Effects of protein tyrosine kinase and protein
tyrosine phosphatase on apical K+ channels in the TAL
Rui-Min
Gu1,
Yuan
Wei1,
John R.
Falck2,
U. Murali
Krishna2, and
Wen-Hui
Wang1
1 Department of Pharmacology, New York Medical College,
Valhalla, New York 10595; and 2 Department of Biochemistry,
University of Texas Southwestern Medical Center at Dallas, Dallas,
Texas 75390
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ABSTRACT |
We have
previously demonstrated that the protein level of c-Src, a
nonreceptor type of protein tyrosine kinase (PTK), was higher in the
renal medulla from rats on a K-deficient (KD) diet than that in rats on
a high-K (HK) diet (Wang WH, Lerea KM, Chan M, and Giebisch G. Am J Physiol Renal Physiol 278: F165-F171, 2000).
We have now used the patch-clamp technique to investigate the role of
PTK in regulating the apical K channels in the medullary thick
ascending limb (mTAL) of the rat kidney. Inhibition of PTK with
herbimycin A increased NPo, a product of channel
number (N) and open probability (Po),
of the 70-pS K channel from 0.12 to 0.42 in the mTAL only from rats on
a KD diet but had no significant effect in tubules from animals on a HK
diet. In contrast, herbimycin A did not affect the activity of the
30-pS K channel in the mTAL from rats on a KD diet. Moreover, addition
of N-methylsulfonyl-12,12-dibromododec-11-enamide, an agent
that inhibits the cytochrome P-450-dependent production of
20-hydroxyeicosatetraenoic acid, further increased
NPo of the 70-pS K channel in the presence of
herbimycin A. Furthermore, Western blot detected the presence of
PTP-1D, a membrane-associated protein tyrosine phosphatase (PTP), in
the renal outer medulla. Inhibition of PTP with phenylarsine oxide
(PAO) decreased NPo of the 70-pS K channel in
the mTAL from rats on a HK diet. However, PAO did not inhibit the
activity of the 30-pS K channel in the mTAL. The effect of PAO on the
70-pS K channel was due to indirectly stimulating PTK because
pretreatment of the mTAL with herbimycin A abolished the inhibitory
effect of PAO. Finally, addition of exogenous c-Src reversibly blocked
the activity of the 70-pS K channel in inside-out patches. We conclude
that PTK and PTP have no effect on the low-conductance K channels in
the mTAL and that PTK-induced tyrosine phosphorylation inhibits,
whereas PTP-induced tyrosine dephosphorylation stimulates, the apical
70-pS K channel in the mTAL.
hypokalemia; hyperkalemia; c-Src; ROMK channel; herbimycin A; phenylarsine oxide
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INTRODUCTION |
THE APICAL K
CHANNELS in the medullary thick ascending limb (mTAL) play an
important role in K recycling across the apical membrane of the mTAL
(6, 8, 27). K recycling is essential for maintaining the
function of the Na-K-Cl cotransporter, and inhibition of the apical K
channels diminishes the transport rate of epithelial NaCl in the mTAL
(6, 8). We have previously demonstrated that the open
probability of the apical K channels in the mTAL was significantly
lower in animals on a K-deficient (KD) diet than that in animals on a
high-K (HK) diet (9). It is conceivable that a decrease in
the activity of the apical K channels is partially responsible for
hypokalemia-induced impairing of the transepithelial transport in the
mTAL (10, 19, 23). Although the mechanism by which K
depletion reduces the activity of the apical K channels is not
completely understood, several factors have been suggested to be
responsible for the effect of K depletion on the apical K channels in
the mTAL. For instance, it was demonstrated that K depletion
significantly decreased the expression of ROMK channels in the cell
membrane of the renal medulla (16). We have also reported
that 20-hydroxyeicosatetraenoic acid (HETE) production increased in the
mTAL harvested from rats on a KD diet and that inhibition of cytochrome
P-450-dependent 20-HETE production significantly increased
the activity of the 70-pS K channel in the mTAL from rats on a KD diet
(9). This suggests that a posttranslational modulation of
channel activity is also involved in downregulating the activity of the
apical K channels.
Furthermore, earlier investigation found that the expression and
activity of c-Src increased significantly in the renal cortex and
medulla in rats on a KD diet (28). Moreover, inhibition of
protein tyrosine kinase (PTK) with herbimycin A significantly stimulated the low-conductance K channel in the cortical collecting duct (CCD) from rats on a KD diet as well as the ROMK1 channel in
Xenopus oocytes coexpressed with c-Src (17,
28). In the present study, we examined the role of PTK and
protein tyrosine phosphatase (PTP) in regulating the apical K channels
in the mTAL. We discovered that stimulation of tyrosine phosphorylation
decreases, whereas stimulation of tyrosine dephosphorylation increases,
the activity of the apical 70-pS K channel but has no effect on the 30-pS K channel in the mTAL.
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METHODS |
Preparation of the TAL.
Pathogen-free Sprague-Dawley rats (50-60 g, either sex) were
purchased from Taconic (Germantown, NY). The animals were pair fed with
either a HK diet (10% wt/wt) or a KD diet (<0.001% wt/wt) (Harlan,
Teklad, Madison, WI) for 10 days before use. The plasma Na and K
concentrations in animals on a different K diet were measured with
flame photometry (Corning 480), and the results are included in
Table 1. The rats were killed by cervical
dislocation after anesthesia with Metofane. The kidneys were removed
immediately, and thin coronal sections were cut with a razor blade. We
used only medullary TALs in the study. The dissection buffer solution contained (in mM) 140 NaCl, 5 KCl, 1.8 MgCl2, 1.8 CaCl2, 5 glucose, and 10 HEPES (pH 7.4 with NaOH) at
22°C. The isolated tubule was transferred onto a 5 × 5-mm cover
glass coated with Cell-Tak (Collaborative Research, Bedford, MA) to
immobilize the tubule. The cover glass was placed in a chamber mounted
on an inverted microscope (Nikon), and the tubules were superfused with
HEPES-buffered NaCl solution composed of (in mM) 140 NaCl, 5 KCl, 1.8 CaCl2, 1.8 MgCl2, and 10 HEPES (pH 7.4). The
mTAL was cut open with a sharpened micropipette to gain access to the
apical membrane. The temperature of the chamber (1,000 µl) was
maintained at 37 ± 1°C by circulating warm water around the
chamber.
Patch-clamp technique.
An Axon 200B patch-clamp amplifier was used to record channel current.
The current was low-pass filtered at 1 kHz using an eight-pole Bessel
filter (902LPF; Frequency Devices, Haverhill, MA) and digitized by an
Axon interface (Digitada 1200). The data 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). Opening and closing transitions were detected using
50% of the single-channel amplitude as the threshold. Channel activity
was defined as NPo, a product of channel number
(N) and open probability (Po). The
NPo was calculated from data samples of 60-s
duration, which were always at the end of each experimental maneuver
and in the steady state. We used the following equation to obtain
NPo
|
(1)
|
where ti is the fractional open time
spent at each of the observed current levels. Because two types of K
channels are present in the apical membrane of mTAL, two types of K
channels sometimes could be detected in the same patches. To avoid the
complexity, we selected those patches with only a single population of
K channels to calculate NPo. If the
low-conductance K channel appeared during the course of the
experiments, we used a ruler to calculate NPo manually. The mean delay time for the effect of herbimycin A or phenylarsine oxide (PAO) was ~10 min. The change in channel activity was clearly related to the application of a given chemical because the
channel activity was quite stable (changes in
NPo were <10% over a 10-min period) in the
absence of herbimycin A or PAO. Moreover, we have selected the results
only if the effect of a given chemical was at least partially reversible.
Western blot.
Protein samples extracted from the renal outer medulla 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 antibodies for c-Src, PTP-1D, PTB-1B, and PTP-1C
were purchased from Transduction Laboratories (Lexington, KY) and were
diluted at 1:1,000. The protein concentration used for immunoblot was
50 µg. The PTPs and c-Src were detected and quantitatively analyzed
by fluorescence phosphorimaging.
Chemicals and experimental solution.
The pipette solution contained (in mM) 140 KCl, 1.8 mM
MgCl2, and 10 HEPES (pH 7.4). Herbimycin A and PAO were
purchased from Sigma (St. Louis, MO) and dissolved in DMSO. The final
concentration of DMSO was <0.1% and had no effect on channel
activity. The chemicals were added directly to the bath to reach the
final concentration. N-methylsulfonyl-12,12-dibromododec-11-enamide (DDMS) was
synthesized at J. R. Falck's laboratory, University of Texas
Southwestern Medical Center at Dallas, and has been shown to
specifically block cytochrome P-450
-hydroxylation of
arachidonic acid (25).
Statistics.
Data are shown as means ± SE. We used paired Student's
t-tests to determine the significance of difference between
the control and experimental periods. Statistical significance was
taken as P < 0.05.
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RESULTS |
To explore the role of PTK in regulating the activity of the
apical K channels in the mTAL, we investigated the effect of herbimycin
A, an inhibitor of PTK, on the activity of the apical 70-pS K channel
and 30-pS K channel, respectively. Figure
1A is a representative
recording showing the effect of herbimycin A (1 µM) on the apical
70-pS K channel in the mTAL from rats on a KD diet. We confirmed the
previous observation that channel activity was low
(NPo = 0.12 ± 0.05) in the mTAL from
animals on a KD diet (9). Moreover, inhibition of PTK
significantly stimulated the 70-pS K channel and increased
NPo to 0.42 ± 0.1 (n = 30)
(Fig. 1, A and B). Herbimycin A also stimulated
the channel activity in the mTAL from rats on a normal diet and
increased NPo modestly from 0.42 ± 0.1 to
0.56 ± 0.1 (n = 6). In contrast, the effect of
herbimycin A on the 70-pS K channel is almost absent in the mTAL from
rats on a HK diet. From inspection of Fig. 1B, it is
apparent that inhibition of PTK slightly increased
NPo from 0.81 ± 0.16 to 0.91 ± 0.12 (n = 19). However, the difference is not significant.

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Fig. 1.
A: channel recording shows effect of 1 µM
herbimycin A on activity of the apical 70-pS K channel in a
cell-attached patch in the medullary thick ascending limb (mTAL) from
rats on a K-deficient (KD) diet. Top trace demonstrates time
course of the experiment, and 2 parts of the trace indicated by numbers
are extended to show the fast-time resolution. The channel-closed level
is indicated by C, and the holding potential is 0 mV. B:
effect of herbimycin A on channel open probability
(NPo) in the mTAL from animals on a KD diet,
normal-K (NK) diet, or high-K (HK) diet. * Data are significantly
different from the corresponding control value. Control bars represent
channel activity obtained in absence of herbimycin A in rats on a KD,
NK, or HK diet, respectively.
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Because inhibition of cytochrome P-450
-oxidation has
been shown to stimulate the activity of the apical 70-pS K channel in
the mTAL from rats on a KD diet (9), we examined the
effect of inhibiting 20-HETE production on channel activity in the
presence of a PTK inhibitor. Figure
2A is a recording
demonstrating that, in the presence of herbimycin A, DDMS, an agent
that inhibits cytochrome P-450
-oxidation, further
stimulated the activity of the 70-pS K channel. This suggests that a
mechanism other than stimulating PTK is involved in mediating the
inhibitory effect of 20-HETE on channel activity. Figure 2B
summarizes the results of 12 experiments showing that inhibition of
cytochrome P-450 monooxygenase further increased
NPo to 0.85 ± 0.1 in the presence of
herbimycin A. Thus it is possible that effects of 20-HETE and PTK are
mediated by at least two different signal transduction pathways.

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Fig. 2.
A: channel recording illustrates effects of
herbimycin A (1 µM) and
N-methylsulfonyl-12,12-dibromododec-11-enamide (DDMS; 5 µM) on the apical 70-pS K channels. The experiment was carried out in
a cell-attached patch from the mTAL of a rat on a KD diet, and the
holding potential was 0 mV. Three parts of the trace from the top
recording indicated by numbers are extended to demonstrate the
fast-time resolution. B: effect of herbimycin A and DDMS on
NPo in the mTAL from rats on a KD diet.
* Data are significantly different from control value. Control bar
represents channel activity calculated in absence of herbimycin A and
DDMS in rats on a KD diet.
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We also examined the effect of herbimycin A on the 30-pS K channel in
the mTAL from rats either on a HK diet or on a KD diet. Figure
3 summarizes the result of 20 experiments
demonstrating that herbimycin A did not increase
NPo of the 30-pS K channel in the mTAL from rats
on a KD diet (control, 0.21 ± 0.1; herbimycin A, 0.25 ± 0.1) or from that in rats on a HK diet (control, 0.82 ± 0.1;
herbimycin A, 0.85 ± 0.1). Therefore, the result suggests that
PTK does not modulate the activity of the 30-pS K channel in the mTAL.

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Fig. 3.
Effect of herbimycin A (1 µM) on activity of the 30-pS
K channel in the mTAL from rats on a KD diet and on a HK diet,
respectively. Experiments were performed in cell-attached patches.
Control represents channel activity in absence of herbimycin A in rats
on a KD diet and on a HK diet, respectively.
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After establishing that an increase in PTK activity may be involved in
mediating the effect of low K intake on the apical 70-pS K channels in
the mTAL, we examined the expression of c-Src, a nonreceptor type of
PTK that is widely distributed in a variety of tissues
(12). Figure 4B
is a typical Western blot showing the expression of c-Src in renal
outer medulla from rats on a HK diet (10 days), normal-K diet, or KD
diet for 10 days. Clearly, the expression of c-Src was significantly
higher in rats on a KD diet (mean increase by 190 ± 20%) than
that in rats on a normal diet. Moreover, the expression of
c-Src decreased significantly by 70 ± 15% (n = 4 rats) in the renal outer medulla from rats on a HK diet compared with
those on a normal diet. Because the tyrosine phosphorylation is
determined not only by PTK but also by PTP (12), we
investigated the expression of three membrane-associated PTP isoforms,
PTP-1B, PTP-1C, and PTP-1D, in the kidney from rats on a HK and on a KD
diet, respectively. We failed to detect the PTP-1B and PTP-1C in the
kidney (data not shown). However, PTP-1D was expressed in the renal
outer medulla and cortex (Fig. 4A). Moreover, its expression
was not significantly altered by K diet. This suggests that the change
in the PTK expression may be an important mechanism to determine the
tyrosine phosphorylation level of the 70-pS K channel in the mTAL.
However, PTP can still play an important role in modifying the
regulation of channel activity by PTK because the interaction of PTP
and PTK determines the tyrosine phosphorylation. This notion is
demonstrated by experiments in which inhibiting PTP reduced the apical
70-pS K channel activity in the mTAL from rats on a HK diet. Figure
5 is a typical recording showing the
effect of PAO on the activity of the 70-pS K channel in the mTAL from
rats on a HK diet. Addition of 1 µM PAO inhibited the activity of the
70-pS K channel, and NPo dropped from 0.82 ± 0.1 to 0.2 ± 0.04 (data not shown). In contrast, PAO had no significant effect on the 30-pS K channel in the mTAL from rats on a HK
diet (Fig. 6), and
NPo was almost identical (control 0.82 ± 0.1, PAO 0.81 ± 0.1). This further supports the notion that the
activity of the 30-pS K channel is not regulated by PTP and PTK. The
effect of PAO on the 70-pS K channel can also be observed in the mTAL
from rats on a KD diet or on a normal diet (data not shown). The effect
of PAO on the 70-pS K channel is produced by inhibiting PTP because
application of PAO had no effect on channel activity in inside-out
patches (data not shown). Moreover, herbimycin A treatment abolished
the inhibitory effect of PAO (Fig.
7A). Figure 7B
summarizes the results of 16 experiments showing that PAO reduced the
channel activity only by 9 ± 1% from 0.95 ± 0.14 to
0.86 ± 0.12 in the presence of herbimycin in the mTAL from rats
on a HK diet.

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Fig. 4.
Western blots show expression of PTP-1D in the renal cortex and
outer medulla from rats on a HK or KD diet (A) and
expression of c-Src in the renal outer medulla in rats on a HK, NK
(control), or KD diet (B). PC, positive control.
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Fig. 5.
Recording showing effect of phenylarsine oxide (PAO; 1 µM) on the apical 70-pS K channel in the mTAL from a rat on a HK
diet. Three parts of the trace indicated by numbers are extended to
show the fast-time resolution. The pipette holding potential was 0 mV.
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Fig. 6.
Recording showing effect of PAO (1 µM) on the 30-pS K
channel in the mTAL from a rat on a HK diet. The experiment was carried
out in a cell-attached patch, and the channel-close level is indicated
by C. Top trace shows time course of the experiment, and 2 parts of the trace are demonstrated at a fast-time resolution. The
holding potential was 0 mV.
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Fig. 7.
A: channel recording demonstrates the effect
of PAO (1 µM) on the 70-pS K channel in the mTAL treated with
herbimycin A. Top trace shows time course of the experiment,
and 2 parts of the trace are demonstrated at a fast time resolution.
The experiment was performed in a cell-attached patch, and holding
potential was 0 mV. B: effect of PAO on the apical 70-pS K
channel in the presence of herbimycin A in the mTAL from rats on a HK
diet. Control bar represents channel activity in the absence of either
herbimycin A or PAO in rats on a HK diet.
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To explore the possibility that the 70-pS K channels are phosphorylated
directly and tyrosine phosphorylation results in an inhibition of
channel activity, we examined the effect of exogenous c-Src on the
70-pS K channel. Figure 8 is a channel
recording showing the effect of c-Src on channel activity in an
inside-out patch. Clearly, c-Src (1 nM) reversibly inhibited the 70-pS
K channel and reduced NPo by 95 ± 5%
(n = 4). From inspection of Fig. 8, it is also apparent
that c-Src did not affect the activity of the 30-pS K channel, which
was present in the same patch.

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Fig. 8.
Effect of exogenous c-Src (1 nM) on activity of the 70-pS
K channel in an inside-out patch. The bath solution contains 100 nM
free Ca2+ and 0.2 mM Mg ATP. Top trace shows
time course of the experiment, and 4 parts of the trace indicated by
numbers are extended to demonstrate the detail of channel activity. The
low-conductance K channel, indicated by an arrow, coexisted in the same
patch and is not affected by c-Src. The activity of c-Src is confirmed
independently in measuring 32P-labeled ATP incorporation
into c-Src-specific substrates.
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 |
DISCUSSION |
The main finding of the present study is that stimulation of PTK
decreased, whereas stimulation of PTP increased, the activity of the
apical 70-pS K channel. The second finding is that PTP and PTK did not
modulate the activity of the 30-pS K channels in the mTAL. We
previously demonstrated that inhibiting PTK increased the number of
low-conductance K channels in the CCD (28), a counterpart
of the 30-pS K channel in the mTAL (2, 11, 27, 32). Also,
we have shown that tyrosine residue 337 in the COOH terminus of ROMK1
is the key site for the effects of PTK and PTP because mutating the
tyrosine residue to alanine abolished the effects of PAO and herbimycin
A on ROMK1 (17). In situ hybridization study has shown
that ROMK1 is located in the CCD, whereas ROMK2 and ROMK3 are expressed
in the mTAL (2). Because the PTK consensus phosphorylation
site (tyrosine residue 318) is also present in ROMK2, the different
response to PTP and PTK must be attributed to the NH2
terminus of ROMK2, which is 19 amino acids shorter than that of ROMK1.
It is possible that the NH2 terminus may be required for
the tyrosine phosphorylation or dephosphorylation. Alternatively, the
NH2 terminus may be important for the downregulation of
ROMK1 channel activity after the tyrosine phosphorylation of the
channel. We have previously demonstrated that the effect of PTK on
ROMK1 is the result of stimulating endocytosis, whereas the effect of
PTP is mediated by exocytosis (17). Therefore, it is
possible that the NH2 terminus may be required for
PTK-induced endocytosis or PTP-induced exocytosis. Relevant to the
possibility that the NH2 terminus of ROMK channels
may have an important function for the channel regulation was our
preceding observation that arachidonic acid blocks ROMK1 but has
no effect on either ROMK2 or ROMK3 (15). Moreover,
mutation of serine residue 4 [a putative protein kinase C (PKC)
phosphorylation site of ROMK1] to alanine can largely abolish the
effect of arachidonic acid on ROMK1. This suggests that the PKC
phosphorylation site plays a key role in rendering ROMK1 the
sensitivity to arachidonic acid. However, it is not known whether the
PKC phosphorylation site is also essential for mediating the effect of
PTK. We need further experiments to explore the role of the
NH2 terminus in mediating the effect of PTK.
In the present study, we used PAO to demonstrate the role of PTP and
herbimycin A to show the effect of PTK. Three lines of evidence
suggested that the effects of PAO and herbimycin A are specific for
inhibiting PTP and PTK, respectively. First, the effect of herbimycin A
was observed in the mTAL only in animals on a KD diet but not from
those on a HK diet. Second, the effect of PAO and herbimycin A was
absent in excised patches. Third, we failed to observe the stimulatory
effect of herbimycin A on channel activity in the mTAL treated with 2 µM PAO (unpublished observation), whereas the effect of PAO was also
abolished in the mTAL treated with herbimycin A. This suggests that the
effect of the two agents is the result of altering the balance of PTP and PTK interaction.
We confirmed the previous observation that the expression of c-Src in
the outer medulla of kidneys was significantly higher from rats on a KD
diet than that from animals on a HK diet (28). Also, we
have shown that the expression of PTP-1D was not changed in the kidney
by dietary K intake. However, it is conceivable that tyrosine
phosphorylation is enhanced in the mTAL from rats on a KD diet because
PTK activity increased severalfold. PTK has been shown to play an
important role in regulating a variety of K channels (3, 22, 31,
34). There are at least two mechanisms by which PTK modulates
the channel activity. First, PTK can alter the channel activity by
modifying endocytosis or recycling. It has been shown that PTK is
involved in regulating the endocytosis of G protein-coupled receptors
(14). Second, PTK can directly phosphorylate the ion
channels and accordingly regulate the channel activity. Relevant to the
first possibility is the observation that PTK decreases the number of
the apical small-conductance K channels in the CCD via endocytosis
(28). However, our data indicate that the effect of PTK
results from a direct phosphorylation of the 70-pS K channel or its
associated proteins because addition of exogenous c-Src blocks the
channel. It was shown that PTK suppresses delayed rectifying K channels
(22), voltage-gated K channels, Kv1.3 (3),
and Ca2+-dependent K channels by direct tyrosine
phosphorylation (20). Moreover, c-Src has been shown to
regulate the N-methyl-D-aspartate channel by
direct association and phosphorylation (31, 34).
There are three types of apical K channels (30-pS, 70-pS, and a
Ca2+-activated maxi-K channel) in the apical membrane of
the mTAL (1, 5, 26, 30). Moreover, the ROMK channel is the
most likely pore-forming subunit of the 30-pS K channel
(18). However, it still has not been established whether
the ROMK channel is also a part of the 70-pS K channel. It is generally
accepted that the 30-pS and 70-pS K channels are mainly responsible for
K recycling. The importance of K recycling in maintaining the function
of the Na-K-Cl cotransporter is best demonstrated by genetic studies in
which defective gene product encoding ROMK channel results in abnormal
renal salt transport (21). Hypokalemia has been shown to
impair the epithelial transport in the mTAL (10, 23). However, the mechanism by which K depletion impairs the epithelial transport in the mTAL is not completely understood. Because the 70-pS K
channel has an important role in K recycling, it is possible that a
diminished apical K recycling is partially responsible for the impaired
NaCl transport in the mTAL during hypokalemia. Moreover, PTK and PTP
should be involved in regulating NaCl transport in the mTAL because
they modulate the activity of the apical K channels. In this regard, it
is possible that increased c-Src activity in the kidney from animals on
a KD diet is partially responsible for impairing epithelial transport
in the mTAL during K depletion.
We have previously demonstrated that inhibition of cytochrome
P-450 monooxygenase increased channel activity
(9). We have now shown that an increase in PTK activity
may also be responsible for suppressing the channel activity in the
mTAL. One possible mechanism by which 20-HETE inhibits the 70-pS K
channel is that the 20-HETE effect may be mediated by stimulating PTK.
It has been reported that the effect of 20-HETE on the
Ca2+-activated K channel is mediated by PTK in smooth
muscle cells (24). Moreover, PTK has been suggested to
mediate the effect of 20-HETE on bicarbonate transport in the mTAL
(7). Finally, Chen et al. (4) have
demonstrated that 14,15-epoxyeicosatrienoic acid, a cytochrome
P-450 epoxygenase-dependent metabolite of arachidonic acid,
stimulates tyrosine phosphorylation in renal epithelial cells
(4). However, the observation that inhibition of
cytochrome P-450
-oxidation can further increase channel
activity in the presence of herbimycin A strongly suggests that the
inhibitory effect of 20-HETE on the 70-pS K channel is produced by a
mechanism other than stimulating PTK. It is most likely that the
effects of 20-HETE and PTK are independent, although we cannot
completely exclude the possibility that the partial effect of 20-HETE
may be achieved by stimulation of PTK.
We have previously demonstrated that the activity of the 70-pS K
channel is inhibited by PKC (29) and activated by protein kinase A (PKA) and cGMP-dependent kinase (13). In the
present study, we have shown that PTK is also an important regulator
for the 70-pS K channel. Moreover, it is possible that the regulation of the 70-pS K channel by PTK may require cross-talk among PKC, protein
kinase G, PKA, and PTK. Further experiments are needed to
explore this possibility. Although increasing PTK activity has an
important role in mediating the effect of dietary K intake on the 70-pS
K channel in the mTAL, it is still not understood how a low K intake
stimulates PTK activity. Because K depletion can cause intracellular
acidosis that has been shown to activate c-Src (33), it is
possible that intracellular acidosis is partially responsible for
increasing c-Src activity during hypokalemia.
We conclude that a low K intake increases the c-Src level in the renal
outer medulla. Increasing PTK activity inhibits, whereas stimulation of
PTP activates, the apical 70-pS K channel in the mTAL.
 |
ACKNOWLEDGEMENTS |
This work is supported by National Institutes of Health Grants
PO1-HL-34300, DK-47402, and GM-31278-0 and by the Robert A. Welch Foundation.
 |
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
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in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 20 February 2001; accepted in final form 17 May 2001.
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