Molecular pathogenesis of inherited hypertension
with hyperkalemia: The Na–Cl cotransporter is
inhibited by wild-type but not mutant WNK4
Frederick H. Wilson*†, Kristopher T. Kahle*†, Ernesto Sabath†‡, Maria D. Lalioti*, Alicia K. Rapson*, Robert S. Hoover§,
Steven C. Hebert§, Gerardo Gamba‡, and Richard P. Lifton*¶
*Departments of Genetics, Medicine, and Molecular Biophysics and Biochemistry, and Howard Hughes Medical Institute, and§Department of Molecular and
Cellular Physiology, Yale University School of Medicine, New Haven, CT 06510; and‡Molecular Physiology Unit, Instituto Nacional de Ciencias Me ´dicas y
Nutricio ´n Salvador Zubira ´n and Instituto de Investigaciones Biome ´dicas, Universidad Nacional Autonoma de Mexico Tlalpan, Mexico City, 14000, Mexico
Contributed by Richard P. Lifton, December 4, 2002
Mutations in the serine-threonine kinases WNK1 and WNK4 [with
no lysine (K) at a key catalytic residue] cause pseudohypoaldoste-
ronism type II (PHAII), a Mendelian disease featuring hypertension,
hyperkalemia, hyperchloremia, and metabolic acidosis. Both ki-
nases are expressed in the distal nephron, although the regulators
and targets of WNK signaling cascades are unknown. The Cl?
dependence of PHAII phenotypes, their sensitivity to thiazide
diuretics, and the observation that they constitute a ‘‘mirror
image’’ of the phenotypes resulting from loss of function muta-
tions in the thiazide-sensitive Na–Cl cotransporter (NCCT) suggest
that PHAII may result from increased NCCT activity due to altered
WNK signaling. To address this possibility, we measured NCCT-
mediated Na?influx and membrane expression in the presence of
wild-type and mutant WNK4 by heterologous expression in Xe-
nopus oocytes. Wild-type WNK4 inhibits NCCT-mediated Na-influx
by reducing membrane expression of the cotransporter (22Na-
influx reduced 50%, P < 1 ? 10?9, surface expression reduced 75%,
P < 1 ? 10?14in the presence of WNK4). This inhibition depends
on WNK4 kinase activity, because missense mutations that abro-
gate kinase function prevent this effect. PHAII-causing missense
mutations, which are remote from the kinase domain, also prevent
inhibition of NCCT activity, providing insight into the pathophys-
iology of the disorder. The specificity of this effect is indicated by
the finding that WNK4 and the carboxyl terminus of NCCT coim-
munoprecipitate when expressed in HEK 293T cells. Together,
these findings demonstrate that WNK4 negatively regulates sur-
face expression of NCCT and implicate loss of this regulation in the
molecular pathogenesis of an inherited form of hypertension.
protein serine-threonine kinases ? hypertension ? thiazide-sensitive Na–Cl
cotransporter ? ion transport ? medical genetics
contributing to morbidity and mortality from stroke, myocardial
infarction, renal failure, and congestive heart failure (1). Its
pathogenesis is largely unknown, resulting in empiric pharma-
cologic therapy. In recent years, genetic approaches investigating
rare Mendelian forms of high and low blood pressure have
provided fundamental insight into mechanisms that contribute
to blood pressure variation (2). These have demonstrated the
causal role of inherited variation in renal salt homeostasis in
blood pressure variation, with mutations in many genes known
to play a role in mediating or regulating renal salt reabsorption
resulting in altered blood pressure.
Pseudohypoaldosteronism type II (PHAII; Online Mendelian
Inheritance in Man database no. 145260) is an autosomal
dominant disease featuring hypertension with hyperkalemia
despite normal glomerular filtration rate; renal tubular acidosis
is a variable associated finding. The clinical features of this
disease are chloride dependent and are also corrected with
thiazide diuretics, specific antagonists of the Na–Cl cotrans-
ypertension is the most common disease in industrialized
societies, affecting ?20% of the adult population and
porter (NCCT) of the distal convoluted tubule (3–7). We have
recently demonstrated (8) that PHAII is caused by mutations in
either of two serine-threonine kinases, WNK1 and WNK4 [with
no lysine (K) at a key catalytic residue]. PHAII-causing muta-
tions in WNK1 are large deletions in the first intron of the gene
that appear to increase WNK1 expression. Mutations in WNK4
are missense mutations in highly conserved segments remote
from the kinase domain (8). Both kinases are present in the
kidney, with their expression confined to the distal convoluted
tubule, connecting tubule, and collecting duct; these nephron
segments are known to play a key role in the regulation of salt,
K?, and pH homeostasis (8). These findings implicate WNK1
and WNK4 in a previously unrecognized signaling pathway that
regulates the balance between Cl?reabsorption versus K?and
H?secretion. Nonetheless, the upstream regulators and the
downstream molecular targets of these kinases are presently
unknown, leaving unresolved the question of their normal
physiologic role and the mechanism by which their mutation
results in the observed PHAII phenotypes.
One attractive target for the WNK kinases is the thiazide-
sensitive NCCT. This cotransporter mediates the apical reab-
sorption of Na?with Cl?and is expressed predominantly in the
distal convoluted tubule (9, 10). Consequently, the expression of
WNK4 and NCCT overlap in epithelial cells of the distal
nephron. Moreover, we have previously shown that loss-of-
function mutations in NCCT cause Gitelman’s syndrome, a
disease featuring a phenotype that is the mirror image of PHAII,
with reduced blood pressure, hypokalemia, and metabolic alka-
losis (11). Coupled with the exquisite sensitivity of PHAII
phenotypes to thiazide diuretics, these observations suggest that
PHAII could result from increased activity of the NCCT due
either to loss of normal inhibition or constitutive activation by
mutant WNK kinases. We now demonstrate that the wild-type
WNK4 kinase is a negative regulator of the thiazide-sensitive
NCCT and that WNK4 mutations found in patients with PHAII
abrogate this inhibitory function. This provides an explanation
by which mutations in WNK4 impart their physiologic effect and
reveals aspects of a new signaling pathway involved in blood
pressure and electrolyte homeostasis.
Assembly of cDNA Constructs. The complete coding sequence of
mouse WNK4 was amplified by PCR from first-strand mouse
Abbreviations: PHAII, pseudohypoaldosteronism type II; WNK, with no lysine (K); NCCT,
Na–Cl cotransporter; EGFP, enhanced GFP; HA, hemagglutinin A.
Data deposition: The sequence reported in this paper has been deposited in the GenBank
database (accession no. AY187027).
†F.H.W., K.T.K., and E.S. contributed equally to this work.
Congress Avenue, Boyer Center for Molecular Medicine 154D, New Haven, CT 06510.
January 21, 2003 ?
vol. 100 ?
kidney cDNA in two overlapping segments of ?2 kb. The
fragments were combined by PCR to yield a full-length WNK4
cDNA that was directly cloned into pcDNA3.1? (Invitrogen) by
ligation into the KpnI and EcoRV sites of the vector. A hem-
agglutinin A (HA) epitope tag was introduced in-frame at the
carboxyl terminus of WNK4 by PCR with ligation into
pcDNA3.1? to generate WNK4-HA. The function of WNK4
with and without the HA epitope was no different in effects on
Na?flux and surface expression (data not shown). Mutant
WNK4-HA constructs (kinase-dead D318A and PHAII Q562E
and E559K) were generated using the QuikChange site-directed
mutagenesis system (Stratagene). A pSPORT1 clone containing
enhanced green fluorescent protein (EGFP) (12) fused in-frame
to the 5? end of rat NCCT as described (13) was used for
quantitation of NCCT expression. cRNA was transcribed in
vitro from linearized plasmids by using the T7 mMESSAGE
mMACHINE system (Ambion, Austin, TX) and quantitated
by UV spectroscopy.
For immunoprecipitation studies, full-length mouse WNK4
was subcloned into pEF1?Myc-His A (Invitrogen), which added
a Myc epitope to the carboxyl terminus of WNK4. A construct
containing the intracytoplasmic carboxyl terminus of NCCT
with the V5 epitope at the C terminus was prepared by ampli-
fication of amino acids 605-1021 of NCCT (GenBank accession
no. X91220) from human kidney cDNA by using specific primers
and cloning the product into pcDNA3.1D?V5-His (Invitrogen).
All constructs were verified by sequence analysis.
Na?Transport Measurements. Oocytes were isolated from adult
Xenopus laevis by using standard procedures (14). Stage V–VI
oocytes were injected with 25 ng of NCCT cRNA alone or
together with 25 ng of wild-type or mutant WNK4-HA cRNA in
in ND96 supplemented with sodium pyruvate (2.5 mM) and
gentamicin (5 mg?ml); on the fourth day oocytes were trans-
ferred to a Cl?-free ND96 medium (96 mM sodium isethionate?
2.0 mM potassium gluconate?1.8 mM calcium gluconate?1.0
mM magnesium gluconate?5.0 mM Hepes/Tris, pH 7.4).22Na?
uptake was assessed in groups of 15–20 oocytes 4 days after
injection as described (15). In brief, oocytes were incubated for
30 min in a Cl?-free ND96 medium with bumetanide (0.1 mM),
followed by a 60-min uptake period in a K?-free NaCl medium
containing ouabain, amiloride, bumetanide, and 2.5 ?Ci (1 Ci ?
37 GBq) of22Na?per ml (NEN; ref. 15). Thiazide sensitivity of
22Na influx was assessed by measuring22Na?uptake in paired
groups of oocytes with or without metolazone (0.1 mM) in the
incubation and uptake media. All experiments were performed
at 32°C. At the end of the uptake period, oocytes were washed
five times in ice-cold uptake solution without isotope to remove
extracellular fluid tracer. After the oocytes were dissolved in
10% SDS, tracer activity was determined for each oocyte by
?-scintillation counting. Flux measurements were made using
26–75 oocytes from at least two frogs in each group, with the
exception of the ‘‘kinase-dead’’ WNK4 group, for which 10
oocytes were studied. For each injection series, the mean22Na-
influx value for NCCT alone was set at 100%, and other values
were expressed as percentage of this value.
Surface Expression Measurements. Oocytes were injected with 40
ng of EGFP-NCCT cRNA alone or together with 40 ng of
wild-type or mutant WNK4-HA cRNA. Oocytes were incubated
for 3–4 days at 18°C in ND96 solution supplemented with
penicillin and streptomycin. Membrane surface expression of
EGFP-NCCT was assayed by laser-scanning confocal micros-
copy with an LSM410 microscope (Zeiss) as described (13).
Excitation was performed at 488 nm, and fluorescent emissions
were detected through a 515- to 565-nm band-pass filter. Fluo-
rescent images of equatorial sections of injected and uninjected
oocytes were captured using a ?10 objective lens. Brightness and
contrast settings were kept constant during imaging of all
oocytes in each injection series, which always included an
EGFP-NCCT-alone control for comparison. Total membrane
fluorescence intensity was calculated for each imaged oocyte by
using SIGMASCAN PRO software (Jandel, San Rafael, CA). Sur-
face expression measurements were made using a total of 45–80
oocytes from at least two different frogs for each experimental
condition. For each injection series, the mean fluorescence value
for NCCT alone was set at 100%, and other values were
expressed as percentage of this value.
Statistical Methods. The significance of differences in22Na influx
and NCCT expression between groups of oocytes was assessed
by two-tailed Student’s t test in which a P value of 0.05 was
Transient Transfections and Immunoprecipitation. HEK 293T cells
were cultured at 37°C under a 5% CO2?95% air atmosphere in
DMEM (Life Technologies, Grand Island, NY) supplemented
with 2 mM L-glutamine, streptomycin, penicillin, 1 mM sodium
by CaPO4precipitation with 10–20 ?g of plasmid DNA, with
cells grown to 70% confluency. After transfection, cells were
incubated in Ultracho media (BioWhittaker) with penicillin and
streptomycin for 48 h; cells were then washed in cold PBS and
lysed at 4°C in lysis buffer [10 mM Tris?HCl, pH 8.0?2.5 mM
MgCl2?5 mM EGTA, pH 8.0?0.5% Triton X-100?1 mM
Na3VO4?50 mM NaF, one tablet of protease inhibitor mixture
(Roche Molecular Biochemicals) per 10 ml of buffer]. Lysates
were cleared by centrifugation, and the supernatant was used for
immunoprecipitation. For each immunoprecipitation, 1 ?g of
mouse monoclonal anti-V5 (Invitrogen), rabbit polyclonal anti-
myc (Santa Cruz Biotechnology), or rabbit polyclonal anti-HA
(CLONTECH) antibody was coupled to 30 ?l of rec-Protein
G-Sepharose (Zymed) for 1 h at 4°C. The resulting antibody-
protein G-Sepharose was resuspended in PBS, added to the
lysates, and incubated overnight at 4°C. Immunoprecipitates
were washed with PBS, and bound protein was eluted by boiling
for 5 min in 2? SDS sample buffer.
Western Blotting. Lysates and immunoprecipitated proteins were
fractionated using 4–15% SDS?PAGE gradient gel electro-
phoresis (Bio-Rad). Proteins were transferred to poly(vinylidene
difluoride) (PVDF) membrane (Bio-Rad) at 100 V and 4°C for
2 h. The membrane was blocked in 5% nonfat dried milk in PBS.
Primary antibodies were diluted in 1% milk in PBS and incu-
bated with the membrane for 1 h at room temperature. Blots
were washed in PBS with 0.1% Tween 20 and probed with an
HRP-conjugated secondary antibody (Zymed) in 1% milk in
PBS for 30 min at RT. The filter was then washed and chemi-
luminescence performed using ECL-Plus (Amersham Pharma-
cia), following standard protocols.
The full-length mouse WNK4 cDNA was cloned as described in
Methods, and the sequence has been deposited in GenBank
(accession no. AY187027). The encoded protein is 86% identical
to human WNK4 and is shorter than the human ortholog by 21
aa (GenBank accession no. AF390018). This cDNA structure is
different from GenBank accession no. XM?109998, which was
deduced from genomic sequence and indicates a coding se-
quence that begins within the highly conserved kinase domain.
To assess the potential effect of WNK4 on Na–Cl flux
mediated by NCCT, we injected Xenopus oocytes with combi-
nations of cRNA of NCCT and wild-type or mutant WNK4 and
determined the resulting22Na influx into oocytes. Injection of
NCCT cRNA alone (Fig. 1) resulted in a 7-fold increase of
Wilson et al.
January 21, 2003 ?
vol. 100 ?
no. 2 ?
metolazone-sensitive22Na influx into oocytes compared with
water-injected controls. Coinjection of NCCT with wild-type
WNK4 resulted in a 50% reduction of the22Na influx seen with
NCCT alone (Fig. 1). This suppression was reproducible in 74
oocytes from five different frogs and was highly statistically
significant (P ? 1 ? 10?9). This finding demonstrates the ability
of wild-type WNK4 to inhibit the activity of the cotransporter.
To determine whether the inhibition of NCCT by WNK4
depends on the catalytic function of the WNK4 kinase domain
and to rule out trivial explanations of this inhibitory effect, we
prepared a kinase-dead mutant of WNK4 in which aspartate 318
in the highly conserved kinase domain is mutated to alanine.
serine-threonine kinases, because the carboxyl group of this
amino acid is essential for Mg2?binding and catalytic function
(16). This mutant WNK4 showed no inhibition of NCCT-
mediated22Na influx, demonstrating the dependence of WNK4
suppression on kinase domain function (Fig. 1).
Is the observed effect of WNK4 on NCCT of physiologic
relevance? Mutations in WNK4 that cause PHAII are clustered
in a short, highly conserved segment remote from the kinase
domain and raise the question as to whether WNK4 harboring
these missense mutations loses the ability to inhibit NCCT-
mediated22Na influx in the oocyte. To address this question, we
have tested the effect of WNK4 harboring the PHAII-causing
missense mutation Q562E (corresponding to the human
Q565E). This mutation is seen only in PHAII and precisely
segregates with the disease in PHAII kindred 13 (8). WNK4
Q562E showed no inhibition of NCCT-mediated22Na influx in
oocytes, demonstrating the functional significance of this mu-
tation (Fig. 1);22Na influx was significantly higher than that seen
with wild-type WNK4 and not significantly different from the
result seen with the kinase-dead WNK4 or in the absence of
WNK4 altogether. Other nearby WNK4 mutations that cause
PHAII, such as E559K (E562K in human WNK4), also show loss
of inhibition of NCCT function (data not shown).
These experiments establish that wild-type WNK4 inhibits
NCCT function, and that PHAII mutations eliminate this effect.
To address the mechanism of this inhibition, we measured
expression of EGFP-tagged NCCT (EGFP-NCCT) in Xenopus
oocytes by using fluorescence confocal microscopy after injec-
tion of NCCT alone or in combination with various WNK4
constructs (Fig. 2). EGFP-NCCT is almost entirely localized to
the plasma membrane (Fig. 2B). This surface expression is
markedly reduced by addition of wild-type WNK4 (Fig. 2C).
Quantitation of this effect indicates a 75% reduction in NCCT
expression in the presence of WNK4 (Fig. 3; P ? 1 ? 10?14). As
was found for22Na influx, this inhibitory effect of WNK4 is
abolished when kinase domain function is disrupted by the
D318A mutation or when WNK4 bears the PHAII-causing
Q562E missense mutation (Fig. 3). Similar results were obtained
with WNK4 E559K (data not shown). These findings indicate
that WNK4 inhibits NCCT function by reducing the amount of
NCCT present at the cell surface.
To address the specificity of the effect of WNK4 on NCCT, we
have asked whether these proteins can be found in a complex in
mammalian cells. We transfected expression plasmids encoding
full-length WNK4 tagged with the myc epitope and the cyto-
plasmic C terminus of NCCT tagged with the V5 epitope into
HEK 293T cells. Immunoprecipitation of NCCT with a mono-
clonal antibody directed against the V5 epitope was performed,
and the precipitated protein complex was fractionated by SDS?
PAGE. Staining with anti-myc antibody revealed a protein in the
immunoprecipitate the size of the myc-tagged WNK4 construct
(Fig. 4A). Detection of this protein depended on transfection of
both plasmids (Fig. 4A) and was not detected in cells coexpress-
ing V5 coupled to other peptides and tagged WNK4 (data not
Oocytes were injected with cRNA encoding NCCT and wild-type or mutant
WNK4;22Na entry was measured as described in Methods. The injected cRNAs
are expressed as a percentage of this value.
in the presence and absence of expression of wild-type WNK4 as described in
Methods. Representative examples of fluorescence seen in the absence of
EGFP-NCCT (A), after expression of EGFP-NCCT alone (B), and after expression
of EGFP-NCCT with wild-type WNK4 (C) are shown. The results demonstrate
Effect of WNK4 on expression of NCCT in Xenopus oocytes. Confocal
and mutant WNK4. Oocytes were injected with cRNA encoding EGFP-tagged
NCCT and wild-type or mutant WNK4; green fluorescence was quantitated by
confocal microscopy of oocytes as described in Methods. The injected cRNAs
are indicated, and the mean and standard error of fluorescence is shown for
each set of oocyte injections; as in Fig. 1, for each set of injections, the mean
Quantitation of expression of EGFP-NCCT in response to wild-type
www.pnas.org?cgi?doi?10.1073?pnas.242735399Wilson et al.
shown). Similarly, in the converse experiment, immunoprecipi-
tation of myc-tagged WNK4 coprecipitated V5-tagged NCCT
(data not shown). Finally, to determine whether the PHAII-
causing mutation Q562E imparts its effect by preventing WNK4
from binding to the NCCT-containing complex, we repeated
immunoprecipitation of V5-tagged NCCT in cells expressing
tagged WNK4 harboring the Q562E mutation. This mutant
WNK4 is still coprecipitated with NCCT (Fig. 4B), indicating
that this mutation does not prevent the entry of WNK4 into a
complex with NCCT.
These studies establish that one function of wild-type WNK4 is
to inhibit activity of the NCCT via reduced cell surface expres-
sion of the cotransporter. Na?-influx studies and measurement
of membrane expression in all cases yield concordant results and
are consistent with all of the inhibitory effects of WNK4 being
attributable to loss of NCCT from the cell surface. This inhib-
itory effect of WNK4 depends on its kinase activity, because
inhibition is lost in kinase-dead WNK4. The coimmunoprecipi-
tation of the C terminus of NCCT and WNK4 indicates that
these proteins can exist together in a complex in mammalian
cells, consistent with the physiologic relevance of WNK4-
mediated inhibition of NCCT; nonetheless, establishing the
precise sites required for this interaction, whether this interac-
tion is direct or indirect, and whether WNK4 directly phosphor-
ylates NCCT will require further investigation. Importantly, the
presence of both NCCT and WNK4 in epithelia of the mam-
malian distal nephron is consistent with the relevance of this
interaction and inhibitory effect of WNK4 (see below). Whether
WNK4 reduces NCCT surface expression by increasing removal
of NCCT from the cell surface or by decreasing its delivery to the
cell surface is presently uncertain. Preliminary experiments
using dominant-negative dynamin (17) or dominant-negative
amphiphysin 1 (18), agents that arrest endocytosis of cell surface
proteins via clathrin-coated pits, demonstrate that down-
regulation of NCCT by WNK4 is unaltered, suggesting that this
pathway is not involved in the mechanism (data not shown).
Mutations in WNK4 that cause PHAII are located outside the
kinase domain and are clustered within a short, well conserved
segment (8). WNK4 bearing these missense mutations distal to
the first coiled-coil domain loses the ability to inhibit NCCT
expression. This finding establishes a specific biochemical con-
sequence of these disease-causing mutations and implicates
unrestrained activity of NCCT in the pathogenesis of PHAII.
This inference is strongly supported by the clinical phenotypes of
PHAII, which can potentially all be explained by increased
NCCT activity (8). Increased NCCT activity is anticipated to
increase net renal salt reabsorption, thereby expanding plasma
volume and raising cardiac output resulting in hypertension; this
pathophysiologic sequence is shared by other Mendelian forms
of hypertension (2). The increased reabsorption of Na?with Cl?
by this cotransporter could reduce the amount of Na?reabsorp-
tion via the electrogenic epithelial Na?channel (ENaC) in the
distal nephron, impairing development of the lumen-negative
potential that is required for normal secretion of K?and H?in
the distal nephron. Furthermore, the exquisite sensitivity of
PHAII phenotypes to thiazide diuretics, specific antagonists of
also consistent with increased NCCT activity playing an impor-
tant role in the pathophysiology of PHAII.
This physiologic explanation for PHAII raises several ques-
tions. First, is NCCT the sole target of WNK4? WNK4 is
expressed in the distal convoluted tubule, the domain of NCCT
expression, but is more strongly expressed in the collecting duct
(8), suggesting that there may be additional targets. Moreover,
WNK4 appears to localize predominantly in the tight junction
complex, raising the question as to whether tight junction
components might also be targets (8). Other possible targets
include regulators or mediators of electrolyte flux such as
paracellular Cl?flux mediators, the Na?channel ENaC, the K?
channel ROMK (involved in distal K?secretion), or subunits of
the apical H?ATPase (involved in distal H?secretion). The
possibility of additional targets is further supported by the
unusual distribution of PHAII-causing mutations. To date, four
different WNK4 mutations have been reported that cosegregate
with PHAII. Three of these cluster within a 4-aa sequence that
is embedded within a highly conserved 10-aa segment distal to
the first coil domain of the protein; the fourth occurs in a
similarly conserved segment just distal to the second coil domain
(8). This highly clustered distribution of mutations is not what
one would expect for general loss of function mutations, which
would be expected to include missense mutations distributed
throughout essential domains of the protein, as well as prema-
ture-termination, frameshift, and splice-site mutations. One
possible resolution of this apparent paradox is that NCCT is only
one of several WNK4 targets. In this case, mutations that knock
out WNK4 function entirely could result in a broader phenotype
than PHAII, and the mutations observed in PHAII kindreds
at NCCT but preserve activity at other targets. Further inves-
tigation to establish the biochemical mechanism of these PHAII
mutations will be required.
A companion question is whether increased NCCT function
alone could effectively impair K?and H?secretion. Patients
with PHAII eventually achieve salt balance, excreting the daily
absorbed salt load, and in this state are delivering a normal salt
load to the collecting duct; this is expected to permit normal K?
and H?secretion. One possible explanation for the observed
impaired K?and H?secretion is suppressed secretion of renin
due to the expanded plasma volume, with the result that
aldosterone secretion is not as high as it would normally be in the
setting of hyperkalemia. A second possibility is raised by the
observation that NCCT can be expressed beyond the distal
convoluted tubule (19), suggesting that in PHAII, the domains
of NCCT and ENaC expression could overlap. In this case,
Full-length wild-type WNK4 tagged with the myc epitope and the C terminus
of NCCT tagged with the V5 epitope were expressed in HEK 293T cells as
described in Methods. Cellular extracts were prepared, and immunoprecipi-
tation was performed with anti-V5 antibodies as described in Methods. The
precipitated protein was subjected to SDS?PAGE on 4–15% gradient gels,
transferred to membrane, and stained with anti-myc antibody. A 170-kDa
protein corresponding to myc-tagged WNK4 is detected in immunoprecipi-
tates from cells expressing both NCCT and WNK4, but not in cells transfected
with only one or neither tagged protein. (B) The experimental protocol is as
in A, except that the Q562E mutation found in PHAII kindred 13 has been
introduced into WNK4; this mutant WNK4 is tagged with the HA epitope
rather than myc, and staining is with anti-HA antibody. Mutant WNK4 is
coprecipitated by anti-V5 antibody only when both tagged NCCT and WNK4
Coimmunoprecipitation of WNK4 and C terminus of NCCT. (A)
Wilson et al.
January 21, 2003 ?
vol. 100 ?
no. 2 ?
NCCT activity could directly attenuate the amount of Na? Download full-text
reabsorbed by ENaC, thereby directly impairing K?and H?
Mutations in the related kinase WNK1 also cause PHAII. In
contrast to the missense mutations in WNK4 that cause disease,
mutations in WNK1 appear to be gain-of-function resulting from
increased expression (8). This observation suggests that WNK1
and WNK4 likely have distinct biochemical mechanisms; how-
ever, the indistinguishable phenotype resulting from mutation in
these two genes suggests that both act in the same pathway.
Comparable experiments in the oocyte system with WNK1 will
consequently be of interest.
Finally, these observations raise the question as to what the
upstream regulators of WNK4 might be. One obvious possibility
is aldosterone, which is known to increase NCCT expression in
rats (20). Part of the mechanism of aldosterone action is
increased transcription of the gene encoding the serine-
threonine kinase SGK (21); interestingly, there are consensus
SGK phosphorylation sites in WNK4, suggesting that phosphor-
ylation of WNK4 by SGK might inhibit its activity, thereby
resulting in increased NCCT expression. A second possibility is
which, interestingly, has the same substrate specificity and can
share targets with SGK (22). These possibilities suggest that
PHAII mutations may define a specific downstream branch
shared by multiple signaling pathways.
We gratefully acknowledge Qiang Leng, Gordon MacGregor, Tony
O’Connell, Ignacio Gime ´nez, and members of the laboratory of Walter
Boron for harvest of Xenopus oocytes; and Norma Va ´zquez for technical
assistance. This work was supported in part by a National Institutes of
Health Specialized Center of Research in Hypertension grant (to R.P.L.)
and National Institutes of Health Grant DK36803 (to S.C.H. and G.G.).
K.T.K. is the recipient of a Howard Hughes Medical Institute Medical
Student Research Fellowship. R.P.L. is an investigator of the Howard
Hughes Medical Institute.
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