Regulation of ROMK1 Channels by Protein-tyrosine
Kinase and -tyrosine Phosphatase*
Zebunnessa
Moral
,
Ke
Dong§,
Yuan
Wei
,
Hyacinth
Sterling
,
Huan
Deng
,
Shariq
Ali
,
RuiMin
Gu
,
Xin-Yun
Huang¶,
Steven C.
Hebert§,
Gerhard
Giebisch§, and
Wen-Hui
Wang
From the
Department of Pharmacology, New
York Medical College, Valhalla, New York 10595, the
§ Department of Cellular and Molecular Physiology, Yale
University School of Medicine, New Haven, Connecticut 06510, and the
¶ Department of Physiology, Cornell University Medical College,
New York, New York 10021
Received for publication, September 21, 2000, and in revised form, December 5, 2000
 |
ABSTRACT |
We have used the two-electrode voltage clamp
technique and the patch clamp technique to investigate the
regulation of ROMK1 channels by protein-tyrosine phosphatase (PTP) and
protein-tyrosine kinase (PTK) in oocytes coexpressing ROMK1 and cSrc.
Western blot analysis detected the presence of the endogenous PTP-1D
isoform in the oocytes. Addition of phenylarsine oxide (PAO), an
inhibitor of PTP, reversibly reduced K+ current by
55% in oocytes coinjected with ROMK1 and cSrc. In contrast, PAO had no
significant effect on K+ current in oocytes injected with
ROMK1 alone. Moreover, application of herbimycin A, an inhibitor of
PTK, increased K+ current by 120% and completely abolished
the effect of PAO in oocytes coexpressing ROMK1 and cSrc. The effects
of herbimycin A and PAO were absent in oocytes expressing the ROMK1
mutant R1Y337A in which the tyrosine residue at position 337 was
mutated to alanine. However, addition of exogenous cSrc had no
significant effect on the activity of ROMK1 channels in inside-out
patches. Moreover, the effect of PAO was completely abolished by
treatment of oocytes with 20% sucrose and 250 µg/ml concanavalin A,
agents that inhibit the endocytosis of ROMK1 channels. Furthermore, the
effect of herbimycin A is absent in the oocytes pretreated with either
colchicine, an inhibitor of microtubules, or taxol, an agent
that freezes microtubules. We conclude that PTP and PTK play an
important role in regulating ROMK1 channels. Inhibiting PTP increases
the internalization of ROMK1 channels, whereas blocking PTK stimulates
the insertion of ROMK1 channels.
 |
INTRODUCTION |
ROMK, a cloned inward rectifying K+ channel from the
renal outer medulla, is a key component of the small conductance
K+ channel identified in the thick ascending limb
and cortical collecting duct
(CCD)1 (1-3). This
conclusion is based on observations that the conductance, open
probability, opening and closing kinetics, and pH sensitivity of ROMK
are similar to that of the native small conductance K+
channel (1, 3, 4). Moreover, both K+ channels are regulated
by protein kinase A and protein kinase C (5-10). A difference between
the native small conductance K+ channel and ROMK is that
ROMK is insensitive to sulfonylurea agents, whereas the native small
conductance K+ channel is inhibited by sulfonylurea agents
(11-13). Three isoforms of ROMK, ROMK1, -2, and -3, have been found in
the rat kidney (14). Based on in situ hybridization, ROMK1
is located in the apical membrane of principal cells in the CCD,
whereas ROMK2 and -3 are expressed at the thick ascending limb (14).
The principal cell in the CCD is responsible for Na+
reabsorption and K+ secretion, which takes place by
K+ entering the cell across the basolateral membrane via
Na,K-ATPase followed by diffusion into the lumen across the apical
membrane through ROMK1-like channels (15).
We have previously demonstrated that inhibition of PTP reduced the
activity of the small conductance K+ channel in the apical
membrane of the CCD of rat kidney (16). Moreover, we have reported that
blocking PTK increased the number of the small conductance
K+ channels in the CCD obtained from rats on a
K+-deficient diet (17). Therefore, we have suggested that
PTK and PTP play a key role in the regulation of the small conductance K+ channel in the rat CCD.
In the present study, we have extended the investigation to explore the
mechanism by which PTK and PTP regulate ROMK1 channels expressed in
Xenopus oocytes. The reasoning for studying ROMK1 is that it
is exclusively expressed in the CCD. We have observed that tyrosine
residue 337 in the C terminus of ROMK1 is essential for mediating the
effects of PTK and PTP on the channel activity.
 |
EXPERIMENTAL PROCEDURES |
Preparation of Xenopus Oocytes--
Xenopus
laevis females were obtained from Nasco (Fort Atkinson, WI).
The method for obtaining oocytes has been described previously (18). We
removed the follicular layer of oocytes under a dissecting microscope
with two watchmaker forceps. After dissection, the oocytes were
incubated over night at 19 °C in a solution containing 66%
Dulbecco's modified Eagle's medium/F12 medium with freshly added 2.5 mM sodium pyruvate and 50 µg/ml gentamycin. Viable
oocytes were selected and micro-injected with ROMK1 cRNA alone (10 ng) or with a mixture of ROMK1 and cSrc(15 ng). The oocytes were incubated at 19 °C in a 66% Dulbecco's modified Eagle's medium/F12 medium, and experiments were performed on days 2-3 after injection. To remove
the vitellin membrane, oocytes were treated with hypertonic solution
containing 220 mM methylglucamine, 220 mM
aspartic acid, 2 mM MgCl2, 10 mM
EGTA, and 10 mM HEPES (pH 7.2).
Patch Clamp Technique--
Patch clamp electrodes were pulled
from glass capillary tubes (Dagan, Minneapolis, MN), with resistance of
4-6 megohms when filled with a solution containing 150 mM KCl, 1 mM CaCl2, 1 mM MgCl2, 5 mM HEPES, pH 7.4. An
Axonpatch200B patch clamp amplifier (Axon, Foster city, CA) was used to
record channel current. Channel currents were low pass-filtered at 1 kHz by an eight-pole Bessel filter (902LPF, Frequency Devices,
Haverhill, MA) and were digitized by a Digidata 1200 interface (Axon
Instruments, Burlington, CA). Data were acquired to a Pentium PC
(Gateway) at a sampling rate of 5 kHz and analyzed with pClamp software
system (6.04, Axon Instruments, Burlington, CA). Channel activity was
defined as NPo, which was calculated from data
samples of 30-60 s duration. We use the following equation to obtain
NPo,
|
(Eq. 1)
|
where ti is the fractional open time spent at
each of the observed current levels.
Two-electrode Whole Cell Voltage Clamp--
A Warner oocyte
clamp OC-725C was used to measure the whole cell K+
current. Voltage and current microelectrodes were filled with 1 M KCl and had resistance of less than 2 megohms. Series
resistance of the pipette was compensated. The current was recorded on
a chart recorder (Gould TA240). To exclude the leaky current, 2 mM Ba2+ was used before and after each maneuver
to determine the Ba2+-sensitive K+ current.
Western Blot--
Approximately 40 oocytes were transferred to a
polypropylene test tube containing 2 ml of buffer (20 mM
Tris, pH 7.5, 0.5 mM EDTA, 0.5 mM EGTA, 25 µg/ml leupeptin, 25 µg/ml aprotinin, 10 mM
-mercaptoethanol, 300 mM sucrose). Oocytes were
homogenized on ice in the following way. They were exposed to a
Polytron mechanical homogenizer for 5 s and then left unstirred
for an additional 5 s. This procedure was repeated three times.
Protein concentration was assayed using the Bradford standard assay,
with bovine serum albumin as a standard. The proteins were
separated by gel electrophoresis in 10% SDS-polyacrylamide and
transferred to a nitrocellulose membrane. Antibodies to cSrc and PTP1D
were obtained from Transduction Laboratories (Lexington, KY).
Fluorescence Localization of ROMK1--
Oocytes were injected
with cRNA encoding cSrc (15 ng) and GFP-ROMK1 (10 ng) or with GFP-ROMK1
alone. Three groups of oocytes (five oocytes/group) were studied: 1)
GFP-ROMK1; 2) cSrc and GFP-ROMK1; and 3) water. We examined the effects
of PAO and herbimycin A on the membrane expression of ROMK1 48 h
after injection with laser scanning confocal microscopy. Five sections
of each oocyte membrane were recorded, and the signal was averaged for
each egg. Oocytes were imaged using a Bio-Rad MRC1000 confocal
microscope. GFP fluorescence was excited at 488 nM with an
argon laser beam and viewed with an inverted Olympus microscope
equipped with a × 20 dry lens. XY scans were obtained at
approximately the midsection of each egg. All images were acquired,
processed, and printed with identical parameters. We used Scion Image
software (Scion Co., Frederick, MD) to determine the GFP fluorescence
intensity before (control) and after adding either PAO or herbimycin A. The background fluorescence taken from water-injected oocytes was
subtracted from the mean value of each measurement.
Preparation of cRNA for Oocyte Injection--
The preparation of
cRNA encoding the ROMK1 channel has been previously described (7). The
GFP-ROMK1 cDNA construct was prepared as follows. Full-length ROMK1
cDNA was made from PCDNA3.1/ROMK1 using the polymerase chain
reaction technique. The forward primer was
TTGTAGGTGGAAGGATCCTGCTACATCTGGGTGTCG, and the reverse primer was
TGGGCCTAAAAGAATTCAGCTGCTGTGCACGACAAC. The 1.2-kilobase cDNA digested with EcoRI and BamHI was cloned into
PEGFPC vector (CLONTECH) cut with the same
restriction enzymes. The sequence of the GFP-ROMK1 construct was
confirmed by sequencing (W. M. Keck Biotechnology Resource Laboratory,
Yale University, New Haven, CT). To make cRNA coding GFP-ROMK1,
cDNA of the fusion protein was subcloned into pSport vector and
transcribed in vitro from the T7 promotor. For preparation
of cRNA of cSrc that was subcloned into the ACC1/EcoRI site
of the pGEm3Z vector, the DNA of cSrc was linearized with EcoRI and transcribed in vitro by using the Sp6
transcription kit (Stratagene, La Jolla, CA).
Experimental Solution and Statistics--
The bath solution for
the patch clamp study and two-electrode voltage clamp was composed of
the following (in mM): 150 KCl, 2.5 MgCl2, 1.8 CaCl2, 1 EGTA, 5 HEPES (pH 7.4). Herbimycin A and phenylarsine oxide were purchased from Sigma and added directly to the bath to reach the final concentration. We present data as
means ± S.E. The Student's t test was used to
determine the significance.
 |
RESULTS |
To study the role of PTK in regulating ROMK1 channels, we
coinjected oocytes with cRNA encoding cSrc and ROMK1, respectively. Fig. 1 is a representative Western blot
showing that cSrc is highly expressed in the oocytes injected with cRNA
of cSrc, whereas the cSrc is present only at negligible levels in
oocytes without injection with cSrc cRNA. We also carried out Western
blot analysis to identify the expression of PTP isoforms. We observed
that PTP1D, a membrane associate PTP isoform, is expressed in
Xenopus oocytes; its expression is not affected by injecting
cSrc RNA. In contrast, expression of PTP-1B and PTP-1C in oocytes was
not detected with the current method (data not shown).

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Fig. 1.
A Western blot shows the expression of cSrc
and PTP-1D in oocytes injected with cSrc + ROMK1 (lanes 1 and 3) and in oocytes injected with ROMK1 alone
(lanes 2 and 4). The protein
concentration in lanes 1 and 2 was 5 µg,
whereas in lanes 3 and 4 it was 10 µg.
PC, positive control.
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After confirming that cSrc is expressed in oocytes injected with cSrc
cRNA, we used the two-electrode voltage clamp technique to study the
role of cSrc in regulating the activity of ROMK1 channels. Fig.
2A is a recording showing the
effect of herbimycin A, an inhibitor of cSrc, on the K+
current in oocytes coexpressing ROMK1 and cSrc. It is apparent that
inhibition of PTK with 1 µM herbimycin A significantly
increases the Ba2+-sensitive K+ current within
10 min. Fig. 2B summarizes results from 38 experiments in
oocytes coexpressing ROMK1 and cSrc. It illustrates that inhibition of
PTK augments the Ba2+-sensitive K+ current by
120 ± 16%, from 6.4 ± 0.9 to 14.1 ± 1.6 µA within 10 min. The effect of herbimycin A was specifically related to the
inhibition of PTK, because the agent had no significant effect on the
Ba2+-sensitive K+ current in the oocytes
expressing ROMK1 alone.

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Fig. 2.
A, a recording showing the effect of 1 µM herbimycin A on the K+ current in oocytes
expressing ROMK1 and cSrc. The current was measured with a
two-electrode voltage clamp. At the end of the experiment,
Ba2+ (arrow) was added to determine the
Ba2+-sensitive K+ current. I,
current. B, a summary showing the time course of the effect
of herbimycin A on the normalized channel activity in oocytes
expressing ROMK1 alone or ROMK1 + cSrc. The asterisks
indicate that the differences are significant in comparison with the
corresponding values obtained in oocytes injected with ROMK1
alone.
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After establishing the role of cSrc in suppressing the activity of
ROMK1 channels, we investigated the effect of PAO, an inhibitor of PTPs
including PTP1D, on ROMK1 channels (19). Fig.
3A is a recording
demonstrating the effect of 1 µM PAO on the
Ba2+-sensitive K+ current in oocytes expressing
ROMK1 and cSrc. Inhibiting PTP significantly decreased K+
current, and the effect of PAO was reversible, because it was observed
in separate experiments that the K+ current returned to the
control value 60 min after removal of PAO (data not shown). Fig.
3B summarizes the results of 22 experiments in which the
effect of PAO was examined. Blocking PTP with 1 µM PAO
reduced the Ba2+-sensitive K+ current by
53 ± 2%, from 21 ± 2 to 10 ± 1 µA. The effect of
PAO was the result of inhibiting PTP, because PAO had no significant effect on channel activity in the oocytes expressing ROMK1 alone.

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Fig. 3.
A, a recording showing the effect of 1 µM PAO on the K+ current in oocytes
expressing ROMK1 and cSrc. After washout of PAO, the pen speed of the
paper recorder was set to zero and resumed to 1 cm/2 mm 10 min later.
This stop-go causes a jump of the trace shown in the figure because of
an increase in K+ current. I, current.
B, a summary showing the time course of the effect of PAO on
the normalized channel activity in oocytes expressing ROMK1 alone or
ROMK1 + cSrc. The asterisks indicate that the differences
are significant in comparison with the corresponding values obtained in
oocytes injected with ROMK1 alone.
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The amino acid sequence of ROMK1 contains the tyrosine residue 337 at
the C terminus, which is within a consensus PTK phosphorylation site.
To test whether tyrosine residue 337 of the C terminus was involved in
mediating the effect of PTK or PTP, we investigated the effect of
herbimycin A or PAO on the ROMK1 mutant R1Y337A in which the tyrosine
residue was mutated to alanine. Fig.
4A summarizes the results of
15 experiments in which the effect of 1 µM herbimycin A
on channel activity in oocytes expressing cSrc and R1Y337A was studied.
Clearly, the effect of herbimycin A was absent in oocytes expressing
the ROMK1 mutant (108 ± 7% of the control K+
current). Furthermore, Fig. 4B demonstrates that PAO had no
significant effect on channel activity (95 ± 5% of the control
value) in oocytes expressing R1Y337A (n = 9). This
strongly suggests that tyrosine residue 337 at the C terminus is
essential for the effects of PTK and PTP on channel activity.

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Fig. 4.
A, the effect of 1 µM
herbimycin A on K+ current in oocytes expressing ROMK1
(R1WT) + cSrc (filled circles) and the ROMK1 mutant
(R1Y337A) + cSrc (open circles), respectively.
Asterisks indicate that the difference between two groups is
significant. B, the effect of 1 µM PAO on
K+ current in oocytes expressing R1WT + cSrc (filled
circles) and R1Y337A + cSrc (open circles),
respectively.
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To explore the possibility that PTK-induced tyrosine phosphorylation of
ROMK1 or its closely associated proteins directly inhibits channel
activity, we examined the effect of exogenous cSrc on the activity of
the ROMK1 channel in inside-out patches. Fig.
5 is a representative recording from four
such experiments showing the effect of exogenous cSrc on ROMK1 channels
in an inside-out patch. The activity of cSrc was independently
confirmed by assessing the phosphorylation rate of a specific peptide
(data not shown). We observed that adding 1 nM cSrc had no
effect on channel activity within 10-15 min (99 ± 4% of the
control value). This does not support the possibility that PTK-mediated
phosphorylation of ROMK1 directly inhibits the channel. The finding
that cSrc did not inhibit the ROMK1 channel in excised patches is,
however, consistent with the previous observation that addition of
exogenous cSrc had no significant effect on the activity of the small
conductance K+ channel in the rat CCD (17).

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Fig. 5.
A channel recording demonstrating the effect
of exogenous cSrc (1 nM) on the activity of ROMK1 channels
expressed in Xenopus oocytes. The experiment was
performed in an inside-out patch, and the holding potential was 0 mV.
The channel closed state is indicated by C and a
dotted line. Three parts of the trace indicated by numbers
are extended to show the fast time resolution.
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Because PTK is involved in the regulation of membrane protein
trafficking (20), it is possible that tyrosine phosphorylation of ROMK1
channels stimulates endocytosis of ROMK1 channels from the cell
membrane. This possibility was tested by experiments in which
the effect of PAO was examined in the presence of 20% sucrose, which
blocks endocytosis. Fig. 6A is
a representative recording showing the effect of 2 µM PAO
on the Ba2+-sensitive K+ current in oocytes
coexpressing ROMK1 and cSrc in the presence or absence of 20% sucrose.
In the presence of 20% sucrose, addition of PAO decreased
K+ current only by a modest 9 ± 1%
(n = 15; Fig. 6B). Fig. 6A also shows that removal of sucrose restored the inhibitory effect of PAO,
which reduced the Ba2+-sensitive K+ current by
50 ± 4%. The decrease in K+ current after the
removal of sucrose was the result of inhibiting PTP, because removal of
sucrose per se had no significant effect on channel activity
in the absence of PAO (data not shown).

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Fig. 6.
A, a recording illustrating the effect
of 2 µM PAO on the Ba2+-sensitive
K+ current in oocytes expressing ROMK1 + cSrc in the
presence or absence of 20% sucrose. The zero current is indicated by a
dotted line. I, current. B, the
time course of the effect of PAO on K+ current in the
presence of 20% sucrose (open circles) and in the absence
of sucrose (filled circles). Asterisks indicate
that the difference between two groups is significant.
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Moreover, we studied the effect of PAO on channel activity in the
presence of concanavalin A, an agent that blocks endocytosis (21).
Treatment of the oocytes with 250 µg/ml concanavalin A completely
abolished the inhibitory effect of PAO (control, 15 ± 0.8 µA;
PAO + concanavalin A, 15.9 ± 1.2 µA; n = 5;
data not shown). This suggested that inhibition of PTP stimulates the
endocytosis of ROMK1 channels.
We have also examined the possibility that inhibition of PTP with PAO
may attenuate the Golgi-dependent secretory pathway of
ROMK1 channels by investigating the effect of PAO in the oocytes treated with 5 µM brefeldin A, an agent that inhibits the
exit of proteins from endoplasmic reticulum (22). Addition of brefeldin A had no effect on K+ current within 30 min. Moreover,
treatment of brefeldin A did not abolish the inhibitory effect of PAO
(data not shown). Because brefeldin A may have no effect on protein
recycling (23), we studied the effect of PAO in the presence of
colchicine, an agent that inhibits microtubules and
microtubule-dependent recycling. Application of 20 µM colchicine had no significant effect on K+
current (Fig. 7A). Moreover,
colchicine failed to block the effect of PAO, because PAO
reduced the Ba2+-sensitive K+ current by
41 ± 5% (n = 5) in the presence of colchicine.
In contrast, blocking microtubules with colchicine abolished the effect
of herbimycin A (Fig. 7B), and channel activity was 105 ± 10% of the control value in the presence of colchicine
(n = 11) in oocytes coexpressing ROMK1 and cSrc. This
suggests that an intact microtubule is required for mediating the
effect of inhibiting PTK on channel activity. This notion is further
supported by experiments in which pretreatment of oocytes with 10 µM taxol blocked the effect of herbimycin A (data not
shown).

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Fig. 7.
A recording showing the effect of 1 µM PAO + 10 µM colchicine (A) and
of 1 µM herbimycin A + colchicine
(B) on K+ current. The zero current
is indicated by a dotted line.I,
current.
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Although the results obtained from the electrophysiological studies
strongly suggest that stimulation of PTK increases endocytosis, whereas
activation of PTP enhances exocytosis, it does not provide direct
evidence to prove the hypothesis. Therefore, we employed laser scanning
confocal microscopy to study the effects of PAO and herbimycin A on the
membrane distribution of ROMK1 labeled with GFP. We first examined the
biophysical properties of GFP-ROMK1 expressed in oocytes. Fig.
8A summarizes results from 10 experiments in which the Ba2+-sensitive K+
current was measured in oocytes expressing GFP-ROMK1. It is apparent that linking GFP to the N terminus of ROMK1 did not affect the expression of ROMK1, because the K+ current (11 ± 1 µA) was similar to that observed in oocytes injected with ROMK1
(14 ± 2 µA; data not shown). Moreover, the
current-voltage curve yields a typically weak inward rectifying
K+ current. Fig. 8B is a channel recording made
in a cell-attached patch from oocytes injected with GFP-ROMK1. The
channel conductance was 38 pS between 0 and 20 mV.

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Fig. 8.
A, a current-voltage curve
showing the Ba2+-sensitive K+ current measured
with a two-electrode voltage clamp in oocytes injected with GFP-ROMK1.
The bath solution contained 150 mM KCl.
I, current. B, a single channel recording
in a cell-attached patch from oocytes injected with GFP-ROMK1. The bath
solution contained 145 mM NaCl and 5 mM
KCl.
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After confirming that GFP-ROMK1 has the same biophysical properties as
those of ROMK1, we examined the effects of PTK and PTP inhibitors on
ROMK1 location in oocytes expressing GFP-ROMK1 alone or GFP-ROMK1 + cSrc. Fig. 9 is a typical recording from five such experiments in which the effect of PAO (1 µM)
on ROMK1 distribution was investigated. Inhibiting PTP did not
significantly change the ROMK1 location in oocytes expressing GFP-ROMK1
alone (Fig. 9A). In contrast, PAO significantly decreased
the membrane fluorescence intensity (48 ± 5%), an index of the
ROMK1 channel density, in the oocytes expressing GFP-ROMK1 and cSrc
(Fig. 9, B and C). Fig.
10 is a representative recording
showing the effect of herbimycin A on ROMK1 distribution in oocytes
injected with GFP-ROMK1 alone (A) and cSrc + GFP-ROMK1
(B). It is apparent that inhibiting PTK increased the
membrane density of ROMK1 channels only in oocytes coinjected with cSrc
but had no effect in oocytes injected with GFP-ROMK1 alone. Fig.
10C summarizes the results of five such experiments showing
that herbimycin A increased the fluorescence intensity by 115 ± 9%.

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Fig. 9.
The effect of PAO (1 µM) on GFP-ROMK1 distribution in the
cell membrane in oocytes expressing GFP-ROMK1 alone
(A) or cSrc + GFP-ROMK1 (B). The
top picture was taken immediately after adding PAO
(control), and the bottom image was collected 30 min after applying
PAO. The bar length represents 60 µm. C, the
effect of PAO on the fluorescence intensity of GFP-ROMK1 in the oocyte
membrane (arbitrary unit).
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Fig. 10.
The effect of herbimycin A (1 µM) on GFP-ROMK1 distribution in the
cell membrane in oocytes expressing GFP-ROMK1 alone
(A) or cSrc + GFP-ROMK1 (B). The
bar length represents 60 µm. The top image was
taken immediately after adding herbimycin A, and the bottom picture was
collected 30 min after applying herbimycin A. C, the effect
of herbimycin A on the fluorescence intensity of GFP-ROMK1 in the
oocyte membrane (arbitrary units).
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To further explore the role of the interaction between PTK and PTP in
regulating ROMK1, we examined the effect of herbimycin A in the oocytes
pretreated with PAO and investigated the effect of PAO in the oocytes
pretreated with herbimycin A. Fig. 11
shows the effects of herbimycin A and PAO in the oocytes treated with PTP and PTK inhibitors, respectively. We observed that
Ba2+-sensitive K+ current in oocytes pretreated
with PAO for 20 min was significantly smaller (5.8 ± 0.9 µA;
n = 12) than that in oocytes treated with herbimycin A
(23.7 ± 2.4 µA; n = 7). Moreover, PAO failed to
reduce the Ba2+-sensitive K+ current (23.1 ± 2.4 µA) in the presence of herbimycin A, whereas herbimycin A had
no significant effect on channel activity (6.1 ± 0.9 µA) in the
presence of PAO. This strongly suggested that the effect of PAO is the
result of stimulating cSrc activity, whereas the effect of herbimycin A
resulted from potentiating the effect of PTP.

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Fig. 11.
The effect of 1 µM PAO or 1 µM herbimycin A on K+
current in oocytes expressing ROMK1 + cSrc. The oocytes were
pretreated with herbimycin A or PAO for 20 min. The current was
measured with a two-electrode voltage clamp.I,
current.
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 |
DISCUSSION |
In the present study we found that inhibiting PTK with herbimycin
A increases K+ current in oocytes expressing ROMK1 channels
and cSrc, whereas blocking PTP with PAO decreases such current.
Moreover, we demonstrated that mutation of tyrosine residue 337 to
alanine abolished the effects of PAO and herbimycin A on channel
activity. This suggests that tyrosine residue 337 is a key site for the
action of PTP and PTK. Three lines of evidence support the conclusion
that effects of PAO and herbimycin A are the result of inhibiting PTP
and PTK, respectively. First, the effect of PAO was absent in the
presence of 20% sucrose or concanavalin A, whereas the effect of
herbimycin A was blocked by colchicine or taxol. Second, neither PAO
nor herbimycin A had an effect on channel activity in inside-out
patches.2 Third, pretreatment
of oocytes with PAO abolished the stimulatory effect of
herbimycin A, whereas herbimycin A treatment completely blocked the
inhibitory effect of PAO. Similar results were also observed in the rat
CCD, in which pretreatment of the CCD with herbimycin A significantly
diminished the effect of PTP inhibitor on the activity of the small
conductance K+ channels (16). This supports the notion that
inhibition of PTK by herbimycin A enhances PTP-induced
dephosphorylation, whereas blocking PTP by PAO potentiates PTK-induced phosphorylation.
Three possible mechanisms could mediate the regulation of ROMK1 by PTK.
First, phosphorylating the tyrosine residue of ROMK1 channels
could directly inhibit channel activity. Second, tyrosine phosphorylation could block exocytosis of ROMK1 channels and reduce the
number of channels on the cell surface. Third, PTK-induced phosphorylation could augment the internalization of ROMK1 channels from the cell membrane.
Relevant to the first possibility are the observations that PTK
suppresses delayed rectifying K+ channels, voltage-gated
K+ channels, Kv1.3, and
Ca2+-dependent K+ channels by
direct tyrosine phosphorylation (24-27). Moreover, cSrc has been shown
to regulate the N-methyl-D-aspartate channel by
direct association and phosphorylation (28). The present study strongly
indicates that the phosphorylation of tyrosine residue 337 is essential
for the effects of PTK and PTP. However, because addition of exogenous
cSrc had no effect on the activity of ROMK1, the hypothesis is not
supported that PTK-mediated phosphorylation of ROMK1 or of its
associated proteins directly blocks the channel. The results rather
suggest that phosphorylation of ROMK1 channels per se does
not directly alter the channel gating of ROMK1. It is more likely that
tyrosine phosphorylation is a necessary step to activate a signaling
cascade that eventually leads to the down-regulation of ROMK1 channels.
Two lines of evidence exclude the second possibility that stimulation
of tyrosine phosphorylation diminishes exocytosis or recycling of ROMK1
channels. First, application of brefeldin A, an agent that inhibits
export of de novo synthesized proteins from endoplasmic
reticulum (22), had no effect on ROMK1. Moreover, treatment of cells
with brefeldin A did not abolish the effect of PAO. This suggests that
stimulation of tyrosine phosphorylation by PAO does not affect a
brefeldin A-sensitive secretory pathway. Second, treatment with
colchicine, an inhibitor of microtubule assembly, or taxol, an agent
that "freezes" the microtubule, neither had an effect on channel
activity nor abolished the effect of PAO. Because microtubules play an
important role in Golgi-independent protein recycling (29), the finding
that the effect of PAO was not changed by colchicine or taxol excluded
the possibility that stimulating PTK decreases
microtubule-dependent recycling.
The observations that the inhibitory effect of PAO was absent in
oocytes treated with either 20% sucrose or concanavalin A indicate
strongly that stimulation of tyrosine phosphorylation increases the
internalization of ROMK1 channels. This conclusion is also supported by
the observation that GFP-ROMK1 membrane location diminished
significantly by PAO treatment. It is possible that the phosphorylation
of tyrosine residue 337 is essential to initiate the internalization of
ROMK1 channels. A large body of evidence indicates that tyrosine
phosphorylation and dephosphorylation play an important role in the
regulation of protein trafficking (20, 30-32). For instance, tyrosine
phosphorylation is involved in mediating the insulin-induced exocytosis
of glucose transporters, e.g. GLUT4 (30). Moreover,
overexpression of cSrc in fibroblasts has been demonstrated to
stimulate the endocytosis of epidermal growth factor receptor (31). In
this regard, it has been reported that cSrc can interact with
actin/filament associate protein, AFAP-110 (32), and phosphorylate
actinin in activated platelets (33). In addition, cSrc has been shown
to mediate epidermal growth factor-induced clathrin phosphorylation,
which is essential for the internalization of epidermal growth
factor receptors (34). Finally, in neuronal cells cSrc can
interact with dynamin and synapsin, which are involved in vesicle
trafficking (35).
In contrast to the effect of PAO, the finding that pretreatment of the
oocytes with taxol or colchicine completely abolished the effect of
herbimycin A suggests that stimulation of tyrosine dephosphorylation
increases exocytosis of ROMK1. Moreover, the experimental results with
confocal microscopy also confirmed that herbimycin A increased the
GFP-ROMK1 density in the cell membrane. Because microtubules have been
shown to be involved in regulating protein trafficking (29), it is
conceivable that increasing tyrosine dephosphorylation enhances the
microtubule-dependent exocytosis of the ROMK1 channel.
Moreover, the finding that colchicine and taxol did not block the
effect of PAO suggests that microtubules may not be involved in
modulating the endocytosis of ROMK1 channels. Relevant to the present
observation is the finding that the cytoskeleton plays a key role in
the exocytotic recruitment of GLUT4 (a glucose transporter) to the
plasma membrane from an intracellular pool, but not in endocytosis
(36).
The physiological significance of PTK-induced regulation of ROMK1
channels has been previously documented. It is well established that
dietary K+ intake plays an important role in the regulation
of renal K+ secretion (15, 37). Because the ROMK1-like
channel is the main K+ channel located in the apical
membrane of the CCD, alteration in ROMK1 channel activity should have a
significant impact on renal K+ secretion. We and others
have observed that the number of the small conductance K+
channel is almost 5-6 times higher in the CCD obtained from rats on a
high K+ diet than that on a K+-deficient diet
(17, 37). The effect of high K+ intake on channel activity
was not the result of increasing the circulating aldosterone level,
because aldosterone perfusion failed to stimulate the activity of the
small conductance K+ channel in the CCD (37). We have
previously reported that the activity and concentration of cSrc, a
nonreceptor type of PTK, were significantly higher in the renal cortex
from rats on a K+-deficient diet than those from rats on a
high K+ diet (17). We have suggested that a low
K+ intake increases the expression and activity of PTK,
which in turn decreases the number of the apical small conductance
K+ channel and suppresses K+ secretion. This
hypothesis was supported by the finding that inhibition of PTK
increased the number of the small conductance K+ channel in
the CCD from animals on a K+-deficient diet (17).
Furthermore, we have also shown that inhibition of PTP reduced the
number of the small conductance K+ channel in the CCD from
rats on a high K+ diet (16).
Although the present study strongly suggests that tyrosine
phosphorylation and dephosphorylation regulate the endocytosis and
exocytosis of ROMK1 channels, the mechanism by which the interaction between PTK and PTP regulates ROMK1 channel trafficking is not completely understood. It is possible that phosphorylation of tyrosine
residue 337 of the ROMK1 channel creates a binding site for proteins
that are involved in ROMK1 trafficking. Further experiments are
required to detect these proteins that interact with the phosphorylated ROMK1 channels. Fig. 12 is a simple
model illustrating the mechanism of the PTK-PTP interaction. An
increase in tyrosine phosphorylation reduces the channel activity by
endocytosis of ROMK1 channels, whereas stimulation of tyrosine
dephosphorylation increases the number of ROMK1 channels by
exocytosis.

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|
Fig. 12.
A cell model illustrating the mechanisms by
which PTK (A) and PTP (B) regulate
ROMK1 channels.
|
|
We conclude that the balance between PTK and PTP activity plays an
important role in the regulation of ROMK1 channels and that tyrosine
residue 337 of the ROMK1 channel is the key site for the effects of PTP
and PTK. The effects of PTK and PTP on channel activity are achieved by
regulating the retrieval or insertion of ROMK1-like channels into the
apical membrane of principal cells in the CCD.
 |
ACKNOWLEDGEMENT |
We thank Dr. L. G. Palmer for constructive
comments during the course of the experiments.
 |
FOOTNOTES |
*
This work was supported by National Institutes of Health
Grants DK 47402 and DK 54983 (to W.-H. W.), DK 17433 (to G. G.), and
DK 37605 (to S. C. H.).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.
To whom correspondence should be addressed: Dept. of
Pharmacology, New York Medical College, Valhalla, NY 10595. Tel.:
914-594-4120; Fax: 914-347-4956; E-mail: wenhui_wang@nymc.edu.
Published, JBC Papers in Press, December 12, 2000, DOI 10.1074/jbc.M008671200
2
W.-H. Wang, unpublished observation.
 |
ABBREVIATIONS |
The abbreviations used are:
CCD, cortical
collecting duct;
PTP, protein-tyrosine phosphatase;
PTK, protein-tyrosine kinase;
PAO, phenylarsine oxide;
GFP, green
fluorescent protein.
 |
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