KCNJ10 gene mutations causing EAST syndrome
(epilepsy, ataxia, sensorineural deafness, and
tubulopathy) disrupt channel function
Markus Reicholda,1, Anselm A. Zdebikb,1, Evelyn Lieberera,1, Markus Rapediusc,1, Katharina Schmidta, Sascha Bandulika,
Christina Sternera, Ines Tegtmeiera, David Pentona, Thomas Baukrowitzc, Sally-Anne Hultond, Ralph Witzgalle,
Bruria Ben-Zeevf, Alexander J. Howieg, Robert Kletab,1, Detlef Bockenhauerb,1, and Richard Wartha,1,2
aDepartment of Physiology, University of Regensburg, 93053 Regensburg, Germany;bDepartments of Physiology and Medicine and Institute of Child Health
andgDepartment of Pathology, University College London, London NW3 2PF, United Kingdom;cDepartment of Physiology, University Hospital Jena, 07743
Jena, Germany;dBirmingham Children’s Hospital, Birmingham B4 6NH, United Kingdom;eDepartment of Anatomy, University of Regensburg, 93053
Regensburg, Germany; andfPediatric Neurology Unit, Edmond and Lilly Safra Children’s Hospital, Sheba Medical Center, Ramat-Gan 46425, Israel
Edited* by Gerhard Giebisch, Yale University School of Medicine, New Haven, CT, and approved June 30, 2010 (received for review March 9, 2010)
Mutations of the KCNJ10 (Kir4.1) K+channel underlie autosomal
recessive epilepsy, ataxia, sensorineural deafness, and (a salt-wast-
ing)renal tubulopathy(EAST) syndrome. We investigated the local-
ization of KCNJ10 and the homologous KCNJ16 in kidney and the
functional consequences of KCNJ10 mutations found in our
patients with EAST syndrome. Kcnj10 and Kcnj16 were found in
necting tubules, and cortical collecting ducts. In the human kidney,
KCNJ10 staining was additionally observed in the basolateral mem-
brane of the cortical thick ascending limb of Henle’s loop. EM of
distal tubular cells of a patient with EAST syndrome showed
reduced basal infoldings in this nephron segment, which likely
reflects the morphological consequences of the impaired salt reab-
sorption capacity. When expressed in CHO and HEK293 cells, the
KCNJ10 mutations R65P, G77R, and R175Q caused a marked impair-
Single-channel analysis revealed a strongly reduced mean open
time. Qualitatively similar results were obtained with coexpression
of KCNJ10/KCNJ16, suggesting a dominance of KCNJ10 function in
native renal KCNJ10/KCNJ16 heteromers. The decrease in the cur-
rent of R65P and R175Q was mainly caused by a remarkable shift of
pH sensitivity to the alkaline range. In summary, EAST mutations of
KCNJ10lead to impairedchannel functionand structural changes in
ent in patients carrying the R65P mutation possibly improves re-
sidual function of KCNJ10, which shows higher activity at
functions of tubular epithelial cells are impaired. Defects of salt
transport in the thick ascending loop of Henle and the distal con-
syndrome(s) and Gitelman’s syndrome, respectively (1). We and
others described a unique autosomal recessive form of Gitelman-
KCNJ10 (2, 3). KNCJ10 (Kir4.1) is expressed in various tissues,
including brain,innerear, eye, and kidney(4,5). Patientssuffering
we called EAST syndrome: epilepsy, ataxia, sensorineural deaf-
ness, and (a salt-wasting) renal tubulopathy. The renal features
loss, activation of the renin-angiotensin-aldosterone system, hy-
pokalemic metabolic alkalosis, hypomagnesemia, and hypo-
In C57BL6 mouse kidney, Kcnj10 is expressed in distal convo-
luted tubules starting from the macula densa down to the early
cortical collecting duct (2, 6). In CD1 mice, Kcnj10 and related
he kidneys play a key role in electrolyte and water homeostasis
of the body. In renal salt-wasting disorders, specific transport
Kcnj16 (Kir5.1) are also found in the cortical thick ascending limb
(7). Kcnj10 and Kcnj16 are localized in the basolateral membrane,
where they establish the hyperpolarized membrane voltage needed
forelectrogenic ion transport (e.g.,Cl−exitand Na+-coupledCa2+
and Mg2+export) (8). Additionally, KCNJ10/KCNJ16 activity is
required for Na+/K+-ATPase pump activity. Basolateral Na+/K+-
ATPases take up K+from the narrow space of the basolateral
invaginations of the plasma membrane. During Na+/K+-ATPase
activity, basolateral K+becomes a rate-limiting factor limiting fur-
ther pump activity. K+efflux through KCNJ10/KCNJ16 allows K+
[so-called “pump-leak coupling” (9–12)]. Although KCNJ10 and
KCNJ16 are not the only K+channels expressed in these nephron
segments, they appear to be critical for the pump-leak coupling,
because human patients and Kcnj10−/−mice display deficits of the
In this study, we have investigated the localization of KCNJ10
in mouse and human kidney and the functional consequences of
syndrome. Notably, the renal biopsy of a patient with EAST syn-
drome disclosed loss of basolateral infoldings of distal convoluted
function resulting in salt wasting. The KCNJ10 mutations G77R
and R199X showed almost complete loss of function. The R65P
mutation and the newly described R175Q mutation resulted in
mutated proteins with small residual function and substantially
changed pH sensitivity with IC50values in the alkaline range. The
changed pH sensitivity in these mutations may therefore have
Localization of KCNJ10 and KCNJ16 in Mouse and Human Kidney.
KCNJ10 and KCNJ16 are inwardly rectifying K+channels ex-
pressed in renal tubules. It has been proposed that both channels
form heterotetramersto build functional channels in native tissues.
Author contributions: M. Reichold, A.A.Z., T.B., S.-A.H., B.B.-Z., A.J.H., R.K., D.B., and R.
Warth designed research; M. Reichold, A.A.Z., E.L., M. Rapedius, K.S., S.B., C.S., I.T., D.P.,
T.B., S.-A.H., B.B.-Z., A.J.H., R.K., and D.B. performed research; M. Reichold, A.A.Z., E.L., M.
Rapedius, K.S., S.B., C.S., I.T., T.B., S.-A.H., R. Witzgall, B.B.-Z., A.J.H., R.K., D.B., and R.
Warth analyzed data; and M. Reichold, A.A.Z., T.B., A.J.H., R.K., D.B., and R. Warth wrote
The authors declare no conflict of interest.
*This Direct Submission article had a prearranged editor.
1M. Reichold., A.A.Z., E.L., M. Rapedius, R.K., D.B., and R. Warth contributed equally to
2To whom correspondence should be addressed. E-mail: richard.warth@vkl.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
| August 10, 2010
| vol. 107
| no. 32 www.pnas.org/cgi/doi/10.1073/pnas.1003072107
We therefore examined the renal localization of both channel
proteins by immunofluorescence. As described previously, in
C57BL6 mouse kidney, Kcnj10 labeling was found in early and late
distal convoluted tubules, starting sharply at the macula densa, and
also in connecting tubules and early cortical collecting ducts.
Aquaporin-2-negative intercalated cells were not labeled by the
KCNJ10 antibody. Using consecutive sections, Kcnj16 showed a
distribution similar to Kcnj10, suggesting that both channels could
formheterotetramers innative tubular cells(Fig.1A–F).Inhuman
kidney, localization distal to the macula densa was similar to that
observed in C57BL6 mouse kidney. However, in addition to this,
staining of Na+2Cl−K+cotransporter (NKCC2) in consecutive
sections (Fig. 1 G and H).
Changes in the Distal Convoluted Tubule in EAST Syndrome. Patients
with EAST syndrome display renal salt wasting and electrolyte
disturbances that resemble the clinical features of impaired distal
tubular salt transport in Gitelman’s syndrome. We investigated
the distal tubular morphology of a patient who had EAST syn-
drome using renal biopsy material. EM of the distal convoluted
tubule revealed a decreased number of mitochondria and re-
duction of basolateral infoldings (Fig. 2). These morphological
data confirm the concept of reduced reabsorptive capacity of the
distal convoluted tubule when basolateral K+efflux is impaired.
Functional Consequences of KCNJ10 Mutations Found in EAST
Syndrome. We have identified mutations of the potassium channel
KCNJ10 that are causative for a renal salt-wasting disease, EAST
syndrome. A first set of experiments using Xenopus oocytes sug-
syndrome: c.194G > C resulting in p.R65P, c.229G > C resulting in
in p.R199X. The KCNJ10 mutation p.R175Q has not been de-
scribed previously. Fig. 3A shows a scheme of human KCNJ10 and
the localization of the above-mentioned mutations. The mutated
KCNJ10 channels were heterologously expressed in CHO cells.
The membrane voltage of cells expressing human WT KCNJ10
was hyperpolarized close to the equilibrium potential of K+(Fig.
3B).Cells expressing R65P> R175Q> G77R werehyperpolarized
expressing cells. R199X-expressing cells were not different from
mutation. Application of Ba2+(5 mM) led to a strong depolar-
ization attributable to inhibition of Ba2+-sensitive KCNJ10 chan-
nels. As expected, the Ba2+-induced depolarization in R199X cells
3B). As a measure of the KCNJ10-specific K+current, the Ba2+-
sensitive outward current was determined (Fig. 3C). To minimize
and Kcnj16 in mouse kidney. A and B, C and D, and E and F are consecutive
sections. (A and B) Markers of distal convoluted tubules [NaCl cotransporter
(NCC), green], connecting tubules and collecting ducts (calbindin, blue), and
principal cells of connecting tubules and collecting ducts [aquaporin-2
(AQP2), red]. (C and D) Kcnj10 (green) was localized in the basolateral mem-
brane of early and late distal convoluted tubules, connecting tubules, and
early cortical collecting ducts. (E and F) Distribution of Kcnj16 (green) was
similar to that of Kcnj10. (G and H) Localization of KCNJ10 in consecutive
sections of human kidney. KCNJ10 labeling (green) was observed in the same
segments as in mouse kidney. In addition, KCNJ10 was found in cortical thick
ascending limbs (arrowheads, NKCC2-positive tubules; asterisk, glomerulus;
M, macula densa).
KCNJ10 and KCNJ16 in the nephron. (A–F) Localization of Kcnj10
syndrome. Electron micrographs of distal convoluted tubule cells of a control
(A) and an EAST patient (B). The patient with EAST syndrome shows a de-
creased number of mitochondria and reduction of basolateral infoldings
(arrows). The apparent difference in the density of mitochondria between A
and B is attributable to slight differences in fixation. (Scale bars: 2 μm.)
Morphological changes of distal convoluted tubule cells in EAST
Reichold et al.PNAS
| August 10, 2010
| vol. 107
| no. 32
endogenous Cl−currents, a clamp voltage of −30 mV was chosen.
Expression of WT KCNJ10 induced large Ba2+-sensitive outward
currents. Currents of mutated channels were reduced (R65P >
R175Q) or not different from those of mock-transfected cells
(G77R and R199X).
In native tissues, KCNJ10 is thought to form heterotetramers
with KCNJ16. Therefore, we tested the effect of KCNJ16 co-
transfection. To avoid contamination with homomeric KCNJ10
channels, KCNJ16 was cotransfected with KCNJ10 in a 10:1
stoichiometry ratio (13, 14). As reported previously, KCNJ16
alone did not hyperpolarize the membrane and did not induce
measurable currents (15). Cells coexpressing WT KCNJ10
with KCNJ16 were strongly hyperpolarized and exhibited a large
Ba2+-sensitive outward current (5) (Fig. 3 B and C). By contrast,
the currents of cells coexpressing mutated KCNJ10 with KCNJ16
were much smaller than those of cells transfected with WT
KCNJ10/KCNJ16, and the membrane voltage was similar to that
of mock-transfected cells. All KCNJ10 mutations found in our
patients therefore led to complete or partial loss of function when
expressed alone or together with KCNJ16.
Effects of KCNJ10 Mutations at the Single-Channel Level. The mutant
channels R65P, R175Q, and, to a lesser extent, G77R showed
the single-channel properties of mutated channels using trans-
fected HEK cells. Using the cell-attached configuration with
a cytosol-like pipette solution, WT KCNJ10-expressing cells
showed large inwardly rectifying currents across the patch mem-
brane. In contrast to this, R65P-, R175Q-, and G77R-expressing
cells showed strongly reduced current amplitudes and slight or no
inward rectification (Fig. 4A). WT KCNJ10 channels showed
clear single-channel levels (25–30 pS) and a high open probability
(70–80%, n = 10) (Fig. 4B). R65P and R175Q showed channel
flickering with no clear single-channel levels. Because of channel
flickering, it was very difficult to determine single-channel con-
ductances of mutant channels. Apparently, they were in the same
range as the WT channels. The channel open probability was
strongly reduced to 20–30% for R65P (n = 11) and to 10–15% for
R175Q (n = 3). In G77R-expressing cells, channel activity was
almost absent and only rare channel events were observed cor-
responding to an open probability of about 0.5% (n = 10).
Coexpression with KCNJ16 resulted in heteromeric KCNJ10/
of homomeric KCNJ10 channels (Fig. 4C). KCNJ10/KCNJ16-
containing patches showed single-channel events of variable cur-
rent amplitude. The most frequently observed amplitude was 50–
70 pS [double the amplitude of homomeric KCNJ10 channels
(15)], but sublevels of smaller size were regularly observed, as
reported previously (13). Heteromers of KCNJ16 with R65P and
R175Q (the mutations with relatively large residual function)
exhibited reduced open probability caused by shortening of the
mean open time. Single-channel amplitude of these channels was
similar to that of KCNJ10/KCNJ16 heteromers, and channel
substates were also observed (Fig. 4C).
pH Sensitivity of Mutated KCNJ10 Channels. KCNJ10 channels are
known to be strongly regulated by variations of cytosolic pH with
activation by alkaline pH and channel inhibition by acidic pH
values. Here, we explored the effects of R65P and R175Q muta-
tions on pH sensitivity of KCNJ10 channels in excised inside-out
patches (Fig. 5). Similar to published data (16), half-maximal ac-
tivity (IC50) of WT KCNJ10 channels expressed in HEK293 cells
was observed at pH 6.3 (Fig. 5 A and B). Channel activity of R65P
at physiological pH was strongly reduced, and the IC50value was
shifted to a more alkaline pH. R175Q channels were almost in-
active in the range of physiological pH, and only small currents
more alkaline pH values, excised membrane patches from Xen-
opus laevis oocytes were used. Similar to the results in HEK293
Fig. 5 C and F) to pH 7.86 (Fig. 5 D and F). The mutant R175Q
experiments. (A) Predicted model of the human KCNJ10 membrane topology
according to Uniprot (ID P78508; available at http://www.uniprot.org/). Four
of these subunits are thought to build a functional channel. KCNJ10 may also
form heteromers with KCNJ16, which has similar topology. The KCNJ10
mutations of the patients with EAST syndrome investigated in this study are
colored in red. (B) Effects of KCNJ10 mutations found in patients with EAST
syndrome on the membrane voltage (Vm) of transfected CHO cells. The group
on the right displays the effect of cotransfection with KCNJ16. White columns
with Ba2+(5 mM). Numbers of experiments are shown in parentheses in C. All
channel (J10). KCNJ16 (J16) alone or in cotransfection with KCNJ10 mutants
did not hyperpolarize the cell membrane. (C) Ba2+-sensitive K+outward cur-
mutated KCNJ10 channels led to a strong reduction in the current (R65P >
R175Q) or to a total loss (G77R, R199X). Cotransfection of KCNJ16 with the
different from WT KCNJ10/KCNJ16; §, different from mock-transfected cells.
Mutated KCNJ10 channels display reduced function in whole-cell
| www.pnas.org/cgi/doi/10.1073/pnas.1003072107Reichold et al.
exhibited large currents at alkaline pH and a dramatic shift of the
IC50to pH 9.35 (Fig. 5 E and F).
Phosphatidylinositol 4,5-Bisphosphate Sensitivity of R175Q. The
KCNJ1 residue R188 (corresponding to R175 of KCNJ10) was
shown to bind phosphatidylinositol 4,5-bisphosphate (PIP2),
which regulates channel activity (17). By competitive binding of
PIP2, polylysine (poly-Lys; 250 mg/L, saturated solution) led to
channel inhibition. The time constant of the poly-Lys inhibition
KCNJ10 (17.89 ± 3.1 s, n = 4), indicating reduced PIP2affinity of
range ofoursolutions exchange rate (∼0.5 s); thus, thereal rate of
inhibition might be even faster (Fig. 5 G and H).
Mutations of human KCNJ10 cause the EAST [or seizures, sen-
sorineural deafness, ataxia, mental retardation, and electrolyte
(2, 3). The cause of the tubulopathy is the loss of KCNJ10 K+
resulting in impaired tubular transport capacity. Here, we inves-
tigated the localization of KCNJ10 in mouse and human kidney
found in our patients who had EAST syndrome.
We found Kcnj10-specific immunofluorescence in the baso-
and early collecting ducts of C57BL6 mouse kidney similar to that
described in rat kidney (4). Kcnj10 expression started at the
Henle’s loop. In contrast to our findings, Lachheb et al. (7) also
observed Kcnj10 expression in the cortical thick ascending limb of
CD1 mice, suggesting strain-dependent variability of Kcnj10 lo-
human kidney. In human cortex, strong KCNJ10 staining was
found in cortical thick ascending limbs, distal convoluted tubules,
and, to a lesser extent, aquaporin-2-positive principal cells of
connecting tubules and cortical collecting ducts. The relatively
broad expression of KCNJ10 in human distal tubules possibly
accounts for the severity of renal salt wasting and electrolyte im-
balance in patients with EAST syndrome and might have an im-
pact on our understanding of ion transport in the cortical thick
In distal convoluted tubules, salt transport occurs mostly trans-
cellularly, requiring high Na+/K+-ATPase pump activity. As a
functional adaptation, these tubular cells are mitochondria-rich
and have deep basolateral infoldings, leading to expansion of the
basolateral membrane surface. The infoldings are decorated with
recycling of K+. Under conditions with lowered transport activity
[e.g., in mice lacking the luminal NaCl cotransporter (18)], the
number of mitochondria andthe depthofbasolateralinfoldings are
strongly reduced. Our previous study suggested that transport in
distal tubules is similarly decreased in patients who have EAST
syndrome (2). Here, we present EM data indicating that distal
convoluted tubular cells of a patient with EAST syndrome exhibit
thissegment(i.e., reducednumberof mitochondriaand basolateral
KCNJ10 channels (J10) showed large inwardly rectifying currents across the patch membrane. In contrast to WT cells, expression of the R65P, R175Q, and G77R
mutants led to a strongly reduced current amplitude (patches showed predominantly nonspecific baseline currents) with diminished inward rectification. (B
and C) Single-channel experiments on transfected HEK293 cells in cell-attached configuration at clamped voltage (Vc) = 0 mV. (B) Cells transfected with WT
KCNJ10 channels showed clear single-channel levels and a high open probability (70–80%). Transfection with R65P and R175Q led to a strongly reduced open
probability with channel flickering, and levels were hardly detectable. In G77R-expressing cells, channel events were rare. (C) Coexpression of KCNJ10 with
KCNJ16 resulted in single-channel events with variable current amplitude. Large amplitudes of about 50–70 pS and sublevels with smaller sizes were observed.
Heteromers of KCNJ16 with R65P and R175Q showed similar single-channel amplitude but reduced mean open probability.
Single-channel properties. (A) Patch current in cell-attached configuration with a cytosol-like pipette solution. HEK293 cells transfected with WT
Reichold et al.PNAS
| August 10, 2010
| vol. 107
| no. 32
infoldings). These morphological changes support the concept of
impaired function of distal convoluted tubules as a cause for salt-
wasting and electrolyte disturbance in patients who have
In patients with EAST/SeSAME syndrome, seven different
disease-relatedmutations havebeendescribedsofar.Inthis study,
mutations expressed in mammalian cell systems and X. laevis
oocytes. One of them (R175Q) has not been described previously.
All the mutations examined showed severely impaired channel
activity. In whole-cell experiments, the residual channel function
wasdependentonthemutationR65P> R175Q>G77R> R199X
(R199X displayed complete loss of function). In cell-attached
experiments, R65P, R175Q, and G77R channels showed flicker-
ing, with a strongly decreased mean open time. In native tissues,
KCNJ10 is thought to form heterotetramers with KCNJ16 (7, 13,
13, 15, 19). Coexpression of KCNJ10 mutants with KCNJ16 pro-
ducedheteromericchannelswith impaired activitysimilar tothose
observed for homomeric KCNJ10 channels. Heteromers of
the mean open time. These data are in agreement with the sub-
stantial loss of channel function in affected tissues of patients with
A characteristic of inwardly rectifying K+channels (KCNJ or
Kir family) is the regulation by intracellular pH (15, 19–23). The
mutation R65P is in close proximity to a lysine residue at position
25) and KCNJ1 channels (23). Interestingly, R65P showed a sig-
nificant change of pH sensitivity (i.e., the IC50values were shifted
toward alkaline pH values). In patches from mammalian cells
expressing R175Q, measurable currents could only be elicited
at pH 8. More alkaline pH values were not applicable in that sys-
tem. Experiments with more robust macropatches from X. laevis
oocytes disclosed a dramatic shift of pH sensitivity in the R175Q
mutant, with an IC50of pH 9.35. The arginine residue R175 is
conserved in all 15 human KCNJ channels. In ROMK [KCNJ1
(26)], the corresponding mutation R188Q also led to a substantial
shift in pH sensitivity (17, 27) and it was reported to reduce the
PIP2sensitivity of ROMK and Kir6.2 channels (17, 27, 28). Using
the PIP2-binding substance poly-Lys, we disclosed a strongly re-
duced PIP2affinity of R175Q. This result suggests that this amino
acid residue, which is conserved in all human inward rectifiers, is
involved in the PIP2regulation of this channel family. In contrast
to the KCNJ10 mutation R175Q of our patient with EAST syn-
drome, the R188Q mutation of ROMK has not yet been found
In the KCNJ10 mutant R65P, the alkaline shift of pH sensitivity
relevant pH range. Hypokalemic metabolic alkalosis is character-
istic of EAST syndrome. Given the shifted pH sensitivity of R65P,
patients with this mutation probably have improved residual chan-
nel function in the alkalotic state. Conversely, conditions that lead
function of mutated KCNJ10 in patients. This will lead to di-
minished renal transport and impaired K+buffering of glial cells,
base balance of patients with EAST syndrome may have a sub-
Patients. Our patients were diagnosed with EAST syndrome as a result of
typical clinical findings (epilepsy, ataxia, sensorineural deafness, and renal
tubulopathy). Genetic and histological studies were approved by the Institute
of Child Health–Great Ormond Street Hospital Research Ethics Committee
and were performed after having obtained parental informed consent.
Mutations in KCNJ10 were confirmed in all our patients, as reported pre-
viously (2). One patient was homozygous for G77R (2); one patient was
compound heterozygous for R65P and R199X (3); and another patient,
a 14-y-old boy of consanguineous offspring, was homozygous for R175Q.
This patient presented with epilepsy from infancy onward and showed
ataxia and sensorineural deafness. Blood chemistry tests documented hy-
pomagnesemia and hypokalemia.
Animal Experiments. C57BL6 mice (3 mo of age) had free access to standard
chow and tap water. The experimental protocols were approved by the local
councils foranimal care andwere conducted according to theGerman law for
animal care and the National Institutes of Health Guide for the Care and Use
of Laboratory Animals.
trace of the pH effect in excised inside-out patch of a KCNJ10-transfected
HEK293 cell (125 mM K+in pipette and bath). For simplicity, the negative
inward current at clamped voltage (Vc) = −20 mV is upwardly reflected. (B)
Summary of experiments as shown in A. The IC50of WT KCNJ10 channels (n =
5) was pH 6.3, and the IC50of the R65P mutant was pH 7.1 (n = 5). R175Q
channels (n = 7) were almost inactive at physiological pH, and only small
currents were detected at pH 8. (C–E) Typical traces for pH regulation in
excised inside-out macropatches of X. laevis oocytes. For simplicity, the
negative inward current at Vc= −80 mV is upwardly reflected. (F) Summary
of experiments as shown in C–E. Currents were normalized to the maximal
currents of the patches. (G) Estimation of channel PIP2affinities by com-
petitive PIP2binding of poly-Lys (250 mg/L, saturated solution) obtained at
pH 8.5. (H) Summary of experiments as shown in G. The time of half-maximal
current inhibition (T50) is depicted (n = 4 each).
KCNJ10 mutations leading to changed pH sensitivity. (A) Typical
| www.pnas.org/cgi/doi/10.1073/pnas.1003072107Reichold et al.
Immunofluorescence. Anesthetized mice (isoflurane) were killed by re-
placement of blood by 0.9% NaCl solution containing 10 IU/mL heparin via
acatheter placedinto theabdominalaorta.Fortissuefixation,mice were then
mM sucrose, 90 mM NaCl, 15 mM K2HPO4, 1 mM EGTA, and 2 mM MgCl2(pH
7.4). The kidneys were removed, incubated in a sucrose solution (170 g/l)
overnight, and frozen in isomethylbutane. Cryosections (5 μm) were mounted
on poly-Lys slides (Kindler). Before incubation with the primary antibodies,
sections were incubated in 0.1% SDS (5 min), rinsed, and blocked with BSA
(50g/L, 15 min). Primary and secondary antibodies were diluted in PBS, pH 7.4,
containing 0.04% Triton X-100 (Sigma) and 0.5% BSA. Primary antibodies
were applied overnight at 4 °C. Polyclonal rabbit antibodies for NaCl cotrans-
porter and NKCC2 were kind gifts from Mark Knepper (National Heart Lung
and Blood Institute, Bethesda, MD) (29, 30). Other antibodies were KCNJ10
(Alomone Labs; Fig. S2) and KCNJ16 (custom-made by Davids, Regensburg,
Germany; Fig. S1), aquaporin-2 (Santa Cruz), and calbindin (Sigma). Appro-
priate Alexa dye-coupled secondary antibodies (Invitrogen) were used. Slides
were washed in PBS (2 × 5 min) and mounted with fluorescence-free glycergel
mounting medium (DakoCytomation).
EM.A renal biopsy was taken at theage of7yfrom aboy with EAST syndrome
and the R65P/R199X mutation. A piece fixed in glutaraldehyde was processed
and sectioned for EM. A control specimen that showed no abnormality from
a boy of the same age who was biopsied for intermittent hema-
turia was handled similarly.
Patch Clamp of Mammalian Cells. For patch-clamp experiments, transient
transfected HEK293 cells (single-channel and pH analysis) and CHO cells
(whole-cell experiments) were used. Patch-clamp recordings were performed
using a custom-made EPC-7–like amplifier (obtained from U. Fröbe, Institute
of Physiology, Freiburg, Germany) and an EPC-10 amplifier (HEKA). The patch
pipette solution was composed of 95 mM K-gluconate, 30 mM KCl, 4.8 mM
Na2HPO4, 1.2 mM NaH2PO4, 5 mM glucose, 2.38 mM MgCl2, 0.726 mM CaCl2,
experiments contained 145 mM NaCl, 1.6 mM K2HPO4, 0.4 mM KH2PO4,
1.3 mM Ca-gluconate, 1 mM MgCl2, 5 mM D-glucose, and 5 mM Hepes. For
excised patches, the bath solution was replaced by the pipette solution.
Macropatches of Oocytes. cRNA from all KCNJ10 constructs, cloned by
inserting PCR amplificates (using primers carrying restriction sites) from pa-
tient DNA into the pTLB expression vector (a kind gift from T. J. Jentsch,
Leibniz-Institut für Molekulare Pharmakologie, Berlin, Germany) using
primers carrying restriction sites, was injected into X. laevis oocytes and
measured 1–2 d after injection. Patch-clamp recordings were done in the
inside-out configuration at a constant membrane voltage of −80 mV under
symmetrical K+conditions (120 mM K+intra/extra). Different pH solutions
were applied to the intracellular side of the membrane via a multibarrel
perfusion system that allowed solution exchange within 1 s. All traces shown
in Fig. 5 are upward reflections of inward currents at −80 mV. To test the
PIP2 sensitivity of KCNJ10, negatively charged PIP2 was clustered by the
polycation poly-Lys (P4158; Sigma). The time course of inhibition was used to
estimate the strength of KCNJ10/PIP2interactions (17).
Statistics. Data are shown as mean values ± SEM from n observations. Paired
as well as unpaired Student’s t test was used as appropriate. Differences
were considered significant if P < 0.05.
ACKNOWLEDGMENTS. We thank Dr. Roger D. G. Malcomson (Birmingham
Children’s Hospital) for EM material and Prof. Dr. Thomas Jentsch (Leibniz-
Institut für Molekulare Pharmakologie, Berlin, Germany) for instruments
and materials. The expert assistance by Helga Schmidt is acknowledged. This
study was supported by Deutsche Forschungsgemeinschaft Grants SFB699 (to
M.R., R. Warth, and R. Witzgall) and BA1793/4-2 (to T.B).
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Reichold et al.PNAS
| August 10, 2010
| vol. 107
| no. 32