. Kidney Int. 1989;35:767–777.
11. Yokota N, Bruneau BG, de Bold ML, de Bold AJ. Atrial natriuretic
factor significantly contributes to the mineralocorticoid escape
phenomenon. J Clin Invest. 1994;94:1938 –1946.
12. Canessa CM, Horisberger J, Rossier BC. Epithelial sodium channel
related to proteins involved in neurodegeneration. Nature. 1993;
361:467– 470.
13. Lingueglia E, Voilley N, Waldmann R, et al. Expression cloning of
an epithelial amiloride-sensitive Na1 channel. FEBS Lett. 1993;318:
95–99.
14. Canessa CM, Schild L, Buell G, et al. Amiloride-sensitive epithelial
Na1 channel is made of three homologous subunits. Nature. 1994;
367:463– 467.
15. Ismailov I, Awayda MS, Berdiev BK, et al. Triple-barrel organiza-
tion of ENaC, a cloned epithelial Na1 channel. J Biolog Chem. 1996;
271:807– 816.
16. Benos DJ, Fuller CM, Shlyonsky VG, et al. Amiloride-sensitive Na1
channels: insights and outlooks. News Physiol Sci. 1997;12:55– 62.
17. Schild L, Canessa CM, Shimkets RA, et al. A mutation in the epi-
thelial sodium channel causing Liddle disease increases channel ac-
March 1998
tivity in the Xenopus laevis oocyte expression system. Proc Natl Acad
Sci USA. 1995;92:5699 –5703.
18. Warnock DG, Bubien JK. Liddle syndrome: clinical and cellular
abnormalities. Hosp Practice. 1994;July:95–106.
19. Bubien JK, Ismailov II, Berdiev BK, et al. Liddle’s disease: abnormal
regulation of amiloride-sensitive Na1 channels by b-subunit mu-
tation. Am J Physiol. 1996;270:C208 –213.
20. Ismailov II, Berdiev BK, Fuller CM, et al. Peptide block of consti-
tutively activated Na1 channels in Liddle’s disease. Am J Physiol.
1996;270:C214 –223.
21. Hansson JH, Schild L, Lu Y, et al. A de novo missense mutation of
the b subunit of the epithelial sodium channel causes hypertension
and Liddle syndrome, identifying a proline-rich segment critical for
regulation of channel activity. Proc Natl Acad Sci USA. 1995;92:
11495–11499.
22. Schild L, Lu Y, Gautschi I, et al. Identification of a PY motif in the
epithelial Na channel subunits as a target sequence for mutations
causing channel activation found in Liddle syndrome. EMBO J.
1996;15:2381–2387.
23. Bork P, Sudol M. The WW domain: a signalling site in dystrophin?
TIBS. 1994;19:531–533.
24. Andre B, Springael J. WWP, a new amino acid motif present in
single or multiple copies in various proteins including dystrophin
and the SH3-binding yes-associated protein YAP65. Biochem Bio-
phys Res Comm. 1994;205:1201–1205.
25. Chen HI, Sudol M. The WW domain of yes-associated protein
binds a proline-rich ligand that differs from the consensus estab-
lished for src homology 3-binding modules. Proc Natl Acad Sci
USA. 1995;92:7819 –7823.
26. Kumar S, Tomooka Y, Noda M. Identification of a set of genes with
developmentally down-regulated expression in the mouse brain.
Biochem Biophys Res Comm. 1992;185:1155–1161.
27. Staub O, Dho S, Henry PC, et al. WW domains off Nedd4 bind to
the proline-rich PY motifs in the epithelial Na1 channel deleted in
Liddle’s syndrome. EMBO J. 1996;15:2371–2380.
28. Zhang JZ, Davletov BA, Sudhof TC, Anderson RGW. Synaptotag-
min I is a high affinity receptor for clathrin AP-2: implications for
membrane recycling. Cell. 1994;78:751–760.
29. Scheffner M, Huibregtse JM, Vierstra RC, Howley PM. The
HPV-16 E6 and E6-AP complex functions as a ubiquintin-protein
ligase in the ubiquitination of p53. Cell. 1993;75:495–505.
30. Huibregtse JM, Scheffner M, Beaudenon S, Howley PM. A family of
proteins structurally and functionally related to the E6-AP ubiq-
uitin-protein ligase. Proc Natl Acad Sci USA. 1995;92:2563–2567.
31. Ciechanover A. The ubiquitin-proteasome proteolytic pathway.
Cell. 1994;79:13–21.
32. Snyder PM, Price MP, McDonald FJ, et al. Mechanism by which
Liddle’s syndrome mutations increase activity of a human epithelial
Na1 channel. Cell. 1995;83:969 –978.
THE AMERICAN JOURNAL OF MEDICINEt Volume 104 309