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ENaC activity is increased in isolated, split-open cortical collecting ducts
from protein kinase C␣knockout mice
Hui-Fang Bao,
1,2
Tiffany L. Thai,
1,2
Qiang Yue,
1,2
He-Ping Ma,
1,2
Amity F. Eaton,
1,2
Hui Cai,
1,2,3
Janet D. Klein,
1,2,3
Jeff M. Sands,
1,2,3
and Douglas C. Eaton
1,2
1
Department of Physiology, Emory University School of Medicine, Atlanta, Georgia;
2
The Center for Cell and Molecular
Signaling, Emory University School of Medicine, Atlanta, Georgia; and
3
Department of Medicine, Renal Division, Emory
University School of Medicine, Atlanta, Georgia
Submitted 23 September 2013; accepted in final form 4 December 2013
Bao HF, Thai TL, Yue Q, Ma HP, Eaton AF, Cai H, Klein JD,
Sands JM, Eaton DC. ENaC activity is increased in isolated, split-
open cortical collecting ducts from protein kinase C␣knockout mice.
Am J Physiol Renal Physiol 306: F309 –F320, 2014. First published
December 11, 2013; doi:10.1152/ajprenal.00519.2013.—The epithe-
lial Na channel (ENaC) is negatively regulated by protein kinase C
(PKC) as shown using PKC activators in a cell culture model. To
determine whether PKC␣influences ENaC activity in vivo, we
examined the regulation of ENaC in renal tubules from PKC␣
⫺/⫺
mice. Cortical collecting ducts were dissected and split open, and the
exposed principal cells were subjected to cell-attached patch clamp. In
the absence of PKC␣, the open probability (P
o
) of ENaC was
increased three-fold vs. wild-type SV129 mice (0.52 ⫾0.04 vs.
0.17 ⫾0.02). The number of channels per patch was also increased.
Using confocal microscopy, we observed an increase in membrane
localization of ␣-, -, and ␥-subunits of ENaC in principal cells in the
cortical collecting ducts of PKC␣
⫺/⫺
mice compared with wild-type
mice. To confirm this increase, one kidney from each animal was
perfused with biotin, and membrane protein was pulled down with
streptavidin. The nonbiotinylated kidney was used to assess total
protein. While total ENaC protein did not change in PKC␣
⫺/⫺
mice,
membrane localization of all the ENaC subunits was increased. The
increase in membrane ENaC could be explained by the observation
that ERK1/2 phosphorylation was decreased in the knockout mice.
These results imply a reduction in ENaC membrane accumulation and
P
o
by PKC␣in vivo. The PKC-mediated increase in ENaC activity
was associated with an increase in blood pressure in knockout mice
fed a high-salt diet.
protein kinase C␣; ENaC; renal tubules; single channels; knockout
mice; hypertension
EPITHELIAL NA CHANNELS (ENaC) are sodium-permeable ion
channels located in the apical membrane of polarized epithelial
cells primarily in the distal nephron, lung, and distal colon. In
the distal nephron, ENaC activity is the rate-limiting step for
Na
⫹
reabsorption (16, 34); therefore, ENaC activity is critical
for the physiological maintenance of systemic Na
⫹
homeosta-
sis and long-term control of blood pressure. Because of its
central role in responding to changes in Na
⫹
uptake, ENaC
activity is tightly regulated; dysregulation of this channel has
been linked to abnormal blood pressure in several genetic
disorders including Liddle’s syndrome (18, 37) and pseudohy-
poaldosteronism type 1 (9, 33, 41).
ENaC can be regulated either by altering the amount of time
the channel spends open (open probability or P
o
) or by altering
the density of functional channels (N) in the apical membrane
of distal nephron epithelial cells. One signaling molecule that
appears to alter ENaC activity is protein kinase C (PKC).
Activation of PKC with phorbol esters reduces ENaC activity
in the apical membrane of A6 cells, an amphibian renal cell
line, and in rat principal cells (15). In contrast to the inhibitory
effect on ENaC due to activating PKC, pharmacologically
inhibiting PKC increases ENaC P
o
(23, 49). A6 cells, on which
many of the experiments described above were performed,
contain several different PKC isoforms; so that it is difficult to
determine which isoform is responsible for the changes in
ENaC activity after stimulation or inhibition of all the PKC
isoforms. There are conflicting reports in the literature about
which isoforms of PKC are present in principal cells of the
mouse cortical collecting duct. One report suggested that, in
mice, there was no PKC present in principal cells (29); subse-
quent work suggested that PKC␣was present, but no other
typical or novel isoforms (22). Therefore, we made use of
PKC␣knockout mice to examine PKC regulation of ENaC in
isolated split-open collecting duct principal cells.
METHODS
Animals. PKC␣
⫺/⫺
mice were initially graciously obtained from
the laboratory of Jeffery Molkentin at the University of Cincinnati
(17) and maintained in house. Control mice were developed from
backcrossing PKC␣
⫺/⫺
mice to SV129 controls 10 times to obtain
littermate wild-type animals. Once established, the wild-type control
line was maintained and used as controls for the PKC␣knockouts.
Mice were kept on a 12:12-h light-dark cycle and fed standard
laboratory chow and tap water ad libitum. Mice fed a high-salt diet
were fed standard laboratory chow containing 8% NaCl in place of
standard chow ad libitum for 14 –21 days before euthanasia. All of our
animal protocols and procedures in this paper were approved by the
Emory Institutional Animal Care and Use Committee.
Blood pressure measurements. Systolic blood pressure and heart
rate were measured by tail cuff as previously described (43). Blood
pressures were measured for 5 consecutive days before and during
weeks 1 and 2of a high-salt diet. Data from the first 2 days of each
cycle were discarded as this was considered a transition period in
which the mice become accustomed to the procedure. Between mea-
surement times, mice were allowed to rest for 2 days to avoid
extraordinarily high stress levels. Blood pressures were measured on
a warmed platform (BP-2000, Visitech Systems), and mice were
allowed to rest on the platform for 15 min before measurement. Five
preliminary measurements were made and discarded to accustom
mice to the procedure. Blood pressures and heart rates are an average
of 10 measurements each day.
Address for reprint requests and other correspondence: D. C. Eaton, Emory
Univ. School of Medicine, Dept. of Physiology, Whitehead Biomedical
Research Bldg., 615 Michael St., Atlanta, GA 30322 (e-mail: deaton
@emory.edu).
Am J Physiol Renal Physiol 306: F309–F320, 2014.
First published December 11, 2013; doi:10.1152/ajprenal.00519.2013.
1931-857X/14 Copyright ©2014 the American Physiological Societyhttp://www.ajprenal.org F309
Downloaded from www.physiology.org/journal/ajprenal (191.101.215.042) on July 22, 2019.
SDS-PAGE and immunoblotting. Freshly isolated kidneys were
minced and washed once with PBS and then homogenized using an
Omni TH homogenizer (Warrenton, VA) in tissue protein extraction
reagent (TPER; Thermo Scientific), both solutions containing protease
and phosphatase inhibitors (Thermo Scientific). Tissue lysates were
centrifuged at 1,000 rpm at 4°C for 10 min to remove debris. The
supernatant was then centrifuged at 18,000 gfor6htosediment a
total membrane fraction; this pellet was suspended in a 150-l lysis
buffer. Protein concentration was calculated for cell and tissue lysates
using the BCA protein assay (Thermo Scientific). Forty micrograms
of total protein prepared in Laemmli sample buffer (Bio-Rad, Hercu-
les, CA) was loaded and resolved on Bio-Rad Any KD gradient gels
using the Criterion or Protean electrophoresis systems (Bio-Rad). The
separated proteins were electrically transferred onto Immobilon-P
transfer membranes (Millipore, Billerica, MA). The membranes were
blocked in 5% wt/vol milk in TBST (Bio-Rad) at room temperature
for 1 h. The membranes were washed once with TBST and then
incubated with primary antibodies at a dilution of 1:1,000 in 5%
wt/vol milk in TBST at 4°C for 8 h. The membranes were washed
three times with TBST for 5-min intervals before being incubated
with horseradish peroxidase-conjugated goat anti-rabbit secondary
antibody at a dilution of 1:5,000 in blocking solution. The membranes
were incubated with SuperSignal Dura Chemiluminescent Substrate
for 5 min before being developed using a Kodak Gel Logic 2200
Imager and Molecular Imaging software (Carestream Health, Roch-
ester, NY). This method was used to detect ENaC subunits (with
in-house antibodies) (1, 4, 8, 40, 46), ERK1/2 (9102, Cell Signaling)
and phosphoERK1/2 (9101a, Cell Signaling). PKC isoforms were
detected with antibodies obtained from Cell Signaling, (In particular,
PKC␣was detected with Cell Signaling no. 9375.)
Antibody production. Restricted segments of the ␣(H554-N643)-
and -C terminus (D566-N647) were subcloned into the pGEX
expression vector. A segment of the ␣-extracellular domain
250
KIGFQ....SNLWMS
347
from a rat was subcloned into a
maltose-binding-protein vector. The constructs were transformed
into competent bacterial cells, induced with IPTG for expression,
and batch purified from inclusion bodies using glutathione Sephar-
ose 4B (2, 3) or an amylose column. A peptide corresponding to
599
CVDNPI...RIQSAF
647
from the Xenopus ␥-subunit was synthe-
sized. The subunit-specific antibodies were raised in rabbits against a
synthetic peptide sequence or fusion proteins described above. Poly-
clonal antibodies against the carboxy terminal domain of ␣-ENaC
(ENaC 59) and -ENaC (ENaC 60) and the extracellular domain of ␣
(890)- and the C-terminal domain of ␥(2102) were generated in
White New Zealand rabbits by Bio-Synthesis (Lewisville, TX). Each
batch of serum was supplemented with sodium azide and evaluated
for specificity and cross-reactivity using protein from the wheat germ
in vitro translation system (Promega) and mouse renal tissue lysates.
Single-channel patch clamp. Renal tubules were manually dis-
sected, and the cortical collecting duct was identified by morphology.
Tubules were placed in physiological saline [(in mM) 140 NaCl, 5
KCl, 1 CaCl
2
, and 10 HEPES adjusted to pH 7.4 with NaOH] in a
plastic dish before being split open to reveal the apical surface of the
cells before single-channel patch clamp as previously described for
patch clamp of cells in culture (6, 40, 45, 46, 49). Briefly, a micro-
electrode was filled with physiological buffer solution in which
lithium was substituted for sodium (in mM: 140 LiCl, 5 KCl, 1 CaCl
2
,
and 10 HEPES adjusted to pH 7.4 with NaOH) and lowered to a single
cell before application of a small amount of suction to achieve a ⬎1
G⍀seal. ENaC were identified by characteristic channel kinetics
(long mean open and closed times ⬎0.5 s) and the current-voltage
relationship of the channel (unit conductance close to 6 pS and a very
positive, ⬎40 mv, reversal potential).
Measurement of plasma aldosterone. Blood samples were taken
from anesthetized mice. Blood samples were extracted with 4⫻
volume of methylene chloride three times before evaporating the
solvent under dry nitrogen. The extracted samples were then prepared
for ELISA according to the manufacturer’s instructions (aldosterone
EIA kit- monoclonal, Cayman Biologicals). Plasma aldosterone was
calculated by comparison with a standard curve prepared from known
concentrations of aldosterone.
In situ biotinylation. For in situ biotinylation, a protocol established
by Frindt and Palmer (12, 14) for biotinylation in rats was modified
for use in mice. Mice were anesthetized by injection of 40 –50 mg/kg
pentobarbital sodium (ip). The abdominal cavity was opened to the
diaphragm, and a butterfly needle was inserted into the abdominal
aorta at the bifurcation of the iliac arteries. The aorta was tied above
the level of the renal arteries, and the left renal vein was cut to allow
exit of perfusate. The mouse was then moved to an ice bath for cold
perfusion. Both kidneys were perfused with PBS for 10 min, after
which the left renal artery and vein were tied and the left kidney was
removed to serve as a nonbiotinylated control. The right renal vein
was then cut, and the right kidney was perfused with PBS containing
0.5 mg/ml sulfosuccinimidyl-2-[biotinamido]ethyl-1,3-dithiopropi-
onate (Pierce) for 15 min, after which a biotin-quenching solution
(PBS in which 25 mM Tris·HCl replaced 25 mM NaCl) was perfused
for 25 min to remove excess biotin. Whole kidneys were homoge-
nized, and protein was extracted in buffer containing 250 mM sucrose
and 10 mM triethanolamine (pH 7.4). The supernatant was then
centrifuged at 18,000 gfor6hat4°Ctosediment a total membrane
fraction; this pellet was suspended in a 150-l lysis buffer. The
membrane fraction was equally loaded on streptavidin beads (high-
capacity neutravadin-agarose resin, Thermo Scientific) and incubated
overnight. Beads were washed with a solution of 150 mM NaCl, 5
mM EDTA, 50 mM Tris, and 1% Triton X-100 (pH 7.4) three times
followed by a high-salt wash (wash buffer with 500 mM NaCl) and
two no-salt washes (10 mM Tris, pH 7.4) to remove unbound protein.
Bound protein was eluted in sample buffer containing 0.5 M DTT.
Biotinylated and unbiotinylated membrane fractions were run on
Bio-Rad Any KD gradient gels and probed with in-house antibodies to
␣-, -, or ␥-ENaC.
Fluorescent immunohistochemistry. Fluorescent immunohistochem-
istry was performed as previously described (43, 45). Briefly, kidneys
were perfused in situ with PBS followed by 2% paraformaldehyde.
Kidneys were removed, put in 20% sucrose solution at 4°C overnight,
dehydrated, and embedded in paraffin wax. Four-micrometer sections
were made, and slices were rehydrated before addition of in-house
ENaC or aquaporin-2 (AQP2) antibodies or commercially available
antibodies against PKC␣(SC-8393, Santa Cruz Biotechnology). An-
tibodies were detected using an appropriate fluorescently conjugated
secondary antibody (Molecular Probes).
Colocalization analysis. To determine whether ENaC subunits
colocalize more strongly with AQP2 in knockout mice compared with
wild-type, kidney slices from wild-type and knockout mice were
stained with rabbit primary antibodies to ␣-, -, or ␥-ENaC and goat
anti-AQP2. Following treatment with fluorescent secondary antibod-
ies, ENaC (green) and AQP2 (red) were examined using confocal
microscopy on an Olympus Fluoview 1000 confocal microscope. To
determine colocalization, we first determined that there was no fluo-
rescence bleed through from the green channel to the red channel or
vice versa. Then we merged images from the green channel and the
red channel; yellow in the merged image was an indication of
colocalization. To produce a more quantitative measure of the colo-
calization, we used the colocalization algorithms in the image analysis
program ImageJ (11, 28, 35). These algorithms examine the merged
images pixel by pixel for red and green intensities and establish
thresholds for intensity below which there is no significant correlation
of red and green. This represents an unbiased method for determining
thresholds. Pixels that have both red and green intensities above the
thresholds are analyzed and recolored white. In addition, the number
and fraction of pixels with red-green colocalization were calculated
and red-green and green-red correlation coefficients were calculated
(Manders coefficients, m
1
and m
2
) according to Eq. 1
F310 PKC␣REGULATES ENaC
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m1⫽
兺is1i·s2i
兺i(s1i)2m2⫽
兺is1i·s2i
兺i(s2i)2(1)
where s1
i
and s2
i
are the red and green intensities of the ith pixel in
the image. Only pixels with significant intensities in both channels
will contribute to the coefficient.
Data analysis and statistics. All data acquisition and analysis were
performed as described previously (45, 47). Data are reported as
means ⫾SE. Statistical analysis was performed with SigmaPlot and
SigmaStat software (Jandel Scientific). Differences between groups
were evaluated with one-way ANOVA or for blood pressure with
two-way ANOVA, and results were considered significant if P⬍
0.05.
We used the FIJI variant of the image-analysis program ImageJ to
analyze changes in Western blots. ImageJ calculates the cumulative
sum of pixel values above baseline for specific bands in a blot. We
averaged these cumulative pixel values for at least three experiments
to determine whether there was a significant difference in band
densities.
RESULTS
Channels in isolated, split-open tubules. Figure 1Ashows an
isolated tubule before (top) and after one end of the tubule from
a wild-type SV-129 mouse kidney was split open. A patch
electrode on a principal cell is visible in the bottom left of the
image. Principal cells were identified by their characteristic
morphology in the split-open tubule. Specifically, in Fig. 1A,
principal cells appear in the Hoffman modulation image to be
large, polygonal, or round cells with concave surfaces; inter-
calated cells have asymmetric shapes with convex, but convo-
luted surfaces. Figure 1Bshows single-channel records from
the patch electrode on the cell in Fig. 1A. The currents are
inward with long mean open and mean closed times charac-
teristic of ENaC. Figure 1Cshows the current-voltage relation-
ship for the channel in Fig. 1B. The inward rectification and the
highly positive reversal potential are also characteristic of
ENaC. The conductance of the channel between ⫺100 and 0 mV
was 13.1 ⫾1.43 pS, similar to that reported in rat connecting
tubules by Frindt and Palmer (13).
PKC
␣
knockout mice. We used mice in which PKC␣is
globally knocked out to study the effect of PKC␣on ENaC
activity in renal principal cells. Western blotting could not
detect PKC␣in kidney lysates from knockout mice (Fig. 2,
left). Immunohistochemistry showed that SV-129 wild-type
mice ubiquitously express PKC␣in the kidney including in
AQP2 (a marker for cortical collecting duct principal cells)-
positive cells (Fig. 2, top right). As expected, the knockout
mice have no detectable PKC␣, and, in particular, have none in
AQP2-positive principal cells (Fig. 2, bottom right). In data not
shown, we blotted renal cortical lysates for at least one isoform
from each of the major PKC types: typical, novel, and atypical.
As expected, we detected low levels of some other PKC
isoforms; but, except for PKC␣, there was no difference in the
isoforms between wild-type and knockout kidneys, implying
that there was no compensation of other PKC isoforms for the
loss of PKC␣.
ENaC activity in PKC
␣
knockout mice on a normal-salt diet.
ENaC activity was recorded from cell-attached patches on
principal cells (as in Fig. 1) from wild-type mice or from
PKC␣knockout mice fed a normal mouse diet (Fig. 3A). The
top two traces are long representative records from tubular
cells from wild-type or knockout tubules. ENaC activity in the
knockout cells is substantially increased above that in the wild-
type. The regions marked 1 and 2 in the top records are
expanded in the two bottom traces, which also show that the
activity of ENaC in knockout mice is substantially higher than
in wild-type mice. Figure 3, B–D, summarizes the results from
patches on a large number of tubules from both wild-type and
knockout mice. Wild-type data are from 31 individual patches;
knockout data are from 21 individual patches. The patches
were from 21 cortical collecting ducts isolated from wild-type
-Vp(mV)
-120
-100
-80
-60
-40
-20
3sec
2pA
BA
C
Voltage (-Vpipette mV)
-120 -80 -40 40
Current (pA) -2.5
-2.0
-1.5
-1.0
-0.5
0
0
20
3sec
0.2pA
Fig. 1. Channels in isolated, split-open tubules.
A: an isolated tubule before (top) and after
splitting open of one end of a tubule from a
wild-type SV-129 mouse kidney. A patch elec-
trode on a principal cell is visible in the bottom
left of the image. Principal cells were identified
by their characteristic morphology. B: single-
channel records from the patch electrode on the
cell in A. The currents are inward with long
mean open and mean closed times characteristic
of epithelial sodium channels (ENaC). Note that
the vertical scale has been expanded 10-fold for
the bottom 2 traces to more easily show the small
unitary currents at these potentials. C: current-
voltage relationship for the channel in B. The
inward rectification and the very positive reversal
potential are also characteristic of ENaC. The line
through the data is the best nonlinear least-squares
fit to the Goldman-Hodgkin-Katz equation and
predicts that intracellular sodium is 12 ⫾6.6 mM
and principal cell sodium permeability is (6.4 ⫾
3.38) ⫻10
⫺7
cm/s. The conductance of the chan-
nel between ⫺100 and 0 mV was 13.1 ⫾1.43 pS,
similar to that reported in rat connecting tubule by
Frindt and Palmer (13).
F311PKC␣REGULATES ENaC
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and 18 cortical collecting ducts from PKC␣knockout mice.
The cortical collecting ducts were obtained from 14 wild-type
mice and 10 PKC␣knockout mice. ENaC activity, measured as
NP
o
, the product of the number of channels (N) times P
o
,is
increased more than threefold in knockout tubules compared
with wild-type (Fig. 3B). When we examined the individual
components of activity, we found that P
o
more than doubled
(Fig. 3C). Given prior reports describing the effects of PKC
inhibitors, this increase in P
o
was not too surprising. What was
somewhat surprising to us was that the number of channels per
PKCα,
,
green, AQP2, red
Wild Type
PKCαKO
PKC WT
98
64
PKC KO
98
64
Fig. 2. PKC␣knockout mice. Left: Western blots of renal cortical lysates from wild-type (WT) and PKC␣knockout (KO) mice showing that the knockout mice
have no PKC␣. The expected molecular weight is 80 –82 KDa. Twenty-five micrograms of lysate were loaded per well. Right: immunohistochemistry shows
that SV-129 WT mice ubiquitously express PKC␣in the kidney including in aquaporin-2 (AQP2; a marker for cortical collecting duct principal cells)-positive
cells (top). The bottom panels show mice in which PKC␣is globally knocked out. As expected, the KO mice have no detectable PKC␣, and, in particular, have
none in AQP2-positive principal cells. Scale bars (red lines) ⫽5m in all panels.
PKCα
α
KO mice
Po
0.0
0.2
0.4
0.6
0.8
Wildtype
Mice
**
0.0
0.5
1.0
1.5
NPo
(Channel activity)
Wildtype
Mice
**
PKC
α
KO mice
0
1
2
3
N (channel density)
Wildtype
Mice
**
PKC
α
KO mice
B
CD
A
1
2
1
2
Fig. 3. ENaC activity in PKC␣KO mice.
ENaC activity was recorded from cell-at-
tached patches on principal cells (as in Fig. 1)
from WT mice or PKC␣KO mice. A:top 2
traces are long representative records from
WT or KO cells (pipette potential is ⫹60
mv). The activity of the KO cell is substan-
tially increased above that of WT. The regions
marked 1 and 2 in the top traces are expanded
10-fold in the bottom traces to emphasize the
difference in the activity of WT and KO cells.
All recordings were made at ⫺60 mV (differ-
ence in potential between the inside of the cell
and the patch pipette. If there is a significant
basal membrane potential, it will add to the
pipette potential). B–D: summary of all single-
channel data. The graph in Bshows that ENaC
activity [NP
o
; measured as the number of
channels (N) times the open probability (P
o
)]
increases over 3-fold in the PKC␣KO mice
compared with WT (P⬍0.001). When the
components of activity are examined individ-
ually, both P
o
(C) and N(D) increase signif-
icantly (P⬍0.01). WT data are from 31
individual patches; KO data are from 21
individual patches. The patches were from 21
cortical collecting ducts isolated from WT
and 18 cortical collecting ducts from PKC␣
KO. The cortical collecting ducts were ob-
tained from 14 WT mice and 10 PKC␣KO
mice.
F312 PKC␣REGULATES ENaC
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patch also increased substantially (Fig. 3D). Therefore, we
decided to more carefully examine this increase in N.
Knocking out PKC
␣
increases the density of ENaC in or
near the apical membrane of principal cells. An increase in the
number of active channels in the apical membrane could be due
to recruitment of new channels to the apical membrane or the
conversion of otherwise silent channels to active channels. In
the latter case, the number of channels in or near the apical
membrane would not change; in the former case, there should
be more ENaC near the apical membrane of principal cells. To
examine which possibility seemed more likely, we used im-
munohistochemistry and confocal microscopy of kidney slices
to examine the number and localization of ENaC subunits in
the apical membranes of AQP2-positive cells. Figure 4 shows
that, in knockout compared with wild-type animals, ␣-ENaC is
more strongly colocalized with AQP2 (indicated in yellow) in,
or very near, the apical membranes of cells that we presume are
principal cells because of the AQP2 staining. Figures 5 and 6
αENaC, AQP2
Wild Type
PKCαKO
Fig. 4. Membrane ␣-ENaC is increased in the prin-
cipal cells of PKC␣knockout mice. We prepared
kidney slices from WT and KO before fixing and
treating with AQP2 and ␣-ENaC antibodies. Subse-
quently, we used secondary antibodies that labeled
AQP2 with a ds-Red monomer and ␣-ENaC with
enhanced green fluorescent protein. Left: 4 panels
are, from bottom right, differential interference con-
trast images, AQP2 in red, merged image, and
␣-ENaC in green. Right: expanded images from the
areas outlined in white in the merged images on the
left.␣-ENaC appears to more strongly colocalize
(yellow) with AQP2 in the principal cells from
PKC␣KO mice than in WT mice. Scale bars (red
lines) ⫽5m in all panels.
βENaC, AQP2
Wild Type
PKCαKO
Fig. 5. Membrane -ENaC is increased in the
principal cells of PKC␣KO mice. We prepared
kidney slices from WT and KO before fixing and
treating with AQP2 and -ENaC antibodies. Sub-
sequently, we used secondary antibodies that la-
beled AQP2 with a ds-Red monomer and -ENaC
with enhanced green fluorescent protein. Left:4
panels are, from bottom right, differential interfer-
ence contrast images, AQP2 in red, merged image,
and -ENaC in green. Right: expanded images
from the areas outlined in white in the merged
images on the left.-ENaC appears to more
strongly colocalize (yellow) with AQP2 in the prin-
cipal cells from PKC␣KO mice than in WT mice.
Scale bars (red lines) ⫽5m in all panels.
F313PKC␣REGULATES ENaC
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show that - and ␥-ENaC are also more strongly associated
with the apical membrane and AQP2 in principal cells from
knockout animals.
Although the yellow color of the merged images in Figs.
4 – 6 suggests that there is more colocalization of ENaC and
AQP2 in the knockout mice, we wanted a more quantifiable
measure of colocalization. Therefore, we used the image-
analysis program ImageJ to quantify colocalization (as de-
scribed in METHODS). Figure 7 shows, for slices from wild-type
and knockout mice, the merged images for AQP2 and each
ENaC subunit; to the right of the merged images are merged
images in which pixels that have significant red and green
intensities have been recolored white. There is more colocal-
ization in the knockout mice than in wild-type. Table 1 gives
actual values for the red-green and green-red overlap and the
number and percentage of pixels with significant overlap.
Association of the red channel (AQP2) with the green channel
(ENaC subunits) was calculated using the colocalization plugin
of ImageJ. The last two columns show that ENaC is signifi-
cantly more likely to be associated with ENaC in the knockout
mice compared with wild-type (P⬍0.001 for all subunits).
Examination of the red-green and green-red localization shows
that AQP2 is almost always associated with an ENaC subunit,
but that ENaC subunits (particularly in wild-type mice) are less
likely to be associated with AQP2. We interpret these results to
imply that ENaC moves in knockout mice to regions of the cell
near AQP2, i.e., near the apical membrane. The percent values
for knockout vs. wild-type are significantly different (P⬍
0.001 for each subunit by z-test).
Although confocal microscopy makes it appear possible that
ENaC is concentrated in the apical membranes of principal
cells in knockout animals, we could not rule out the possibility
that ENaC subunits were very close to the inner surface of the
apical membranes, but not actually in the membrane as parts of
active channels. Therefore, we adapted an approach previously
used in rats (12, 14) to mice in which we perfused one kidney
with biotin to label proteins on the luminal surface of the
tubules and confirm that ENaC is actually in the apical mem-
brane (Fig. 8A). After precipitating biotin-labeled proteins
from kidney lysates, we resolved the avidin precipitates and
blotted for the three ENaC subunits (Fig. 8B). The quantity of
each of the subunits was increased in the PKC␣knockout
mice, confirming that indeed ENaC was increased in the apical
membranes of principal cells as we had shown in the confocal
images (Fig. 8C).
Plasma aldosterone in PKC
␣
knockout mice is not signifi-
cantly different from wild-type mice. The increase in P
o
and
channel density could be explained if plasma aldosterone levels
were substantially elevated in PKC␣knockout mice. There-
fore, we took blood samples from 24 mice (10 wild-type and 7
knockout animals on a normal diet; 3 wild-type and 4 knockout
on a high-salt diet). After sample preparation, plasma aldoste-
rone was determined using a commercially available enzyme-
linked immunoassay kit. As we anticipated, because a normal
mouse diet contains moderate amounts of salt and a high-salt
diet even more, aldosterone levels were low but not signifi-
cantly different in wild-type or knockout mice [normal diet
wild-type ⫽0.57 ⫾0.14 nM (n⫽10) vs. knockout ⫽0.76 ⫾
0.20 nM (n⫽7); high-salt diet wild-type ⫽0.54 ⫾0.24 nM
(n⫽3) vs. knockout ⫽0.33 ⫾0.21 nM (n⫽4; means ⫾SE)].
Therefore, differences in aldosterone concentration cannot ex-
plain the increase in ENaC activity in PKCa knockout mice.
ERK1/2 activity is reduced in PKC
␣
knockout mice. ENaC
density in the apical membrane is controlled by the activity of
the ubiquitin ligase Nedd4-2 (39, 53). The ability of Nedd4-2
to ubiquitinate ENaC and promote internalization is augmented
by PKC-mediated ERK1/2 phosphorylation of ENaC (7).
Therefore, we examined ERK1/2 phosphorylation as a measure
of ERK1/2 activity in wild-type and PKC␣knockout mice. We
found that the ERK1/2 amount was unchanged, but phosphor-
ylation was significantly reduced in knockout mice (Fig. 9).
γ
γ
ENaC, AQP2
Wild Type
PKCαKO
Fig. 6. Membrane ␥-ENaC is increased in the
principal cells of PKC␣knockout mice. We pre-
pared kidney slices from WT and KO before fixing
and treating with AQP2 and ␥-ENaC antibodies.
Subsequently, we used secondary antibodies that
labeled AQP2 with a ds-Red monomer and ␥-
ENaC with enhanced green fluorescent protein.
Left: 4 panels are, from bottom right, differential
interference contrast images, AQP2 in red, merged
image, and ␥-ENaC in green. Right: expanded im-
ages from the areas outlined in white in the merged
images on the left.␥-ENaC appears to more
strongly colocalize (yellow) with AQP2 in the prin-
cipal cells from PKC␣KO mice than in WT mice.
Scale bars (red lines) ⫽5m in all panels.
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This presumably explains the increased channel density of
ENaC in the apical membrane of principal cells.
Blood pressure is increased in PKC
␣
knockout mice. If the
increase in ENaC activity in knockout mice is accompanied by
increased distal nephron sodium transport, then one might
expect that the blood pressure of knockout mice would be
elevated above that in wild-type mice. In particular, challeng-
ing the mice with a high-sodium diet should increase blood
pressure. Figure 10 shows that when blood pressure was
measured by tail cuff (as described in METHODS), wild-type
mice challenged with a high-salt diet (8% NaCl) for 2 wk had
little if any change in blood pressure while blood pressure in
knockout mice increased dramatically.
ENaC activity in PKC
␣
knockout mice on a high-salt diet.
ENaC activity was recorded from cell-attached patches on
principal cells (as in Figs. 1 and 3) from wild-type mice or
from PKC␣knockout mice fed a high-salt diet (Fig. 11A). The
top two traces are long representative records from tubular
cells from wild-type or knockout tubules. ENaC activity in the
knockout cells is substantially increased above that in the
wild-type. Figure 11, B–D, summarizes the results from
patches on a large number of tubules from both wild-type and
knockout mice. Wild-type data are from 33 individual patches;
knockout data are from 42 individual patches. The patches
were from 11 cortical collecting ducts isolated from WT and 15
cortical collecting ducts from PKC␣KO. The cortical collect-
α
β
γ
merged co-localized overlay merged co-localized overlay
Green = ENaC Red = AQP2 White = Co-localized ENaC and AQP2
Fig. 7. ENaC subunits are in closer association with AQP2 in principal cells of KO animals than in WT animals. Kidney slices were stained with rabbit anti-ENaC
subunit antibodies and goat anti-AQP2 antibody. Following treatment with appropriate fluorescent secondary antibodies, ENaC subunits (green) and AQP2 (red)
were examined by confocal microscopy using an Olympus FV-1000 confocal microscope. The images in the 2 left columns are images from WT animals. The
left image of the pair is a composite merged image of the red and green channels. From top to bottom are images for each of the 3 ENaC subunits. The yellow
pixels in the composite image show the close association of an ENaC subunit with AQP2. The second column on the left is an image of the same slice as the
merged image that was analyzed for colocalization of red and green pixels using a quantitative algorithm (colocalization threshold plugin in the ImageJ program;
see METHODS). Pixels that have an intensity for both green and red above the green and red thresholds represent colocalization and are recolored in white. The
other areas of the image represent a traditional merge of the green and red channels. Yellow areas may represent additional colocalization, but the intensities
in the red and green channels are lower than the white highlighted pixels. Two right columns are the merged image and colocalized image for each of the 3 ENaC
subunits for KO mice, respectively. There are significantly more white pixels in slices from KO animals than in WT animals (P⬍0.001 by z-test; see Table1).
Scale bars ⫽5m in all panels.
Table 1. Colocalization of ENaC subunits with AQP2 in wild-type and knockout mice
Images AQP2 with ENaC ENaC with AQP2 Number of Colocalized Pixels % Colocalized pixels
Manders coefficients
␣Knockout AQP2 vs. ␣⫺ENaC 0.915 0.849 6,673 15.6%
WT AQP2 vs. ␣⫺ENaC 0.951 0.568 3,196 7.66%
Knockout AQP2 vs. ⫺ENaC 0.923 0.801 4,162 9.88%
WT AQP2 vs. ⫺ENaC 0.896 0.668 1,099 2.61%
␥Knockout AQP2 vs. ␥⫺ENaC 0.955 0.802 7,197 13.6%
WT AQP2 vs. ␥⫺ENaC 0.910 0.773 1,345 2.58%
ENaC, epithelial Na channel; AQP2, aquaporin-2; WT, wild-type.
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ing ducts were obtained from four wild-type mice and four
PKC␣knockout mice. ENaC activity, measured as NP
o
, the
product of the number of channels (N) times P
o
, is approxi-
mately doubled from 0.170 ⫾0.0386 in wild-type to 0.327 ⫾
0.0566 in knockout (P⫽0.033) (Fig. 11B). When we exam-
ined the individual components of activity, we found that P
o
increased ⬃50% from 0.101 ⫾0.0180 to 0.152 ⫾0.0186 (P⫽
0.028) (Fig. 11C). The number of channels per patch also
increased by a small but significant amount (Fig. 11D) from
1.35 ⫾0.097 to 1.67 ⫾0.095 (P⫽0.023). Interestingly, in the
knockout but not the wild-type animals, we observed several
patches with more channels than we could easily measure
(⬎12). This implies that NP
o
,N, and P
o
might have all been
underestimated in the knockout animals.
DISCUSSION
The effect of PKC on distal nephron ENaC has been previ-
ously described in the literature (7, 15, 23, 23, 40, 49). With a
few exceptions, previous reports using cultured cells have
generally shown that PKC activity inhibits ENaC. Interest-
ingly, despite the role of PKC as a protein kinase, ENaC does
not appear to be directly phosphorylated by PKC (48, 49).
Therefore, PKC must act indirectly to phosphorylate one or
more ENaC-regulatory proteins. The purpose of this work was
to investigate which proteins might be modulated by PKC to
alter ENaC activity.
We were particularly interested in the PKC␣knockout
animals because, while there are several different renal PKC
isoforms, principal cells appear to contain mostly the ␣-iso-
form. Originally, it was reported that there were no PKC
isoforms in principal cells (29). Subsequently, it was shown
that principal cells did contain PKC␣but no other conventional
isoforms (or ␥) or novel isoforms (␦,ε,,or) (22).
However, there is evidence for at least one atypical isoform ()
(19); however, the atypical isoforms usually are associated
with regulation of nuclear gene expression so that only the
␣-isoform appears relevant to membrane signaling. Therefore,
we concentrated on PKC␣signaling mechanisms. In our work,
we also show (Fig. 2) that PKC␣is ubiquitously expressed in
the kidney of wild-type mice (including AQP2-positive prin-
Alpha
Beta
Wildtype
Mice
PKCα
α
KO mice
Wildtype
Mice
PKC
α
KO mice
αENaC
68 kDa
βENaC
95 kDa
Gamma
Wildtype
Mice
PKC
α
KO mice
γENaC
67 kDa
Descending
aorta
Perfusion cannula
ligations
0
2
4
6
8alpha
WT PKCαKO
Band Density
(in thousands)
*
0
10
20
30
Band Density
(in thousands)
gamma
WT PKCαKO
*
0
5
10
15
20
25 beta
WT PKCα KO
Band Density
(in thousands)
*
Fig. 8. In situ biotinylation of mouse kidney. A: schematic of the method. Mice were anesthetized by injection of 80 –90 mg/kg pentobarbital sodium (ip). The
abdominal cavity was opened to the diaphragm, and a butterfly needle was inserted into the abdominal aorta at the bifurcation of the iliac arteries. The aorta was
tied above the level of the renal arteries, and the left renal vein was cut to allow exit of the perfusate. Both kidneys were perfused with PBS for 5 min, after
which the left renal artery and vein were tied and the left kidney was removed to serve as a nonbiotinylated control. The right renal vein was then cut, and the
right kidney perfused with PBS containing 0.5 mg/ml sulfosuccinimidyl-2-[biotinamido]ethyl-1,3-dithiopropionate (Pierce) for 5 min, after which a biotin-
quenching solution was perfused for 25 min to remove excess biotin. B: biotinylated ENaC subunits. Whole kidneys were homogenized, and protein was
extracted. A membrane fraction was equally loaded on streptavidin beads and incubated overnight. After washing, protein was eluted with sample buffer and
resolved on gels and detected with ENaC-specific antibodies. The amount of each of the subunits was greater in the PKC␣KO mice than WT. C: quantification
of the amounts of ENaC subunits. Mean densitometric analysis is shown of 3 typical experiments for each subunit. We used ImageJ to quantify the blots. The
program calculated the cumulative sum of the pixel values above the background for specific bands. Asterisks indicate significant differences in KO density
compared with wild-type (by t-test: ␣-ENaC: P⫽0.026; -ENaC: P⫽0.046; by rank sum test: ␥-ENaC: P⫽0.029).
F316 PKC␣REGULATES ENaC
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cipal cells) but is not detectable in knockout mice, confirming
the previous work of Madsen and coworkers (22).
ENaC P
o
is increased in PKCa knockout mice. Previous
work examining the effects of PKC activation or inhibition on
single ENaC all suggested that there was an effect on channel
P
o
: PKC activation decreased P
o
; PKC inhibition increased P
o
(5, 7, 23, 24, 40, 48, 49). Therefore, it was not surprising to us
that in principal cells in which PKC was knocked out P
o
should
be increased. It was interesting, however, that knocking out
only one isoform, PKC␣, was sufficient to produce an increase
in P
o
as large as any seen in previous work using inhibitors that
inhibited all typical and most novel isoforms of PKC. How-
ever, in retrospect, given that PKC␣appears to be the only
isoform present in AQP2-positive principal cells, the signifi-
cant effect of knockout on P
o
might be expected. On the other
hand, in other sodium-transporting epithelia that express mul-
tiple PKC isoforms (such as in the lung), the effect of knocking
out only PKC␣might have much more complicated effects.
The mechanism by which P
o
is increased likely involves
phosphatidylinositol 4,5-bisphosphate (PIP
2
) interaction with
the channel. That ENaC gating depends upon PIP
2
has been
well known for a long time (20, 25–27, 30, 50, 52). Recently,
however, Alli et al. (1) have shown that the local concentration
of PIP
2
in the membrane is controlled by association with the
apical membrane of a specialized protein, myristoylated ala-
nine-rich C-kinase substrate (MARCKS; or the very similar
MARCKS-related protein). The ability of MARCKS to control
the local concentration of PIP
2
in the membrane near ENaC
depends upon the state of MARCKS phosphorylation: 1) when
dephosphorylated MARCKS associates with the membrane
and membrane PIP
2
concentrations are elevated, which leads to
increased ENaC P
o
; and 2) when phosphorylated MARCKS
leaves the membrane and enters the cytosol, which reduces
PIP
2
concentrations near ENaC, after which ENaC activity
decreases. The primary kinase that phosphorylates MARCKS
is PKC␣. Thus, in the absence of PKC␣, MARCKS remains
associated with the membrane and increases PIP
2
concentra-
tions near ENaC and ENaC P
o
increases (as we observed).
ENaC membrane density is increased in PKCa knockout
mice. Besides an increase in P
o
, we also observed an increase
in the membrane density of ENaC. While it might be possible
that increased PIP
2
in the apical membrane alone could stabi-
lize ENaC and allow an accumulation in the membrane, other
mechanisms appear to contribute. Activation of PKC with
phorbol esters is known to reduce ENaC subunit protein in the
membrane (7, 40). It is also known that phosphorylation of
ENaC by the active, phosphorylated form of ERK1/2 does
promote interaction of ENaC with the ubiquitin ligase (7)
Nedd4-2. Nedd4-2 ubiquitination promotes ENaC removal
from the membrane and internalization, reducing the overall
levels of ENaC in the membrane (21, 32). PKC can phosphor-
ylate and activate ERK1/2 (7, 10). We showed that in the
absence of PKC␣the active phosphorylated form of ERK1/2
was significantly reduced, thereby leading to reduced ENaC
internalization and to the increase in membrane ENaC we
observed.
Blood pressure is elevated in PKCa knockout mice. One
might expect that if there are substantial increases in distal
nephron sodium reabsorption, there would be a concomitant
increase in blood volume and blood pressure. One previous
study did not observe any significant difference in blood
pressure between wild-type and PKC␣knockout mice (43).
However, those experiments were done on mice fed a normal-
salt diet, which might not reveal a sodium balance problem.
Even mice with a mutation which produces constitutively
active ENaC do not have noticeably elevated blood pressure in
the absence of a high-salt challenge (31). Therefore, we fed our
mice an 8% sodium chloride diet and did observe a significant
increase in blood pressure in the knockout animals. We con-
Systolic Blood Pressure
(mm Hg)
100
120
140
160
180
Wild Type
PKC KO
*
Fig. 10. Blood pressure is increased in PKC␣knockout mice. When blood
pressure was measured by tail cuff (as described in METHODS), WT mice
challenged with a high-salt diet (8% NaCl) had little if any change in systolic
blood pressure, while blood pressure in KO mice increased significantly
(marked with asterisk; n⫽4/group). P⬍0.001 for week 2 on a high-salt for
KO vs. WT by 2-way ANOVA.
ERK1/2 phosphoERK1/2
Band Density
(in thousands)
0
10
20
30
40
Wild type
PKC-αKO
Wild type
Wild type
PKC-αKO
PKC-αKO
2/1KREohpsohp2/1KRE
*
Wild type
Wild type
PKC-αKO
PKC-αKO
α-tubulin α-tubulin
Fig. 9. Active ERK is reduced in PKC␣KO mice. We measured total ERK1/2
and phosphoERK in kidney lysates (each lane represents a separate animal).
We used ImageJ to quantify the blots using a area that included both ERK1 and
ERK2 bands. The program calculated the cumulative sum of the pixel values
above the background for specific bands. The mean values of the densitometry
results from 3 separate experiments show that total ERK is the same (t-test,
P⫽0.59), but phosphoERK is significantly reduced in KO compared with WT
mice (t test, P⬍0.001).
F317PKC␣REGULATES ENaC
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firmed using single-channel measurements that ENaC activity
in knockout animals was increased and could, therefore, ac-
count for the high blood pressure.
Figure 12 shows a schematic diagram of PKC signaling in
wild-type and knockout mice. The situation in wild-type mice
is shown in Fig. 12A. PKC␣is active and phosphorylates
MARCKS protein. When phosphorylated, MARCKS leaves
the membrane and does not sequester and present PIP
2
to
ENaC, causing ENaC P
o
to decrease. Active PKC also phos-
phorylates ERK, which in turn phosphorylates ENaC. ERK
phosphorylation of ENaC promotes Nedd4-2 interaction with
and ubiquitination of ENaC, with subsequent internalization.
This reduces the apical density of ENaC. The situation in
PKC␣knockout mice is shown in Fig. 12B. PKC is absent, and
MARCKS protein is not phosphorylated. Therefore, MARCKS
remains associated with the membrane and presents PIP
2
to
ENaC to increase ENaC P
o
. ERK is also not phosphorylated so
that ENaC internalization is reduced and apical membrane
ENaC is increased.
A recent genome-wide association study (44) found that
polymorphic changes in only a very limited number of genes
were associated with increases in blood pressure. The strongest
linkage involved polymorphisms in the gene for PKC␣. The
study provides no information about the specific effects that the
polymorphisms have on PKC activity. We speculate based on
our results that the polymorphisms lead to decreases in PKC
activity. We will be interested in expressing specific PKC
mutations in heterologous expression systems that also express
ENaC to determine the effect of the polymorphisms on ENaC
activity.
Our work has shown an important role for PKC␣in the
regulation of ENaC with a potential for significant hyperten-
sion in animals or patients with reduced PKC activity. One
therapeutic agent in common use, rapamycin, is a PKC inhib-
PKCα
α
KO mice
Po
Wildtype
Mice
N (channel density)
Wildtype
Mice
PKC
α
KO mice
*
B
CD
A
0.0
0.2
0.4
NPo
(Channel activity)
Wildtype
Mice
PKC
α
KO mice
0.00
0.05
0.10
0.15
0.20
0.0
0.4
0.8
1.2
1.6
2.0
**
C
-Vp = -60mv
PKC-
α
knock out mice
Control mice
C
10sec
1pS
High salt 2 weeks
Fig. 11. ENaC activity from tubules in high-salt
diet mice. ENaC activity was recorded from cell-
attached patches on principal cells (as in Figs. 1
and 3) from WT mice or PKC␣KO mice. A:top
trace is a representative record from WT, and
bottom trace from a KO cell. The activity of the
KO cell is substantially increased above that of
the WT. All recordings were made at ⫺60 mV
(difference in potential between the inside of the
cell and the patch pipette. If there is a significant
basal membrane potential, it will add to the pi-
pette potential). B–D: summary of all single-
channel data The graph in Bshows that ENaC
NP
o
[measured as the number of channels (N)
times the P
o
] increases ⬃50% in the PKC␣KO
mice compared with WT (P⫽0.033). When the
components of activity are examined individually,
both P
o
(C) and N(D) increase significantly (P⬍
0.03). WT data are from 33 individual patches; KO
data are from 42 individual patches. The patches
were from 11 cortical collecting ducts isolated from
WT and 15 cortical collecting ducts from PKC␣
KO. The cortical collecting ducts were obtained
from 4 WT mice and 4 PKC␣KO mice.
β
βγ
α
PKC
Nedd4-2
ENaC
Internalization
(-)
PIP
2
ERK1/2
(+)
(+)
MARCKS
P
P
P
βγ
α
PKC
Nedd4-2
ENaC
Internalization
PIP
2
ERK1/2
MARCKS
Fig. 12. Schematic diagram of PKC signaling in
WT and KO mice. A: situation in WT mice. PKC␣
is active and phosphorylates myristoylated alanine-
rich C-kinase substrate (MARCKS) protein. When
phosphorylated, MARCKS leaves the membrane
and does not sequester and present phosphatidyl-
inositol 4,5-bisphosphate (PIP
2
) to ENaC, causing
a decrease in ENaC P
o
. Active PKC also phosphor-
ylates ERK, which in turn phosphorylates ENaC.
ERK phosphorylation of ENaC promotes Nedd4-2
interaction with and ubiquitination of ENaC, with
subsequent internalization. This reduces the apical
density of ENaC. B: situation in PKC␣KO mice.
PKC is absent, and MARCKS protein is not phos-
phorylated. Therefore, MARCKS remains associ-
ated with the membrane and presents PIP
2
to
ENaC to increase ENaC P
o
. ERK is also not
phosphorylated so that ENaC internalization is
reduced and apical membrane ENaC is increased.
F318 PKC␣REGULATES ENaC
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itor (49) and does commonly induce hypertension (42). Given
the examination of PKC inhibitors for a variety of clinical
disorders (36, 38, 51), it seems appropriate to understand the
mechanisms by which the inhibitors can produce excessive
renal sodium transport and hypertension.
GRANTS
This work was supported by National Institutes of Health Grants R37
DK037963 to D. C. Eaton, R01 DK89828 to J. M. Sands, R01-DK067110 to
H.-P. Ma, and T32 DK07656 and AHA 13POST16820072 to T. L. Thai.
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by the authors.
AUTHOR CONTRIBUTIONS
Author contributions: H.-F.B., H.-P.M., H.C., J.D.K., J.M.S., and D.C.E.
provided conception and design of research; H.-F.B., T.L.T., and Q.Y. per-
formed experiments; H.-F.B., T.L.T., Q.Y., A.F.E., and D.C.E. analyzed data;
H.-F.B., T.L.T., H.-P.M., J.D.K., J.M.S., and D.C.E. interpreted results of
experiments; H.-F.B., T.L.T., A.F.E., and D.C.E. prepared figures; H.-F.B.,
T.L.T., H.-P.M., A.F.E., H.C., J.D.K., J.M.S., and D.C.E. edited and revised
manuscript; H.-F.B., T.L.T., Q.Y., H.-P.M., A.F.E., H.C., J.D.K., J.M.S., and
D.C.E. approved final version of manuscript; D.C.E. drafted manuscript.
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