Kinase regulation of hENaC mediated through a region in the
COOH-terminal portion of the
-subunit
Kenneth A.
Volk,
Russell F.
Husted,
Peter M.
Snyder, and
John B.
Stokes
Department of Internal Medicine, University of Iowa and
Department of Veterans Affairs Medical Center, Iowa City, Iowa 52242
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ABSTRACT |
In an effort to gain insight into how
kinases might regulate epithelial Na+ channel (ENaC)
activity, we expressed human ENaC (hENaC) in Xenopus oocytes
and examined the effect of agents that modulate the activity of some
kinases. Activation of protein kinase C (PKC) by phorbol ester
increased the activity of ENaC, but only in oocytes with a baseline
current of <2,000 nA. Inhibitors of protein kinases produced varying
effects. Chelerythrine, an inhibitor of PKC, produced a significant
inhibition of ENaC current, but calphostin C, another PKC inhibitor,
had no effect. The PKA/protein kinase G inhibitor H-8 had no effect,
whereas the p38 mitogen-activated protein kinase inhibitor, SB-203580
had a significant inhibitory effect. Staurosporine, a nonspecific
kinase inhibitor, was the most potent tested. It inhibited ENaC
currents in both oocytes and in M-1 cells, a model for the collecting
duct. Site-directed mutagenesis revealed that the staurosporine effect
did not require an intact COOH terminus of either the
- or
-hENaC
subunit. However, an intact COOH terminus of the
-subunit was
required for this effect. These results suggest that an integrated
kinase network regulates ENaC activity through an action that requires
a portion of the
-subunit.
epithelial sodium channel; protein kinase C; staurosporine; mutation; heterologous expression; M-1 cells; oocyte
expression
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INTRODUCTION |
THE EPITHELIAL SODIUM CHANNEL (ENaC) resides in the
apical membrane of Na+-transporting epithelia in the distal
nephron, distal colon, lung, and other epithelia (14). In its fully
functional state, it is composed of three homologous subunits (4, 5,
25). Its major function is to provide the rate-limiting step to
transepithelial Na+ transport. Thus it plays a central role
in regulating fluid homeostasis and blood pressure. The recent
demonstration that activating and inactivating mutations in this
channel produce hypertension and hypotension, respectively, prove its
central importance in these functions (32).
Na+ absorption through ENaC is regulated through a number
of mechanisms including steroid-induced channel synthesis,
phosphorylation by intracellular kinases, methylation, and ionic
effects (14). The role of kinases in the rapid (minutes) regulation of
ENaC function has been well recognized for many years, but the
molecular mechanisms remain elusive. One possibility is that direct
phosphorylation of one or more of the ENaC subunits is responsible for
regulation of its activity. A recent report demonstrated that two of
the three ENaC subunits (
and
, but not
) can be
phosphorylated in vivo by treatment with protein kinase A (PKA),
protein kinase C (PKC), insulin, or aldosterone (35). The demonstration
of direct phosphorylation of ENaC subunits does not exclude the
possibility that phosphorylation of other proteins participates in ENaC
regulation. Intracellular kinases might phosphorylate proteins that
associate with ENaC, or phosphorylation cascades might produce effects
that are only distantly related to channel phosphorylation.
One of the difficulties encountered by investigators attempting to
dissect the mechanisms responsible for kinase-mediated regulation of
ENaC is that specific kinase activators and inhibitors have different
effects in different tissues. For example, whereas PKC is generally
considered to be an inhibitor of ENaC (13, 16, 24), this deduction
overlooks the significant differences in response to phorbol esters in
different tissues (6, 11).
The purpose of the present experiments was to begin to develop a system
where kinase activity could be systematically studied to dissect the
molecular mechanisms responsible for ENaC regulation. We chose the
Xenopus oocyte because of its versatility and simplicity in
evaluating effects of heterologously expressed proteins. The results
provide a basis for suspecting a complex network of kinases interacting
to regulate ENaC activity.
 |
METHODS |
Channel expression in Xenopus oocytes.
The human ENaC (hENaC)
-,
-, and
-subunits used in these
experiments have been described previously (28, 40). Specifically, we
used the
-1 subunit as defined by Thomas et al. (40). The coding
regions for the hENaC subunits were subcloned into the PGEM-HE plasmid.
This plasmid has been engineered for use in the Xenopus oocyte
cRNA expression system (23). It contains a T7 promoter site upstream
from the 5'-untranslated region (UTR) and 3' UTR of
Xenopus
-globin mRNA. The hENaC subunit coding sequence was
inserted into the multiple cloning site between the
-globin UTRs. We
have found that generating hENaC cRNA using the PGEM-HE construct
results in enhanced hENaC expression in oocytes compared with cRNA
containing no UTRs (unpublished observations). Plasmids were amplified
using the JM109 strain of Escherichia coli (Promega), purified
by CsCl banding, and linearized with a specific restriction enzyme
(Nhe I or Sph I) that cuts just downstream to the
-globin 3' UTR. The mMessage mMachine (Ambion) T7 in vitro
transcription kit was used to produce capped cRNA from each construct.
The integrity of the cRNAs was evaluated by agarose gel electrophoresis
and quantitated by densitometry. The cRNAs were diluted in water so that 50-nl injections with a Drummond Nanoject oocyte injector carried
1 ng of each subunit.
The rat ENaC (rENaC) subunit cDNA clones have been described previously
(28). The coding regions of the
- and
-subunits were subcloned
into PGEM-HE, whereas the coding region of the
-subunit was in vitro
transcribed from the PCR-Script vector (Stratagene).
All of the oocyte currents were measured from cRNA-injected oocytes
except for the truncation study shown in Fig. 7. For these currents,
nuclear injections of cDNA constructs composed of the wild-type or
mutated hENaC subunits in the pMT3 vector were used. The cDNA
constructs for the wild-type subunits and truncation mutants have been
described (37). Oocytes were injected with 0.2 ng of cDNA of each hENaC subunit.
ROMK1 (rat) (18) was provided by Dr. Jason Xu (Vanderbilt University)
in the pSport vector. This construct was amplified, purified, and
transcribed in vitro as described above; 1 ng of ROMK1 cRNA was
injected into oocytes.
Xenopus oocyte handling and current measurement.
Mature female Xenopus laevis were housed in the University of
Iowa animal facility in dechlorinated tap water at 18-20°C. Stage V and VI oocytes were removed from toads that were anesthetized in an ice-cold 2 mg/ml tricaine solution. The oocytes were
defolliculated with collagenase and stored overnight at 18°C in
frog Ringer solution consisting of 115 mM NaCl, 2.5 mM KCl, 1.8 mM
CaCl2, 10 mM HEPES, 5 mM sodium pyruvate, and 100 U/ml
penicillin-streptomycin (pH 7.35). After 12-24 h of recovery from
the collagenase treatment, healthy oocytes were injected with cRNA from
-,
-, and
-hENaC. Ringer solution was changed daily. Whole
cell hENaC currents were measured 48-72 h after cRNA injections.
Measurements were made in frog Ringer solution using an OC-725C oocyte
voltage-clamp amplifier (Warner Instruments). The pCLAMP software suite
(Axon Instruments) was used for amplifier control and data
collection/analyses. All recordings were performed at room temperature.
Amiloride-sensitive currents were derived by subtracting currents
recorded in 10-33 µM amiloride from preamiloride currents. Whole
cell capacitance was determined electronically using the automated
voltage-step protocol and current transient analysis of the pCLAMP program.
Whole cell hENaC currents heterologously expressed in Xenopus
oocytes typically "run down" while being measured in
voltage-clamp configuration (1). Although the rundown varies from cell
to cell, we have found that the rate is fairly consistent in each oocyte. To accurately determine a drug-induced percent change in
current, the run-down rate for each oocyte was determined. During a 3- to 5-min control period before drug application, the stable rate of
rundown was recorded and subsequently fit by linear regression. This
regression line was extrapolated throughout the remainder of the
experiment. Percent changes in current magnitude were calculated using
the extrapolated current value as control.
Whole cell ROMK1 current measurement was performed under the conditions
described (18). The recording bath solution was the same as the frog
Ringer solution described above, except that it contained 60 mM NaCl/60
mM KCl in place of 115 mM NaCl, and the CaCl2 was 0.3 mM.
Short-circuit current measurements.
M-1 cells were obtained from Dr. Géza Fejes-Tóth (Dartmouth
Medical School) and cultured as previously described (31). Briefly,
cells were seeded at confluent density on Millicell PCF filters
pretreated with human placental collagen. The filters were grown 3 days
in DMEM/Ham's F-12 supplemented with insulin (5 µg/ml), transferrin
(5 µg/ml), triiodothyronine (5 nM), hydrocortisone (50 nM), sodium selenite (10 nM), gentamicin (50 µg/ml), BSA (10 g/l),
and dexamethasone (5 nM). The monolayers were grown 1 day in the same
medium without albumin and steroids and then 1 day in albumin- and
serum-free media with the addition of 100 nM aldosterone plus 100 nM
dexamethasone. Measurements of transepithelial voltage, resistance
(Rt), and short-circuit current
(Isc) were conducted at 37°C as described (31).
The bathing solution used for examining the staurosporine effect
contained (in mM) 140 NaCl, 5 KCl, 10 HEPES, 1 mM MgCl2,
1.5 mM CaCl2, 1 mM Na2HPO4, and 5 mM glucose. Benzamil-sensitive currents were derived by subtracting
currents recorded in 10 µM benzamil from prebenzamil currents.
Materials.
Mature female X. laevis were purchased from Xenopus I
(Dexter, MI) or Nasco (Fort Atkinson, WI). Benzamil, phorbol
12-myristate 13-acetate (PMA), and 4
-PMA were purchased from RBI
(Natick, MA). Staurosporine, chelerythrine chloride, calphostin C, H-8, KN-93, SB-203580, and 1-oleoyl-2-acetyl-sn-glycerol (OAG) were obtained from Biomol (Plymouth Meeting, PA). All other chemicals were
purchased from Sigma Chemical (St. Louis, MO). The Millicell filters
were purchased from Millipore (Bedford, MA).
 |
RESULTS |
PKC stimulation enhances ENaC currents.
Our initial intent was to study the mechanism of PKC inhibition of ENaC
currents expressed in Xenopus oocytes as was reported in Ref.
1. To accomplish this, we injected the cRNAs for hENaC
,
, and
into Xenopus oocytes and recorded currents while applying 80-100 nM PMA in the bathing solution. Surprisingly, when the magnitude of the whole cell currents was <2 µA at
60 mV, PMA caused a reproducible increase in current. A representative example of
this effect is shown in Fig.
1A. Peak enhancement
typically occurred ~7-9 min after exposure to PMA. Figure
1B demonstrates that the percent increase in current induced by
PMA was inversely related to the magnitude of the current measured
before PMA addition. The smallest baseline currents (<500 nA) were
typically at least doubled in magnitude while larger currents were only
modestly increased if there was any increase at all. The results were
similar whether human or rat ENaC cRNA was injected. Water-injected
oocytes had no amiloride-sensitive currents and did not respond to PMA (data not shown). It is important to note that 10 µM amiloride consistently reduced the baseline and PMA-enhanced inward currents from
hENaC-expressing oocytes to magnitudes similar to the
amiloride-insensitive currents recorded from water-injected oocytes
(from 0 to
200 nA at
60 mV). These observations strongly
suggest that both the baseline currents and the PMA-enhanced currents
were carried through hENaC.

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Fig. 1.
Phorbol 12-myristate 13 acetate (PMA) enhances whole cell currents
recorded from Xenopus oocytes injected with cRNA for human
epithelial Na+ channel (hENaC) -, -, and
-subunits. A: each data point represents average
current during a 500-ms hyperpolarization to 60 mV from a
holding voltage of 0 mV. By convention, the flow of positive ions into
the oocyte is shown as negative current. Current values were recorded 5 s apart; 2 min were allowed for establishment of baseline current
values and run-down rate; 80 nM PMA and 10 µM amiloride were added to
the bathing solution at the indicated times. B: relationship
between the PMA-induced current increase and pre-PMA current
magnitude.
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To address whether the increase of hENaC current by PMA was mediated
through PKC, 4
-PMA, a PMA analog that does not activate PKC, was
applied in the bathing solution. Figure 2
shows that 100 nM 4
-PMA did not stimulate hENaC currents in an
oocyte where subsequent application of PMA enhanced current as
expected. We also tested the effect of a PKC-activating diacylglycerol
analog, OAG. OAG, at 5 µM, also increased hENaC current with a time
course nearly identical to PMA (data not shown).

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Fig. 2.
4 -PMA does not stimulate hENaC currents. Whole cell inward currents
were recorded at 60 mV every 5 s. Each data point represents
average current during a 500-ms hyperpolarization. Additions were made
to the bathing solution as indicated by arrows; 100 nM 4 -PMA did not
enhance current in the same oocyte where subsequent exposure to 80 nM
PMA caused a typical increase in current. This was a consistent finding
in 7/7 experiments.
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Effect of kinase inhibitors.
The PMA and OAG results suggested that PKC might be involved in the
minute-to-minute regulation of hENaC activity in Xenopus oocytes. However, since PMA treatment enhanced hENaC current only when
the baseline current was <2 µA, we suspected that PKC might be
maximally active in oocytes expressing large hENaC currents. We also
considered the possibility that other kinases might play a role in
regulating (increasing) hENaC currents. This notion derives in part
from experiments showing variability in the response to PKC agonists
(6, 11). We therefore performed experiments using kinase inhibitors
with varying specificities for PKC and other known kinases. A kinase
inhibitor profile for hENaC activity would serve two purposes. The PKC
inhibitors would test the hypothesis that PKC was involved in acute
regulation of hENaC activity, and other kinase inhibitors could
identify molecules that might also participate in the regulation of hENaC.
Figure 3 shows the effects of several
kinase inhibitors on hENaC currents. Chelerythrine and calphostin C are
PKC inhibitors with different potencies and selectivities for PKC
isoforms (17, 19, 20). Chelerythrine, but not calphostin C,
significantly reduced hENaC currents (P < 0.05). This
difference was probably not due to the higher dose of chelerythrine,
since both chelerythrine and calphostin C were applied at 15-20
times their reported IC50 doses.

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Fig. 3.
Selected kinase inhibitors reduce amiloride-sensitive currents.
Currents were recorded at 60 mV from oocytes incubated in
various kinase inhibitors for 30-90 min. Concentrations were 100 nM staurosporine, 10 µM chelerythrine, 100 µM SB-203580, 1 µM
calphostin C, 1 µM H-8, and 1 µM KN-93. Each bar represents the
average of the amiloride-sensitive inward currents that were normalized
to the inhibitor-free control currents of that day. Numbers in
parentheses represent numbers of oocytes used to calculate average and
SE. * Significantly different from control (P < 0.05).
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Another kinase inhibitor that has been used to inhibit PKC activity is
staurosporine. It is now known that staurosporine is not selective for
PKC, but rather it blocks a broad range of serine/threonine and
tyrosine kinases (2, 12, 27, 39, 42). Incubation of oocytes in 100 nM
staurosporine for 30-90 min produced 84% inhibition of
amiloride-sensitive current. Also shown in Fig. 3 are the effects of
three other kinase inhibitors. H-8 (15), which inhibits both PKA and
protein kinase G, did not inhibit hENaC current. The
Ca2+-calmodulin kinase II inhibitor, KN-93 (26), also had
no effect. A p38 mitogen-activated protein (MAP) kinase inhibitor,
SB-203580 (10), significantly reduced hENaC currents (55% inhibition).
Staurosporine-sensitive current.
The striking inhibitory effect of staurosporine on hENaC currents in
Xenopus oocytes prompted further investigation. First, we asked
whether the reduction in inward current at
60 mV was due to the
inhibition of the Na+-selective hENaC. In the vehicle
control experiments, nearly all of the current was amiloride sensitive;
therefore, it was reasonable to conclude that the
staurosporine-sensitive channel was hENaC. A further test of this
conclusion was the determination of the current-voltage relationships
and calculated reversal potentials (Erev) for the
control and staurosporine-treated whole cell currents. As shown in Fig.
4, the reversal potential of the control
currents is approximately +5 mV. Oocytes treated with staurosporine had smaller inward currents at all voltages negative to +5 mV and smaller
outward currents at all voltages positive to +10 mV. The extrapolated
Erev for the staurosporine group was approximately
2 mV. This current reduction profile and
Erev shift toward negative values caused by
staurosporine are consistent with the inhibition of a
Na+-selective channel. These results eliminate the
possibility that endogenous K+ or Cl
channel activities or alterations in intracellular ion concentrations could explain the staurosporine-induced inward current reductions at
60 mV.

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Fig. 4.
Staurosporine reduces currents at all voltages and shifts the reversal
potential in the negative direction. Whole cell currents were elicited
with various voltage steps from a holding potential of 0 mV.
Representative current traces for 2 groups, control (B) and 100 nM staurosporine (C), are shown at right. Dashed lines,
zero current level. A: comparison of average current-voltage
(I-V) relationships (control, n = 47; staurosporine,
n = 43). Extrapolated reversal potential, defined as voltage
where the I-V relationship intercepts the abscissa, is shifted
toward negative values in the staurosporine-treated group.
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We considered endocytosis as a potential mechanism for the reduction of
the hENaC currents by staurosporine. PKC stimulation is known to
enhance endocytosis in oocytes, leading to reduced function of both
expressed and endogenous transport proteins (30). To address this
issue, oocytes were injected with the cRNA encoding the epithelial
K+ channel, ROMK1, and Ba2+-sensitive currents
were measured. Figure 5 shows that there
was no effect of staurosporine on the magnitude of
Ba2+-sensitive currents in these oocytes. In addition,
whole cell capacitance was measured for 10 oocytes expressing hENaC
before and 10 min after 100 nM staurosporine treatment. This treatment reduced the inward currents at
60 mV by ~50%. The capacitance after staurosporine (237 ± 6 nF) was not different from pretreatment values (229 ± 5 nF) by paired analysis.

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Fig. 5.
Staurosporine does not inhibit ROMK1 Ba2+-sensitive
K+ currents. Oocytes were injected with 1 ng of rat ROMK1
cRNA. After 72 h incubation in frog Ringer solution, 2.5 mM
Ba2+-sensitive currents were measured in a modified frog
Ringer solution containing 60 mM KCl and 0.3 mM CaCl2.
Average I-V relationship for 5 control oocytes is compared with
that for 5 oocytes incubated for 30-60 min in 100 nM
staurosporine.
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A representative time course of the staurosporine inhibition of hENaC
currents in oocytes is shown in Fig. 6. We
have corrected for rundown by linear extrapolation through the
prestaurosporine currents as described in METHODS and shown
in Fig. 6. Staurosporine induced an immediate decrease in the magnitude
of the current. At 10 min, the current was reduced to approximately
one-half of the extrapolated prestaurosporine current.

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Fig. 6.
Staurosporine inhibition of hENaC currents is immediate. Currents were
elicited with voltage steps to 60 mV from a holding potential of
0 mV every 5 s. Each data point represents average current during a
500-ms hyperpolarization. After a 3-min control period, during which a
constant rate of rundown was established, 100 nM staurosporine was
added to the bathing solution. Dashed line, best-fit regression line to
the pretreatment current values; , extrapolated staurosporine-free
current value at the time of the 33 µM amiloride addition.
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Staurosporine-sensitive region of hENaC.
We hypothesized that the staurosporine effect was mediated via an
intracellular portion of the hENaC complex. We examined the COOH
terminus because this region is involved in regulating ENaC activity
(33, 34, 37). First, we mutated each of the three subunits to remove
the COOH-terminal portions beginning just downstream of the M2
membrane-spanning domain. These three truncations,
S594X,
R566X, and
K576X represent similar structural modifications and
allow expression of channel activity. Figure 7 shows that truncation of the
- or
-subunit resulted in enhanced baseline currents. These results are
consistent with reports demonstrating that elimination of the PPPXY
motif increases Na+ transport and causes Liddle's syndrome
(33, 34, 37). However, in contrast to
and
, truncation of the
COOH terminus of
resulted in control currents that were not larger
in magnitude than wild-type currents. This result is consistent with
our previous report (37). Staurosporine inhibited currents when
or
were truncated. However, staurosporine did not inhibit hENaC
currents when a truncated
construct was expressed. These results
suggest that the staurosporine-sensitive region is located in the COOH
terminus of the
-subunit and not the
- or
-subunit.

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Fig. 7.
Truncation of intracellular COOH terminus of -hENaC, but not - or
-hENaC, eliminates the staurosporine effect. Amiloride-sensitive
currents were measured from 4 groups of cDNA-injected oocytes 48 h
after nuclear injection. Open bars, average staurosporine-free currents
at 60 mV that have been adjusted for rundown as shown in Fig. 6;
solid bars, average currents 10 min after bath application of 100 nM
staurosporine. Numbers in parentheses represent number of oocytes used
to calculate average and SE. * Statistical significance between
control and staurosporine currents at P < 0.05 using a paired
t-test.
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Staurosporine inhibits M-1 cell Isc.
The M-1 mouse collecting duct epithelial cell line has been used
extensively to study amiloride-sensitive Na+ transport
through ENaC (7, 21, 22, 38). Based on the strong and rapid inhibitory
effect of staurosporine on heterologously expressed hENaC, we
hypothesized that there were staurosporine-sensitive kinases involved
in short-term ENaC regulation in cells that naturally express ENaC. We
therefore asked whether staurosporine inhibited Na+
transport in the M-1 cell line by measuring Isc
while applying staurosporine. Figure 8
demonstrates the dose-dependent inhibition of Na+ transport
in M-1 cell monolayers by staurosporine.

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Fig. 8.
Staurosporine inhibits M-1 cell monolayer short circuit currents
(Isc). Each bar represents average fraction of
benzamil-sensitive current in each group. Values were determined 30 min
after solution change containing no staurosporine (control), 100 nM
staurosporine, or 1 µM staurosporine. Baseline currents were
10-20 µA/cm2, and postbenzamil currents were
1-2 µA/cm2. Numbers in parentheses represents
numbers of monolayers in each group. * Significant difference from
control (P < 0.05) using ANOVA and Newman-Keuls multiple
comparisons.
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 |
DISCUSSION |
The present results demonstrate that kinases endogenous to the
Xenopus oocyte can participate in the regulation of hENaC
activity. Activation of the PKC system stimulates the activity of hENaC (Fig. 2), while several kinase inhibitors, including chelerythrine, a
relatively specific inhibitor of PKC, inhibit hENaC activity (Fig. 3).
Staurosporine, a nonspecific kinase inhibitor, had the most dramatic
effect of the inhibitors tested and produced a rapid reduction in
Na+ current (Fig. 6). The effect of staurosporine seems to
be relatively specific for hENaC activity, as there was no effect on
the ROMK1 K+ channel (Fig. 5). This kinase regulation
appears to be effected via a specific region of the hENaC complex.
Whereas truncation of the COOH-terminal regions of the
- and
-subunits did not alter the inhibitory effect of staurosporine (Fig.
7), truncation of the analogous region of the
-subunit eliminated
this staurosporine effect. We infer from these results that a domain
within this region of the
-subunit plays an important role in the
kinase-mediated regulation of hENaC.
These results might appear to conflict with those of Awayda et al. (1)
who reported that phorbol esters inhibit ENaC currents in oocytes.
However, the time course of these studies were different; these authors
examined the effects after 30 min of exposure, whereas our data report
the effects after only 10 min of exposure. It is likely that the timing
and specific experimental conditions influence the nature of the
reaction to phorbol esters. The response to phorbol esters is also
somewhat dependent on the specific oocyte. As demonstrated in Fig. 1,
those with larger hENaC currents show a smaller response to phorbol
esters. We interpret these results to indicate that there may be a
variety of factors in the oocyte that influence the magnitude of hENaC
currents. The effect of an inhibitor of p38 MAP kinase, SB-302580 (Fig.
3), supports this interpretation. In addition, it has been recently
reported that overexpression of a steroid-induced kinase, sgk,
increases ENaC currents in oocytes (9, 29). The activity of such
kinases might vary from oocyte to oocyte.
What is the relevance of effects of protein kinases on hENaC function
in Xenopus oocytes to mammalian epithelial cells? This question
touches on the relevance of all heterologous expression systems and
must be addressed in any effort to integrate isolated molecular actions
into models of cell and organ function. In this regard, staurosporine
inhibits Na+ current in the cortical collecting duct
(CCD) cell line, M-1 (Fig. 8). Thus its actions in the
oocyte might have relevance to regulation of ENaC in intact cells.
However, the action of kinases on ENaC function is much more varied
than is commonly appreciated. For example, activation of PKA in rat CCD
produces a sustained increase in Na+ transport, whereas
similar maneuvers applied to the rabbit CCD produces inhibition of
Na+ transport (3, 8). In addition, and perhaps more
relevant for the present experiments, phorbol esters can either
increase or decrease Na+ transport depending on the tissue
examined (11). These divergent effects appear to be tissue, rather than
species, specific. Amphibian skins from two different species
demonstrate enhancement of Na+ transport in response to
phorbol esters, whereas urinary bladders from these species respond by
decreasing Na+ transport (6). One possible explanation for
this difference in response is different PKC isoforms in the various
tissues (6). It is also possible that activation of the resident PKC
might participate in a cascade with other endogenous protein kinases to
produce the final effect on ENaC function.
What inferences can we make about the mechanism(s) of kinase regulation
from these experiments? First, it appears that there is endogenous
kinase activity (in the oocyte and in M-1 cells) that participates in
maintaining an active ENaC; the response to the protein kinase
inhibitors demonstrates this point. Second, PKC may play a role in this
activity, but it cannot explain the entire effect. The response to
phorbol esters is modest and dependent on the baseline current.
Furthermore, relatively specific inhibitors of PKC did not reduce the
Na+ current to values similar to those of the more
nonspecific kinase inhibitor, staurosporine. Third, this effect of
staurosporine is not mediated via generalized endocytosis (41). If
endocytosis were a prominent feature, we would have expected that all
membrane proteins, including ROMK1, would be reduced in activity.
Furthermore, the oocyte capacitance was not altered by staurosporine.
Fourth, this kinase effect is not mediated solely through the COOH
termini of
- or
-hENaC; truncation of these regions did not
prevent staurosporine from inhibiting the Na+ current. Thus
the recent demonstration that these regions can be phosphorylated (35)
apparently does not explain the actions of staurosporine reported here.
Finally, in contrast to the
- and
-subunits, the COOH terminus of
the
-subunit does seem to play a role in producing the staurosporine
effect. This result is even more intriguing in the context of the
failure of kinases to phosphorylate this region under the same
conditions where
- and
-subunits were phosphorylated (35). Thus
these kinase-dependent effects may not be mediated directly via
phosphorylation of ENaC.
A review of the literature on the effects of mutations of the
-ENaC
subunit identifies clues as to the location of the region that might be
responsible for the staurosporine effect. First, two groups have
reported that mutation of the key amino acids of the COOH-terminal
PPPXY motif of any one of the three subunits will produce an increase
in Na+ current (33, 37). We note that this result may not
be as simple as it first appears, as not all investigators agree about the importance of this motif in the
-subunit (36). Nevertheless, this motif within the
-subunit seems to participate in restraining ENaC activity. Second, a truncation of the rENaC
-subunit at the
P646 position (a COOH-terminal deletion beginning 25 amino acids
upstream of the PPPXY motif) produces an increase in current (34). It
seems likely that this effect is due to the elimination of the PPPXY
motif. Third, a truncation of the hENaC
-subunit at the S594
position (just 3' to the second membrane-spanning domain)
produces a decrease in Na+ current (Fig. 7 and Ref. 37).
Integrating these results leads to the conclusion that the effect of
staurosporine may be mediated through the region of the
-subunit
somewhere between the second membrane-spanning region and the PPPXY
motif. Inspection of this region does not provide obvious candidate domains.
In conclusion, we have demonstrated that kinase inhibitors can reduce
Na+ transport in M-1 cells and ENaC activity in
Xenopus oocytes. These results suggest that endogenous kinases
stimulate ENaC activity. The specific kinase(s) responsible for this
effect is not clear at present, but the aggregate data suggest that
several are involved. The action of these kinases appears to be
mediated through the COOH-terminus of the
-subunit.
 |
ACKNOWLEDGEMENTS |
This research was supported in part by National Institutes of
Health Grants DK-52617 and HL-55006 and by a grant from the Department
of Veterans Affairs.
 |
FOOTNOTES |
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: J. B. Stokes,
Dept. of Internal Medicine, E300GH, Univ. of Iowa, Iowa City, IA 52242 (E-mail: john-stokes{at}uiowa.edu).
Received 12 October 1999; accepted in final form 29 December 1999.
 |
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