Effect of dietary K intake on apical small-conductance K
channel in CCD: role of protein tyrosine kinase
Yuan
Wei,
Peter
Bloom,
Daohong
Lin,
Ruimin
Gu, and
Wen Hui
Wang
Department of Pharmacology, New York Medical College, Valhalla, New
York 10595
 |
ABSTRACT |
We have used Western blot
to examine the expression of cSrc protein tyrosine kinase (PTK) and
protein tyrosine phosphatase (PTP)-1D in the renal cortex, and the
patch-clamp technique to determine the role of PTK in mediating the
effect of dietary K intake on the small-conductance K (SK) channel in
the cortical collecting duct (CCD). When rats were on a K-deficient
(KD) diet for 1, 3, 5, and 7 days, the expression of cSrc increased by
40, 90, 140, and 135%, respectively. In contrast, the expression of cSrc in the renal cortex from rats on a high-K (HK) diet for 1, 2, and
3 days decreased by 40, 60, and 75%, respectively. However, the
protein level of PTP-1D was not significantly changed by dietary K
intake. The addition of 1 µM herbimycin A increased
NPo, a product of channel number (N)
and open probability (Po) in the CCD from rats
on a normal diet or on a KD diet. The increase in
NPo was 0.30 (normal), 0.45 (1-day KD), 0.65 (3-day KD), 1.55 (5-day KD), and 1.85 (7-day KD), respectively.
Treatment of the CCD with herbimycin A from rats on a KD diet increased
NPo per patch from the control value (0.7) to
1.4 (1-day KD), 1.6 (3-day KD), 2.6 (5-day KD), and 3.5 (7-day KD),
respectively. In contrast, HK intake for as short as 1 day
abolished the effect of herbimycin A. Furthermore, the expression
of ROMK channels in the renal cortex was the same between rats on a KD
diet or on a HK diet. Moreover, treatment with herbimycin A did not
further increase NPo in the CCDs from rats on a
HK diet. We conclude that dietary K intake plays a key role in
regulating the activity of the SK channels and that PTK is
involved in mediating the effect of the K intake on channel activity in
the CCD.
hyperkalemia; hypokalemia; protein tyrosine phosphatase 1D; renal
potassium secretion; cortical collecting duct
 |
INTRODUCTION |
THE CORTICAL COLLECTING
DUCT (CCD) IS RESPONSIBLE FOR K secretion and Na reabsorption.
For K secretion, K enters the cell through basolateral Na-K-ATPase and
then diffuses into the lumen via the apical K channels
(6). Although two types of K channels, Ca2+-activated K channel and the small-conductance K (SK)
channel, are identified in the apical membrane of principal cells of
the CCD, it is generally agreed that the SK channel is mainly
responsible for K exit across the apical membrane (3-4, 7,
10, 18). It is well established that hormones such as
aldosterone and vasopressin play an important role in the regulation of
K secretion (6). In addition, dietary K intake plays a key
role in modulating renal K secretion: an increase in K intake
stimulates, whereas a low-K intake inhibits, K secretion
(6). The effect of dietary K intake on K secretion is, at
least in part, achieved by modifying the number of the SK channels in
the CCD. We and others (13, 18) have shown that the number
of SK channels increased by three to four times in the CCD from rats on
a high-K (HK) diet than from those on a normal diet. The effect of HK
intake on channel activity is not mediated by aldosterone because
infusion of aldosterone failed to increase the number of the SK
channels (13). In previous studies, we have demonstrated
that inhibition of protein tyrosine kinase (PTK) increased the number
of the SK channels in the CCD obtained from rats on a K-deficient (KD)
diet (21). Moreover, blocking protein tyrosine phosphatase
(PTP) reversibly decreased the number of SK channels in the CCD from
animals on a HK diet (22). Also, we have demonstrated that
the protein level and activity of cSrc are significantly higher in the
renal kidneys from rats on a KD diet than from those on a HK diet. We
have proposed that an increase in tyrosine phosphorylation of SK
channels reduces, whereas an increase in tyrosine dephosphorylation
augments, the number of SK channels. In the present study, we have
extended our investigation by examining changes in protein levels of
cSrc and PTP-1D in the renal cortex from rats on a HK diet or on a KD
diet. We have also investigated the effect of inhibiting PTK on channel
activity to determine the relationship between cSrc expression and
channel activity in the CCD.
 |
METHODS |
Preparation of CCDs.
Pathogen-free Sprague-Dawley rats of either sex (5 wk) were used in the
experiments and were purchased from Taconic Farms, (Germantown, NY).
The animals were put on a HK diet (wt/wt, 10%) or on a KD diet (Harlan
Teklad, Madison, WI) for different days before use. The weight of the
rats used for experiments was ~100 g. The rats were killed by
cervical dislocation, and the kidneys were removed immediately. Several
thin slices of the kidney (<1 mm) were cut and placed in an ice-cold
Ringer solution until dissection. The dissection was carried out at
room temperature, and two watch-make forceps were used to isolate the
single CCD. To immobilize the tubules, we placed them on a 5 × 5-mm cover-glass coated with Cell-Tak (Becton Dickinson, Bedford, MA)
and then transferred them 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 around the chamber. The CCD
was cut open with a sharpened micropipette to expose the apical membrane.
Patch-clamp technique.
An Axon model 200A patch-clamp amplifier was used to record channel
current. The current was low-pass filtered at 1 KHz by an eight-pole
Bessel filter (model 902LPF; Frequency Devices, Haverhill, MA) and
digitized by an Axon interface (Digitada 1200). Data were acquired by
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) that was calculated
from data samples of 60 s duration in the steady state as follows:
|
(1)
|
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 the K current at several
cell membrane potentials.
Tissue preparation for Western blot.
Five to six rats were used for each set of experiments to examine the
effect of dietary K intake on cSrc, cYes, and PTP levels in
the renal cortex. The rats were from the same clone and were the same
age. Five days after receiving them, we started to feed the rats with a
different K diet according to the protocol. The plasma Na and K
concentrations were measured with flame photometry (Corning 480) in the
rats on a normal chow, on a KD diet for 7 days, or on a HK diet for 3 days (Table 1). The rats were killed on
the same day, and the renal cortex was cut and homogenized. The tissue
was suspended in RIPA solution (1:8 ratio, wt/vol) containing (in mM)
10 NaCl, 1 NaF, 1% NP-40 (tergitol), 50 Tris · HCl, 1% Triton
(×100), 0.1% SDS, 1.5 NaVO4, 1 sodium malybdate, 1 paranitrophenyl-phenyl phosphate, and 1 EDTA. For every 125-mg tissue
sample, we added a 25-µl cocktail inhibitor of proteases (Sigma, St
Louis, MO). The samples were left on ice for 15 min and were followed
by homogenization. The homogenized tissue sample was incubated in the
presence of DNAse (1 µl) at 4°C for 60 min and followed by
centrifuging at 1,790 rpm for 10 min at 4°C. We have taken the
supernatant for measuring protein concentrations. We measured protein
concentrations of the whole cell extract twice. Only if the difference
between two assays was less than 5% did we consider that the
measurement was accurate. Moreover, we stained the gel with Comassie
blue to confirm that the amount of proteins was loaded equally. Also,
we performed the same Western blot three times for each sample and
normalized the results compared with the control value obtained from
rats on a normal diet. If the difference among three measurements was
less than 10%, we considered the results reliable. Protein samples
extracted from the kidney cortex were separated by electrophoresis on
8% SDS-polyacrylamide gels and transferred to nitrocellulose
membranes. The membranes were blocked with 10% nonfat dry milk in
Tris-buffered saline (TBS), rinsed, and washed with 1% milk in
Tween-TBS. The PTP-1D, PTP-1B, and PTP-1C antibodies were purchased
from Transduction Laboratories (Lexington, KY) and were diluted at
1:1,000. The cSrc and cYes antibodies were obtained from
Santa Cruz (Santa Cruz, CA) and were diluted at 1:1,000 and 1:500,
respectively. The antibody of cSrc recognizes the epitope corresponding
to amino acids 509-533 in the COOH-terminus of cSrc. In addition,
the cSrc antibody has a cross-reaction with cYesp62 and
Fynp59, two members of the Src family. The cYes
antibody reacts with the epitope corresponding to amino acids
3-18 in the NH2-terminus of cYes and has no
cross-reaction with other Src PTK. The immunogen of the PTP-1D antibody
is located between amino acids 1 and 177 in the
NH2-terminus and has no cross-reaction with other PTPs. The
ROMK antibody was obtained from Alomone Laboratories (Jerusalem,
Israel) and has been previously characterized by Mennitt et al.
(11). The protein concentration used for the immunoblot was 50 µg. The proteins were detected and quantitatively analyzed by
fluorescence phosphorimaging.
Experimental solutions and statistics.
The pipette solution contained (in mM) 140 KCl, 1.8 MgCl2,
and 10 HEPES (pH = 7.4). The bath solution was composed of (in mM)
140 NaCl, 5 KCl, 1.8 CaCl2, 1.8 MgCl2, 5 glucose, and 10 HEPES (pH = 7.4). Herbimycin A was purchased from
Biomol (Plymouth Meeting, PA) and dissolved in the DMSO solution. The
final concentration of DMSO was less than 0.1% and had no effect on
channel activity.
Data are shown as means ± SE, and paired or unpaired Student's
t-test was used to determine the significance between the
two groups. Statistical significance was taken as P < 0.05.
 |
RESULTS |
We have previously demonstrated that dietary K intake can affect
the expression of cSrc (21, 22). In the present study, we
have extended the investigation to examine the time course of the cSrc
expression in the kidneys from rats that were maintained on a KD diet
from 0 (control), 1, 3, 5, and 7 days. Figure
1A is a typical Western blot
showing the expression of cSrc in the renal cortex from animals on a KD
diet for 0, 1, 3, and 5 days. It is apparent that the expression of
cSrc increased progressively by prolonged KD adaptation. The expression
of cSrc was 140 ± 15% (1 day KD, n = 6 rats),
190 ± 46% (3 day KD, n = 6 rats), and 240 ± 60% (5 day KD, n = 6 rats) of the control value,
respectively. The cSrc expression in the renal cortex in rats on a KD
diet for 7 days was not different from that for 5 days (235 ± 33%, n = 5 rats) (data not shown). We also
investigated the expression of cYes, a member of the cSrc
family, in the renal cortex from rats on a KD diet. From inspection of
Fig. 1B, it is clear that the protein level of
cYes progressively increased when the animals were on a KD
diet (1-day, 135 ± 13%; 3-day, 170 ± 20%; and 5-day KD,
190 ± 20% of the control value, n = 4 rats).
However, the expression of PTP-1D was not affected by dietary K intake
(Fig. 1C, 1 day, 95 ± 13%, 3 days, 115 ± 22%,
and 5 days, 110 ± 20% of the control value, n = 6 rats). Moreover, we did not detect the expression of PTP-1B and
PTP-1C in the renal cortex (data not shown).

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Fig. 1.
Three representative Western blots show the expression of cSrc
(A), Yes (B), and protein tyrosine
phosphatase (PTP)-1D (C) in the renal cortex from rats on a
normal (control), 1-day K-deficient (KD), 3-day KD, and 5-day KD diet,
respectively. The protein concentration of each lane was 50 µg. PC,
positive control.
|
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After finding that dietary K intake affected the expression of cSrc, we
examined the effect of herbimycin A on the SK channels to determine
whether its effect was correlated with the cSrc level. We confirmed the
previous finding that inhibiting PTK increased the number of SK
channels in the CCD from rats on a KD diet. Figure 2 is a recording illustrating the effect
of 1 µM herbimycin A on the SK channel in a CCD from a rat on a KD
diet for 5 days. Inhibiting PTK opened a K channel in a cell-attached
patch within 10-15 min. The measurement of channel conductance (35 pS) and analysis of kinetics revealed that the K channel was a typical SK channel. However, the effect of herbimycin A on channel activity depends on the duration of K depletion because the addition of herbimycin A increased the channel activity only in one out of nine
cell-attached patches from rats on a KD diet for 1 day (Table 2). A prolonged time for KD adaptation
progressively increased the responsiveness of the SK channels to
herbimycin A. Table 2 summarizes the results of experiments in which
the effect of herbimycin A on the SK channels was examined. The
positive response of the SK channels to the agent was observed in 3 out
of 10 patches (3-day KD), 4 out of 8 patches (5-day KD), and 16 out of
22 patches (7-day KD), respectively. Figure
3 shows that inhibiting a PTK-induced increase in NPo/patch was 0.30 ± 0.3 (normal, n = 9), 0.45 ± 0.3 (1-day KD,
n = 9), 0.65 ± 0.3 (3-day KD, n = 10), 1.55 ± 0.5 (5-day KD, n = 8), and 1.85 ± 0.3 (7-day KD, n = 22), respectively. The data represent a mean increase in NPo,
including the experiments in which the response to herbimycin A was
negative (zero increase).

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Fig. 2.
A
channel recording showing the effect of herbimycin A (1 µM) on the
small-conductance K (SK) channel in a cell-attached patch in the
cortical collecting duct (CCD) from rats on a KD diet for 5 days. The
top trace shows the time course, and 2 parts of the trace
are extended to demonstrate the fast time resolution. C, channel-closed
level.
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Fig. 3.
The effect of herbimycin A (1 µM ) on the activity of
the SK channels in the CCD from rats on a normal 0-day KD, 1-day KD,
3-day KD, 5-day KD, and 7-day KD diet, respectively. All data are
significantly different from that of untreated tubules. Values are
means ± SE. NPo, a product of channel number
(N) and open probability (Po).
*Significantly different from the value obtained from rats on a normal
diet (0-day KD), P < 0.05.
|
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Our findings and those of others (15) confirmed that
NPo was not significantly different between rats
on a normal diet or on a KD diet and were 0.75 ± 0.2 (normal,
n = 33 patches), 0.7 ± 0.2 (1-day KD,
n = 46 patches), 0.65 ± 0.15 (3-day KD,
n = 50 patches), 0.62 ± 0.15 (5-day KD,
n = 31 patches), and 0.6 ± 0.15 (7-day KD,
n = 43 patches), respectively. However, treatment of the CCD with herbimycin A (1 µM) for 20 min increased the
NPo/patch to 1.3 ± 0.2 (normal,
n = 47 patches), 1.4 ± 0.3 (1-day KD,
n = 71 patches), 1.60 ± 0.2 (3-day KD,
n = 78 patches), 2.6 ± 0.3 (5-day KD,
n = 49 patches), and 3.5 ± 0.4 (7-day KD,
n = 67 patches), respectively (Fig.
4).

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Fig. 4.
NPo in the tubules treated with
herbimycin A or in the untreated CCDs from rats on a normal 0-day KD,
1-day KD, 3-day KD, 5-day KD, and 7-day KD diet, respectively. All data
obtained from the tubules treated with herbimycin A are significantly
higher than that in untreated counterparts. Values are means ± SE. *Effect of herbimycin A in the CCDs from rats on a KD diet for more
than 5 days is significantly larger than that from rats on a control
diet (0-day KD), P < 0.05.
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In previous experiments, we observed that inhibiting PTK with
herbimycin A cannot increase the channel activity in the CCD from rats
on an HK diet for 10 days (21). Furthermore, we have demonstrated that the cSrc expression decreased in the renal cortex from rats on a HK diet (21). We have now expanded our
study to examine the time course of cSrc expression in the renal cortex from rats on a HK diet. Figure 5 is a
Western blot demonstrating the protein level of cSrc and PTP-1D in the
renal cortex from rats on a HK diet for 1, 2, and 3 days, respectively.
The protein level of cSrc in the renal cortex dropped by 40 ± 10% (1-day HK, n = 4 rats), 60 ± 11% (2-day HK,
n = 4 rats), and 75 ± 15% (3-day HK,
n = 4 rats). In contrast, we did not observe a
significant change in PTP-1D levels. This is consistent with previous
observations that the PTP-1D expression was not affected by an HK
intake.

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Fig. 5.
Western blots show the expression of cSrc (A)
and PTP-1D (B) in the renal cortex from rats on a normal
1-day high-K (HK), 2-day HK, and 3-day HK diet, respectively. The
protein concentration of each lane was 50 µg. The same membrane was
used for detecting PTP-1D after the cSrc antibody was stripped from the
membrane.
|
|
We confirmed that NPo increased by ~4-fold,
from 0.75 ± 0.2 to 3.4 ± 0.4 in the CCD from rats on an HK
diet for 24 h (15). This increase was not the result
of changing the protein level of the SK channels. Figure
6 is a Western blot demonstrating that the ROMK channel protein in the renal cortex and renal medulla in rats
on a KD diet was even slightly higher (120 ± 18%) than those on
a HK diet, although the difference is not significant (n = 4 rats). It is generally believed that the ROMK
channel is the key component of the native SK channels in the CCD and
in the thick ascending limb (14, 20). We have also
investigated the effect of herbimycin A on the activity of the SK
channels in the CCD from rats on a HK diet. The addition of herbimycin A (1 µM) failed to increase the number of the SK channels in the CCD
from rats on a HK diet (Table 2). Moreover, treatment of the tubule
with herbimycin A did not increase the channel activity. Figure
7 summarizes the results of experiments
in which NPo was measured after the tubules were
treated with herbimycin A (1 µM) for 20 min. It is apparent
that the effect of herbimycin A on channel activity was completely
absent in the CCD from rats on an HK diet. The
NPo from the CCD treated with herbimycin A was 3.5 ± 0.4 (1-day HK, n = 33 patches), 3.6 ± 0.3 (2-day HK, n = 21 patches), and 3.7 ± 0.3 (3-day HK, n = 27 patches), which are not significantly
different from the corresponding values from untreated CCDs.

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Fig. 6.
Western blot showing the expression of the ROMK channel
in the renal cortex and medulla from rats on an HK diet and on a KD
diet (LK) for 7 days, respectively. The protein concentration was 50 µg for each lane.
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Fig. 7.
The NPo in the tubules treated
with 1 µM herbimycin A or in the untreated CCDs from rats on a normal
0-day HK, 1-day HK, 2-day HK, and 3-day HK diet, respectively. Values
are means ± SE. *Significantly different from corresponding
untreated CCDs, P < 0.05.
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 |
DISCUSSION |
The main finding of the present study is that the protein levels
of cSrc increased progressively when the rats were maintained on a KD
diet whereas they decreased when the rats were on a HK diet. Moreover,
we have demonstrated that the effect of herbimycin A on channel
activity was augmented progressively in proportion to the increase in
cSrc levels. We confirmed Palmer's observation that the basal level of
NPo in the CCD from rats on a KD diet for 7 days
was not significantly different from that in rats on a normal diet
(13). However, the effect of herbimycin A on the channel
activity in the CCD from rats on a normal chow was significantly smaller than that observed in the rats on a KD diet for 7 days. Moreover, treatment of the tubule with herbimycin A did not increase channel NPo in the CCD from rats on a normal
chow to the extent that was observed in the CCD from rats on a HK diet.
In contrast, inhibiting PTK increased NPo in the
CCDs from rats on a KD diet for 7 days to the same level as those
observed in rats on a HK diet. This suggests that the longer the
animals are on a KD diet, the larger the PTK-sensitive pool of SK channels.
We speculate that there could be three pools of SK channels in the CCD
under physiological conditions. The first group of SK channels is open
and actively involved in K exit across the apical membrane. The second
group of SK channels is phosphorylated by PTK and internalized or
subjected to endocytosis. The third group of SK channels is not
phosphorylated by PTK; however, it remains inactive. When rats are on
the KD diet, the expression levels and activity of cSrc increase.
Therefore, the tyrosine phosphorylation of SK channels by PTK is
enhanced, and the population of the second group is expanded.
Accordingly, inhibiting PTK with herbimycin A increases the number of
SK channels in the CCD from rats on a KD diet. In contrast, when the
animals are on an HK diet, the number of the active SK channels
(pool 1) must increase. Moreover, because the PTK level
falls, the population of SK channels that are phosphorylated by PTK is
diminished. In this case, inhibition of PTK cannot stimulate the
channel activity. In addition, it is possible that the number of the
silent SK channels that are not phosphorylated by PTK may also
decrease. Also, it is conceivable that only a small fraction of SK
channels are phosphorylated by PTK in the CCD from rats on a normal
diet. This view is supported by the observation that inhibiting PTK can
only modestly increase the channel activity.
Also, we found that the expression of ROMK channels in the renal cortex
and medulla from animals on a HK diet was not significantly different
from that in those on a KD diet. Because ROMK channels are located in
the CCD and cortical thick ascending limb (1), this
suggests that the expression of ROMK channels in the CCD may not be
affected by dietary K intake. Interestingly, it was recently reported
that the ROMK protein level in the membrane fraction decreased in the
renal cortex and medulla in kidneys from rats on a KD diet
(11). Because we used the homogenized tissue to detect the
expression of ROMK channels in the renal cortex and medulla, the
difference in the ROMK channel expression between the membrane fraction
and the whole homogenized tissue strongly suggests that a significant
fraction of ROMK channels are located in intracellular compartments.
Our previous observation that inhibiting PTK increased
NPo in the CCD from rats on a KD diet for 10 days to the same extent as that observed in rats on a HK diet indicated
that a low-K-intake-induced decrease in channel activity is mediated by
a PTK-dependent endocytosis (21). This notion was further
supported by experiments in which inhibiting PTP reduced the number of
the SK channels in the CCD from rats on a HK diet, and the effect could
be blocked by 20% sucrose that blocks the endocytosis
(22). Recently, we further demonstrated that stimulation
of PTK increases the endocytosis of ROMK1 in oocytes expressing cSrc
and ROMK1 (12). Therefore, our data strongly suggest that
PTK plays a key role in mediating the effect of dietary K intake on the
K secretion.
It has been shown that the channel activity increased significantly
when rats were on a HK diet for as few as 6 h (15). The stimulatory effect of HK intake on the SK channels is not the
result of increasing transcription of the gene encoding ROMK channels
(5) or increasing the expression of ROMK channels (11). Also, the effect of HK intake cannot be mimicked by
aldosterone infusion (13). Although we did not measure the
level of cSrc in the renal cortex 6 h after the rats were on an HK
diet, it is unlikely that a decrease in PTK expression and
dephosphorylation of the SK channels by PTP could account for the
HK-induced increase in the channel activity. This conclusion is based
on the observation that inhibition of PTK in the CCD from rats on a
normal diet did not mimic the effect of an HK diet on channel activity.
Therefore, it is possible that the rapid increase in channel activity
observed in the CCD from rats on a HK diet for 6 h may have
resulted from opening the previously silent SK channels that were not
phosphorylated by PTK and that were located in the cell membrane. This
hypothesis that the ROMK channel expression in the membrane fraction is
not significantly different between the animals on a normal diet or an
HK diet is supported by Mennitt et al. (11). However, the signal transduction pathway activating the silent SK channels in
response to an increase in K intake is still unclear. Because cAMP-dependent protein kinase [protein kinase A (PKA)] has been shown
to play an important role in stimulating SK channels acutely (2,
19), it is possible that PKA may be involved in mediating the
effect of an HK intake on channel activity in the CCD. In addition to
PKA, we have previously demonstrated that protein kinase C (PKC) and
calmodulin-dependent kinase II are also involved in regulating the SK
channels (9, 19). Therefore, it is conceivable that an HK
diet may stimulate channel activity by downregulating PKC and
calmodulin-dependent kinase activity. It is likely that PTK is
responsible for modulating the SK channels chronically in response to
changing K intake whereas PKA/PKC is involved in regulating channel
activity acutely. Further experiments are needed to prove this hypothesis.
In the present study, we confirmed the previous finding that PTP-1D is
the major isoform of PTP. In addition, the expression of PTP-1D is not
significantly regulated by K intake. Because the tyrosine
phosphorylation and dephosphorylation are controlled by an interaction
between PTP and PTK (8, 16), changes in PTK expression and
activity should be mainly responsible for altering the tyrosine
phosphorylation process: an increase in PTK levels should favor
tyrosine phosphorylation whereas a decrease in PTK should facilitate
tyrosine dephosphorylation. However, changes in PTP activity can still
have an important role in regulating the tyrosine
phosphorylation/dephosphorylation process. For instance, we have
previously demonstrated that inhibiting PTP decreased the
NPo of the SK channels in the CCD from rats on
an HK diet and that the effect of phenylarsine oxide was abolished by
inhibiting PTK (22). This suggests that PTK in the CCD
from rats on an HK diet is still active. Therefore, inhibiting PTP can
still enhance the PTK-dependent tyrosine phosphorylation in the CCD
from rats on an HK diet. Thus it is still possible that the activity of PTP-1D is different in the CCD from rats on a HK diet or on a KD diet.
Also, as only cSrc and cYes have been closely examined in
this study, it is conceivable that other isoforms of PTK are involved
in mediating the effect of dietary K intake on channel activity. We
have chosen cSrc and cYes in the present study because both
PTKs are the most abundant in the Src family and are ubiquitously distributed in different tissues such as the kidney (17).
It is well established that cSrc has been demonstrated to play an important role in regulating a variety of cell functions including modulating channel activity (17). The mechanism by which a
low-K+ intake increases the cSrc PTK level is not clear. K
depletion has an effect on cell proliferation and possibly could change the population of collecting duct cells in the kidney. Although we
could not completely exclude the possibility that increasing cSrc PTK
levels are partially the result of growing collecting duct cells, it is
most likely that increasing cSrc levels are the result of stimulating
cSrc expression in the previously existing cells. This conclusion is
based on our observation (Wang W, unpublished observations) that an
increase in protein expression in the renal cortex from rats on a KD
diet can only be observed in <5% of protein bands detected by
Comassie blue staining gel compared with control samples. This
suggests that increasing cSrc PTK expression in the renal cortex from
rats on a KD diet did not result from a nonspecific stimulation of
protein synthesis.
We conclude that the interaction between PTK and PTP plays a key role
in mediating the effect of dietary K intake on the SK channels in the
CCD. K depletion increases the expression of PTK and stimulates
tyrosine phosphorylation. In contrast, HK intake decreases the PTK
levels and augments tyrosine dephosphorylation.
 |
ACKNOWLEDGEMENTS |
This work is supported by National Institute of Diabetes and
Digestive and Kidney Diseases grant DK-47402.
 |
FOOTNOTES |
Address for reprint requests and other correspondence: W. H. Wang, Dept. of Pharmacology, New York Medical College, Valhalla, NY
10595 (E-mail wenhui_wang{at}nymc.edu).
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. Section 1734 solely to indicate this fact.
Received 11 December 2000; accepted in final form 6 April 2001.
 |
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Am J Physiol Renal Fluid Electrolyte Physiol 281(2):F206-F212
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