RAPID COMMUNICATION
Protein tyrosine kinase regulates the number of renal secretory K
channels
Wenhui
Wang1,
Kenneth M.
Lerea2,
Mary
Chan1, and
Gerhard
Giebisch3
1 Department of Pharmacology,
2 Department of Anatomy and Cell Biology, New
York Medical College, Valhalla, New York 10595; and
3 Department of Cellular and Molecular
Physiology, Yale University School of Medicine, New Haven,
Connecticut 06510
 |
ABSTRACT |
The
apical small conductance (SK) channel plays a key role in K secretion
in the cortical collecting duct (CCD). A high-K intake stimulates renal
K secretion and involves a significant increase in the number of SK
channels in the apical membrane of the CCD. We used the patch-clamp
technique to examine the role of protein tyrosine kinase (PTK) in
regulating the activity of SK channels in the CCD. The application of
100 µM genistein stimulated SK channels in 11 of 12 patches in CCDs
from rats on a K-deficient diet, and the mean increase in
NPo, a product of channel number (N) and
open probability (Po), was 2.5. In contrast,
inhibition of PTK had no effect in tubules from animals on a high-K
diet in all 10 experiments. Western blot analysis further shows that the level of cSrc, a nonreceptor type of PTK, is 261% higher in the
kidneys from rats on a K-deficient diet than those on a high-K diet.
However, the effect of cSrc was not the result of direct inhibition of
channel itself, because addition of exogenous cSrc had no effect on SK
channels in inside-out patches. In cell-attached patches, application
of herbimycin A increased channel activity in 14 of 16 patches, and the
mean increase in NPo was 2.4 in tubules from rats
on a K-deficient diet. In contrast, herbimycin A had no effect on
channel activity in any of 15 tubules from rats on a high-K diet.
Furthermore, herbimycin A pretreatment increased NPo per patch from the control value (0.4) to 2.25 in CCDs from rats on a K-deficient diet, whereas
herbimycin failed to increase channel activity
(NPo: control, 3.10; herbimycin A, 3.25) in the CCDs from animals on a high-K diet. We conclude that PTK is involved in
regulating the number of apical SK channels in the kidney.
potassium depletion; high-potassium intake; potassium secretion; cortical collecting duct
 |
INTRODUCTION |
THE CORTICAL COLLECTING DUCT (CCD) plays a key role in
the hormone-regulated Na reabsorption and K secretion (8, 9). There are
two types of cells in the collecting duct epithelium, principal and intercalated cells, and the principal cells are responsible for Na reabsorption and K secretion (8, 9). The K secretion
is a two-step process: 1) K enters the cell across the
basolateral membrane via a Na-K-ATPase; 2) K is then secreted into the lumen across the apical membrane through the SK channel. Therefore, SK channels provide the major route for K exit across the
apical membrane in the CCD (6, 12, 21, 22). Thus changes in channel
open probability or channel number could significantly affect renal K
secretion (20). The K secretion is regulated by hormones such as
aldosterone and vasopressin and by acid-base balance (1, 8, 18, 19). In
addition, dietary K intake plays an important role: a high-K intake
stimulates whereas a low-K intake suppresses K secretion (4, 13, 18,
24). The dietary effect on the K secretion is partially achieved by changing the apical K conductance (14, 20). Several studies have
demonstrated that the number of SK channels in the apical membrane
increased by 100-200% in the CCDs obtained from rats on a high-K
diet in comparison with tubules from rats on a normal K diet (14, 21).
The effect of high-K intake on channel number is not the result of
increasing the circulating level of aldosterone, because the number of
SK channels does not change in CCDs isolated from animals on a low-Na
diet (14), a maneuver which significantly increases the level of
aldosterone. Moreover, a high-K diet does not increase transcription of
SK channels, since the mRNA encoding ROMK channels, which are closely
related to apical SK channels in the CCD (5, 10, 21), was the same in
CCDs from animals on a high-K or a normal K diet (7). This suggests
that the effect of a high-K diet on the number of SK channels is
achieved by a posttranslation modulation. In the present study, we
explore the role of PTK in mediating the effect of K diet on the
activity of the apical SK channels in the rat CCD.
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METHODS |
Preparation of rat CCD. The experiments were carried out in the
isolated CCDs from kidneys of pathogen-free Sprague-Dawley rats. The
animals were obtained from Taconic (Germantown, NY) and
maintained either on a high-potassium diet (10%) or a
potassium-deficient diet (Harlan Teklad, Madison, WI) for 10 days
before use. The weight of animals used for experiments was between 100 and 120 g. After euthanasia, the abdomen of the animal was cut open,
and the two kidneys were rapidly removed. Several thin slices of the kidney (<1 mm) were cut and placed on the ice-cold Ringer solution until dissection. The dissection was carried out at room temperature, and two watchmakers forceps were used to isolate the single CCD. The
tubules were placed onto a 5-mm × 5-mm cover glass coated with
Cell-Tak (Becton-Dickinson, Bedford, MA), to immobilize the tubules,
and then transferred to a chamber (1,000 µl) mounted on an inverted
Nikon microscope. The CCDs were superfused with HEPES-buffered NaCl
solution, and the temperature of the chamber was maintained at 37 ± 1°C by circulating warm water surrounding the chamber. The CCD was
cut open with a sharpened micropipette to expose the apical membrane.
Patch-clamp technique. We used an Axon model 200A patch-clamp
amplifier to record channel current. The current was low-pass filtered
at 1 kHz using an eight-pole Bessel filter (model 902LPF; Frequency
Devices, Haverhill, MA) and digitized at a sampling rate of 44 kHz
using a modified Sony PCM-501ES pulse code modulator and stored on
videotape (JVC-HR-J400U). For analysis, data stored on the tape were
collected to an IBM-compatible Pentium computer (Gateway 2000) at a
rate of 4 kHz and analyzed using the pClamp software system 6.04 (Axon
Instruments, Burlingame, CA). Channel activity was defined as
NPo, a product of channel number (N) and open probability (Po), which was calculated from
data samples of 60-s duration in the steady state as follows:
where
ti is the fractional open time spent at
each of the observed current levels. The slope conductance of the channel was calculated by measurement of K current at several cell
membrane potentials.
Western blot. Protein samples extracted from the kidney cortex
and outer medulla were separated by electrophoresis on 8%
SDS-polyacrylamide gels and transferred to nitrocellulose membrane. The
membranes were blocked with 10% nonfat dry milk in Tris-buffered
saline (TBS), rinsed, and washed with 1% milk in Tween-TBS. The cSrc antibody was purchased from Santa Cruz (Santa Cruz, CA) and was diluted
at 1: 2,500. The protein concentration used for immunoblot was 100 µg. The cSrc was detected and quantitatively analyzed using
fluorescence phosphorimaging.
Measurement of the activity of cSrc tyrosine kinase. Kidney
extracts prepared in RIPA buffer [150 mM NaCl, 50 mM Tris (pH 7.4), 50 mM
-glycerophosphate, 50 mM NaF, 1 mM sodium orthovanadate, 2.5 mM EDTA, 5 mM EGTA, 0.5 mM dithiothreitol, 1% Triton X-100, 0.5%
sodium deoxycholate, 0.1% SDS, 100 µg/ml phenylmethylsulfonyl fluoride, 5 µg/ml aprotinin, 5 µg/ml leupeptin, and 2 µg/ml
pepstatin] were diluted to 1 mg/ml with the buffer. The extract
(300 µl) was immunoprecipitated overnight using cSrc antibody (1:200
dilution) coupled to protein A-Sepharose. The Sepharose
pellet was suspended in 50 µl of 10 mM HEPES (pH 7.4). For protein
tyrosine kinase (PTK) assays, the Sepharose pellet was
incubated in a total volume of 30 µl containing 100 µM
[32P]ATP (1 cpm/fmol), 12 mM magnesium acetate,
2 mM MnCl2, 0.3 mM dithiothreitol, 0.5 mM sodium
orthovanadate, 0.5 mM ammonium molybdate, and 2 mM R-R-Src
peptide (Arg-Arg-Leu-Ile-Glu-Asp-Ala-Glu-Tyr-Ala-Ala-Gly) (3).
Reactions were quenched by adding 75 mM phosphoric acid, and 15 µl of
cocktail were placed on phosphocellulose
paper. The amount of 32P incorporated was
assessed using a liquid scintillation counter. In addition, 10 µl of
the Sepharose pellet were boiled and used for immunoblot analysis. The
protein tyrosine activity was normalized in comparison to the relative
kinase concentration determined by the Western analysis using
fluorescence phosphorimaging technique.
Experimental solution and statistics. The pipette solution
contained (in mM) 140 KCl, 1.8 MgCl2, and 10 HEPES (pH
7.4). The bath solution for cell-attached patches was composed of (in
mM) 140 NaCl, 5 KCl, 1.8 CaCl2, 1.8 MgCl2, and
10 HEPES (pH 7.4). For inside-out patches, the bath solution had the
same composition as those in cell-attached except that free
Ca2+ was reduced to 100 nM. Genistein and herbimycin A were
purchased from Biomol (Plymouth Meeting, PA) and chemicals were
dissolved in the DMSO solution. The final concentration of DMSO was
less than 0.1% and had no effect on channel activity. Partially
purified preparation of active p60-Src was obtained from Upstate (Lake Placid, NY). The activity of this enzyme was confirmed using R-R-Src peptide in in vitro kinase assays.
Data are shown as means ± SE, and an unpaired Student's
t-test was used to determine the significance between two
groups. Statistical significance was taken as P < 0.05.
 |
RESULTS |
Figure 1 shows the mean
NPo of the apical SK channel per patch in
cell-attached patches in CCDs obtained from animals on either a high-K
or a K-deficient diet. The mean NPo in 88 patches
was 3.1 ± 0.4 in the CCD obtained from rats on a high-K diet but
decreased sharply to only 0.40 ± 0.05 (n = 67) in the CCD
isolated from animals on a K-deficient diet. To explore the role of PTK
in regulating the apical SK channels, we examined the effect of 100 µM genistein on the activity of the SK channels. Figure
2 is a representative recording of channel
activity in a cell-attached patch in the CCD obtained from a rat on a
K-deficient diet. It is apparent that the inhibition of PTK increases
the activity of apical SK channels. We studied the biophysical
properties of the channel shown in Fig. 2, and the result is
demonstrated in Fig. 3. Figure 3A
is a recording in an inside-out patch with 140 mM KCl in the pipette
and 5 mM KCl in the bath, showing that the channel has a high open
probability (0.90) that is not voltage dependent between 20 and
40 mV. The channel has one open time (15 ms) and one closed time
(0.6 ms) (Fig. 3B). A long closed state could be observed when
cell membrane potential hyperpolarized. Since events were not
enough to make a curve fitting, we did not include these
long closed times. The current-voltage (I-V) curve yields the
slope conductance of 38 pS between 20 and
40 mV (Fig.
3C). Thus we confirmed that the newly appeared channel is the
SK channel. Table 1 summarizes the effect
of 100 µM genistein on channel activity. Inhibition of PTK increased
the channel activity in CCDs from animals on a K-deficient diet in 11 of 12 patches, and the mean increase in NPo was 2.5 ± 0.2. In contrast, application of genistein failed to activate the
SK channel in all 10 patches in CCDs obtained from high-K-adapted rats.
This suggests that PTK activity may be higher in CCDs from rats on a
K-deficient diet than those on a high-K diet.

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Fig. 1.
Mean NPo of each patch observed in cortical
collecting duct (CCD) obtained from rats on a high-K diet or a low-K
diet. NPo is a product of channel number
(N) and open probability (Po). *Significant
difference between two groups (P < 0.01).
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Fig. 2.
A representative channel recording showing effect of 100 µM genistein
on the channel activity in CCD from rats on a low-K diet. Experiments
were performed in a cell-attached patch; "C" indicates the
channel closed level. Top trace: time course of effect of
genistein; the 3 parts of the trace indicated by 1-3 are
extended, below, to show to fast time resolution.
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Fig. 3.
Channel induced by genistein in Fig. 2 is recorded in the inside-out
configuration. A: channel current at different voltages.
B: open and closed time histograms of SK channel. C:
current-voltage (I-V) curve of SK channel.
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This inference is confirmed by Western blot analysis, which shows the
presence of cSrc in both renal cortex and outer medulla (Fig.
4). Kidneys from rats on a K-deficient diet
express a higher level of cSrc than those on a high-K diet. Figure
5A summarizes the results obtained
from six experiments. The level of cSrc in the renal cortex and outer
medulla increased by 261 ± 20% in rats on a K-deficient
diet compared with those on a high-K diet. We also used
R-R-Src peptide as substrate (3) to measure the activity of cSrc in the kidneys and found that the specific activity of cSrc
obtained from rats on a K-deficient diet is significantly higher (61 ± 8%) than those on a high-K diet (Fig. 5B).

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Fig. 4.
A Western blot shows that antibody to cSrc p60 recognizes a 60-kDa band
corresponding the cSrc protein in renal cortex and outer medulla (OM)
from rats on both a high-K and a low-K diet.
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Fig. 5.
A: relative intensity of cSrc protein levels in kidney from
high-K and low-K adapted rats. Intensity was determined using
fluorescence phosphorimaging and normalized to fluorescence intensity
of rats on a high-K diet. B: relative activity of cSrc in
kidneys from rats on both a high-K and a low-K diet measured with
R-R-Src peptides. Activity was normalized to the level in high-K diet
rats. *Significant difference between two groups (P < 0.01).
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To examine whether cSrc can inhibit the SK channel, we tested the
effect of exogenous purified preparation of active cSrc on the SK
channel in inside-out patches. The activity of this exogenous enzyme
was confirmed in in vitro kinase assay by measuring the phosphorylation
rate of R-R-Src peptides. Figure 6 is a
representative channel recording from three experiments showing that
the effect of cSrc on channel activity in the CCD from rats on a high-K
diet. Addition of 1 nM exogenous cSrc failed to inhibit the SK channel in an inside-out patch within 15-20 min. This suggests that cSrc cannot inhibit the SK channel directly.

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Fig. 6.
Top: channel recording showing effect of 1 nM exogenous cSrc on
channel activity in an inside-out patch. Exogenous cSrc was added
directly into bath solution for 15-20 min. Bottom:
all-points-histogram obtained in absence of (control) or in presence of
cSrc (cSrc). NPo is the same in both cases.
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To further confirm that the effect of genistein is the result of
inhibition of PTK, we have examined the effect of herbimycin A, an
agent which is more specific inhibitor of PTK including cSrc. Figure
7 is a representative channel recording
showing the effect of 1 µM herbimycin A on the SK channels in the CCD
obtained from rats on a K-deficient diet. As in the case of genistein, herbimycin A stimulated the SK channels in 14 of 16 experiments in CCDs
isolated from rats on a K-deficient diet: mean increase in
NPo was 2.4 ± 0.2 (Table
2). In contrast, herbimycin A had no effect
on the channel activity in the CCD from high-K adapted rats in any of
15 experiments (Table 2). In addition, effects of herbimycin A can only
be observed in cell-attached patches but not in excised patches (data
not shown). This indicates that the effect of herbimycin A is not the
result of inhibiting the membrane-bound PTK and in turn activating SK
channels. That an increase in PTK activity may be partially responsible
for the low channel activity observed in the CCD obtained from rats on a K-deficient diet is further supported by experiments in which channel
activity was assessed in the presence of herbimycin A (Fig.
8A). Pretreatment of CCDs with 1 µM herbimycin (20-30 min) significantly increased the mean
NPo from the control value (0.40) to 2.2 ± 0.2 per patch (n = 55) in the CCD obtained from rats on a
K-deficient diet. In contrast, the mean NPo of 30 patches carried out on those on a high-K diet was 3.25 ± 0.4, which is not significantly different from the
NPo observed under the control conditions (Fig.
8B). This strongly indicates that the activity of PTK plays a
key role in regulating the apical SK channel density and that an
increase in the activity of PTK such as cSrc lowers the number of the
apical SK channel.

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Fig. 7.
A channel recording showing effect of 1 µM herbimycin A on channel
activity in CCDs obtained from rats on the low-K diet. Experiment was
carried out in a cell-attached patch, and pipette holding potential was
40 mV. Top trace: time course of experiments (initial
part of channel current is out of scale because of saturation of the
amplifier). The 2 parts of the trace indicated by 1 and
2 are extended, below, at a fast time resolution. Channel
closed level is indicated by "C," and each short line indicates
channel current level.
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Fig. 8.
Effect of herbimycin A treatment on mean NPo per
patch in CCD from rats on a K-deficient diet (A) and in CCD
from high-K diet rats (B).
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 |
DISCUSSION |
The main finding of the present investigation is that inhibition of PTK
stimulates the activity of apical SK channels in CCDs from animals on a
K-deficient diet. We have demonstrated that the level of cSrc, which is
the most abundant and highly expressed PTK in a variety of
cells, increased significantly in kidneys from rats on a
K-deficient diet in comparison with the those from rats on a high-K
diet. However, it is possible that cSrc is not the only isoform of PTKs
responsible for regulating the number of apical K channels in the CCD.
A careful study is required to determine isoforms of PTKs that are
involved in regulation of the activity of SK channels. In addition,
tyrosine phosphorylation is determined by both PTK and protein tyrosine
phosphatases. Therefore, it is conceivable that protein tyrosine
phosphatase may be also involved in regulating the activity of SK
channels. Further experiments are needed to investigate the role of
protein tyrosine phosphatase in mediating the effect of K intake on K secretion.
The mechanisms by which a high intake of K augments the apical K
conductance and SK activity have been investigated previously. Two
findings argue against direct involvement of aldosterone. First, a
low-Na diet, known to stimulate aldosterone secretion, has no effect on
the number of apical SK channels (14). Second, the acute administration
of aldosterone does not increase the number of SK channels (14). A
permissive role of aldosterone in optimizing K secretion during chronic
K loading has been reported (14) and is most likely to involve
stimulation of Na-K-ATPase. The present data suggest an
aldosterone-independent effect of K intake on apical SK channels. Our
data support the view that a decrease in PTK levels and activity in the
CCD from rats on a high-K diet increases the number of apical SK
channels, whereas an increase in PTK activity from rats on a
K-deficient diet depletes the apical membrane of SK channels. The
mechanism by which PTK inhibits SK channels is not known. Since the
effect of herbimycin A/genistein occurs quite rapidly, within
20-30 min, it is thus unlikely that changes in protein synthesis
are involved.
There are at least two possibilities by which PTK can regulate the
activity of SK channels. First, PTK phosphorylates the SK channel and
inhibits channel activity. PTK has been shown to be involved in
suppressing a delayed rectifying K channel and Kv1.3 (2,
11) and in activating Ca2+-dependent K channels and delayed
rectifier K channels in Schwann cells by direct tyrosine
phosphorylation (16, 17). Although this mechanism is not supported by
our observations that the addition of exogenous cSrc did not affect
channel activity in inside-out patches, we cannot exclude the
possibility that PTKs other than cSrc can phosphorylate the SK channels
and inhibit channel activity. Alternatively, the phosphorylation of the
SK channel by cSrc may require some cytosolic factors that are absent
in excised patches. Therefore, we need further experiments to examine
whether ROMK channels can be phosphorylated on tyrosine residue and
whether channel activity is inhibited by tyrosine phosphorylation.
Another possibility is that PTK reduces the insertion of SK channels or
increases the internalization of SK channels. It is of interest that
cSrc has been shown to interact with several signaling agents such as
dynamin and synapsin that control membrane protein traffic (5, 15) and
that overexpression of cSrc enhances endocytic internalization of
epidermal growth factor receptor in fibroblasts (23).
The mechanism by which PTK activity increases in tubules from animals
on a K-deficient diet is not clear. It has been demonstrated that
acidosis stimulates the cSrc activity in proximal tubules (25). Since K
depletion induces cytosolic acidosis ,which decreases the apical K
conductance in the CCD, it is possible that changes in cell pH are
involved in the regulation of PTK activity. Thus a common mechanism may
be responsible for reduction of apical K conductance of the CCD from
animals with acidosis or on a low-K diet. We conclude that PTK is
involved in mediating effects of K intake on the apical K secretory
channels in the CCD and that PTK may be a member of the
aldosterone-independent signaling which regulates K secretion.
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ACKNOWLEDGEMENTS |
We thank Dr. Robert W. Berliner for helpful discussion during the
course of these experiments.
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FOOTNOTES |
This work was supported by National Institute of Diabetes and Digestive
and Kidney Diseases Grant DK-47402.
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: W. Wang, Dept.
of Pharmacology, New York Medical College, Valhalla, NY 10595 (E-mail:
wenhui_wang{at}nymc.edu).
Received 20 July 1999; accepted in final form 12 October 1999.
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