Department of Pharmacology, New York Medical College, Valhalla, New York 10595
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ABSTRACT |
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We used confocal microscopy, patch-clamp, and biotin-labeling techniques to examine the role of soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins in mediating the effect of inhibition of PTK on ROMK1 trafficking in HEK-293 cells transfected with c-Src and green fluorescent protein (GFP)-ROMK1. Inhibition of c-Src with herbimycin A significantly decreased the tyrosine phosphorylation level of ROMK1. Patch-clamp studies demonstrated that addition of herbimycin A increased the activity of ROMK1 in cell-attached patches. Confocal microscopic imaging showed that herbimycin A decreased the intracellular intensity of GFP-ROMK1. The biotin-labeling technique demonstrated that the inhibition of c-Src increased surface ROMK1 by 110%. In contrast, inhibition of c-Src did not increase the K channel number in HEK cells transfected with R1Y337A, a ROMK1 mutant in which tyrosine residue 337 was mutated to alanine. This suggests that tyrosine residue 337 is essential for the herbimycin A-induced increase in surface ROMK1 channels. To determine whether SNARE proteins are involved in mediating exocytosis of ROMK1 induced by the inhibition of c-Src, we examined the effect of herbimycin A on ROMK1 trafficking in cells treated with tetanus toxin. The incubation of cells in a medium containing tetanus toxin abolished the herbimycin A-induced increase in the number of surface ROMK1. In contrast, inhibition of c-Src still increased the numbers of surface ROMK1 in cells treated with boiled tetanus toxin. We conclude that tyrosine dephosphorylation enhances the exocytosis of ROMK1 and that SNARE proteins are required for exocytosis induced by inhibition of PTK.
c-Src; soluble N-ethylmaleimide-sensitive factor attachment protein receptor proteins; protein tyrosine phosphatase
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INTRODUCTION |
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ROMK1 IS AN INWARDLY RECTIFYING K channel located in the apical membrane of the cortical collecting duct (CCD) (9, 33) and is responsible for renal K secretion (5, 28). K secretion is a two-step process: K enters the cell via the basolateral Na-K-ATPase and is secreted into the lumen across the apical membrane through the K channels. Although two types of K channels have been identified in the apical membrane of the CCD (2, 3), it is generally believed that the small-conductance K (SK) channel is a main contributor to apical K conductance (14, 20, 21, 27). A large body of evidence indicates that ROMK1 is a key component of the SK channel identified in native tissue (4, 14, 19, 27).
Several studies have demonstrated that the number of functional ROMK-like SK channels in the apical membrane of the CCD varies and depends on dietary K intake: an increase in K intake augments the number of SK channels in the CCD (13, 29, 31). In contrast, low-K intake reduces the number of ROMK channels in the cell membrane in the renal cortex and outer medulla (8). Our previous studies demonstrated that PTK and protein tyrosine phosphatase (PTP) play an important role in mediating the effect of low-K intake on the number of the ROMK-like SK channels in the CCD (28, 30, 31). We have reported that inhibition of PTK increases the activity of the SK channels in the apical membrane of the rat CCD (28). The effect of inhibition of PTK is possibly mediated by increasing SK channel insertion because this depends on the intact cytoskeleton (32). This speculation has further been suggested by confocal microscopy, demonstrating that inhibition of c-Src increased the membrane density of green fluorescent protein (GFP)-tagged ROMK1 in oocytes injected with ROMK1 and c-Src (10). However, confocal microscopic imaging of oocytes cannot clearly separate the ROMK1 channels located in the cell membrane from those in the submembrane. Moreover, the mechanism of membrane insertion of ROMK1 may be different between oocytes and mammalian cells. Finally, it is not known whether soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins are required for exocytosis of ROMK1 induced by inhibition of c-Src and other types of PTKs. In the present experiments, these questions were investigated by examining the effect of inhibition of PTK on the insertion of ROMK1 in HEK-293 cells transfected with ROMK1 and c-Src.
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METHODS |
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Construction of GFP-ROMK and c-Src. ROMK1 or mutant ROMK1 (R1Y337A) was cloned into the EcoR1 and BamH1 sites of PEGFPC1 (Clontech, Palo Alto, CA) using primers 5'-TGGGCCTAAAAGAATTCAGCTGCTGTGCAGACAAC (sense, nt 81-115) and 5'-TTGTAGGTGGAAGGATCCCTGCTACATCTGGGTGTCG (antisense, nt 1310-1346) to amplify PCR fragments of ROMK1 or the mutant that was subcloned into PCDNA3.1. The coding sequence of c-Src was cut from pGEM vector with HinDIII and EcoRI and ligated into pCDNA3.1 expression vector (Invitrogen). All vector sequences were confirmed by automated DNA sequencing at the William Keck Biotechnology Laboratory at Yale University.
Transfection of HEK-293 cells. HEK-293 cells were plated in 35-mm dishes and transfected with 1 µg of ROMK1 or R1Y337A and 1 µg of c-Src using 7 µl of LT1 reagent (PanVera, Madison, WI) according to instructions provided by the manufacturer. To study the effect of tetanus toxin on ROMK1 trafficking, 30 nM tetanus toxin or boiled toxin was added at the same time as the transfection. Tetanus toxin was obtained from Calbiochem (San Diego, CA). The experiments were carried out 2 days after transfection. The successful rate for cell transfection was between 60 and 70%.
Confocal microscopy. The method using the confocal microscope has been previously described (24). Briefly, a Bio-Rad MRC 1000 confocal microscope was used in the study. GFP fluorescence was excited at 488 nm with an argon laser beam and viewed with an Olympus microscope equipped with a ×60 oil lens. All images were acquired, processed, and printed with identical parameters before and after herbimycin A treatment.
Biotinylation, immunoprecipitation, and Western blot analysis. Changes in surface ROMK after herbimycin A treatment were quantitated by labeling cells with cell-impermeant sulfo-NHS-biotin (Pierce) according to the protocol provided by the manufacturer. After biotinylation, the cells were washed 2× with PBS and trypsinized with trypsin-EDTA. They were pelleted by centrifugation for 5 min at 10,000 rpm, washed 2× with PBS, and lysed with cold RIPA buffer (1× PBS, 1% Igepal CA-630, 0.1% SDS, 0.5% deoxycholate) supplemented with 1 mM sodium molybdate, 1 mM sodium fluoride, 1 µM PMSF, and 100 µl of protease inhibitor cocktail/ml (Sigma) of lysis buffer. After clarification, total protein concentrations were determined with a Bio-Rad (Bio-Rad Laboratories, Richmond, CA) protein assay kit, and aliquots of lysates containing equal amounts of protein were immunoprecipitated overnight with 1 µg of anti-GFP (Clontech) monoclonal antibody and 20 µl of protein A/G-agarose (Santa Cruz Biotechnology, Santa Cruz, CA). After collection of the immune complex by centrifugation and a washing 2× with PBS, proteins were resolved by electrophoresis on 10% SDS gels and transferred to PVDF membranes (Bio-Rad). The membranes were blocked with 5% milk in Tris-buffered saline, and the biotin-labeled GFP-ROMK1 proteins were detected using NeutrAvidin horseradish peroxidase (Pierce). To determine the effect of herbimycin A on the tyrosine phosphorylation level of ROMK1, PY20 antibody (Santa Cruz Biotechnology), which reacts with the tyrosine-phosphorylated proteins, was used to detect the phosphorylated ROMK1. Changes in biotin-labeled surface ROMK1 proteins or the tyrosine-phosphorylated ROMK1 levels were normalized with corresponding total ROMK1 protein, which was determined with ROMK antibody (Alomone Laboratories, Jerusalem, Israel). The density of the band was determined using Alpha DigiDoc 1000 (Alpha Innotech, San Leandro, CA).
Patch-clamp technique.
An Axon 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 (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, which was calculated from data samples of 30-s duration in the steady state as
follows
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(1) |
Experimental solution and statistics. To examine the role of SNARE proteins in the regulation of ROMK1 exocytosis, 30 nM tetanus toxin or boiled toxin was added to the incubation media at the same time the cells were transfected with ROMK1 and c-Src.
The bath solution for the patch-clamp study was composed of (in mM) 140 NaCl, 5 KCl, 1.8 MgCl2, 1.8 CaCl2, and 10 HEPES (pH 7.4). The pipette solution was composed of (in mM) 140 KCl, 1.8 MgCl2, and 10 HEPES (pH 7.4). Herbimycin A was purchased from Sigma (St. Louis, MO) and added directly to the bath to reach the final concentration. We present data as means ± SE. Student's t-test was used to determine significance. ![]() |
RESULTS |
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We used HEK-293 cells transfected with the GFP-tagged ROMK1 and
c-Src to study the effect of herbimycin A, an inhibitor of PTK, on
ROMK1 trafficking. A previous study demonstrated that the GFP-tagged
ROMK1 had the same biophysical properties of ROMK1 (10,
24). Figure 1,
A-C, is a representative confocal image from
14 experiments showing the effect of herbimycin A on ROMK1 distribution
in living HEK-293 cells transfected with GFP-ROMK1 and c-Src. Under
control conditions, a large number of GFP-ROMK1 channels accumulated in
the perinuclear region. Addition of 1 µM herbimycin A decreased the
intracellular location of ROMK1 channels and increased the number of
ROMK1 channels in the cell membrane. This is evidenced by the
observation that the cell diameter appears to be larger in the presence
of the PTK inhibitor than before addition of herbimycin A. The effect
of herbimycin A on ROMK1 channel trafficking was specific because it
did not have a significant effect on ROMK1 channel distribution in
HEK-293 cells transfected with GFP-ROMK1 alone (Fig. 1,
D-F). Also, there is no significant change
in ROMK1 location within 15 min in the absence of herbimycin A in cells
transfected with ROMK1 and c-Src (data not shown).
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After finding that the inhibition of c-Src lowered the distribution of
ROMK1 in the intracellular compartment, we used the biotin-labeling
technique to examine whether herbimycin A increases the number of ROMK1
channels in the cell membrane. Figure 2
is a Western blot analysis showing the effect of herbimycin A treatment on the surface localization of ROMK1 in HEK-293 cells transfected with
GFP-ROMK1 and c-Src. The ROMK1 channels were harvested by immunoprecipitation of the cell lysate with GFP antibody after a 15-min
incubation of cells with 1 µM herbimycin A. The surface-localized ROMK1 (71 kDa) labeled with biotin was detected by neutravidin (A), and total ROMK proteins were recognized by ROMK
antibody (B). The level of surface-located ROMK1 channels
was normalized compared with total ROMK protein. It was calculated that
inhibition of c-Src significantly augmented the biotin-labeled fraction
of ROMK1 by 110 ± 20% (n = 7) compared with
those from untreated cells. In addition to the 71-kDa band, biotin also
labeled a low-molecular-mass band (60-65 kDa). However, the nature
of the low-molecular-mass band is not clear. This band cannot be a
glycosylated ROMK1 because the molecular mass of glycosylated ROMK1
should be greater than 71 kDa. This low-molecular-mass protein
may be a ROMK1-associated membrane protein or a degraded ROMK1 protein
that cannot be recognized by ROMK antibody. Further study is clearly
required to determine the nature of the protein.
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The notion that the inhibition of c-Src increased the ROMK1 channel
number in the cell membrane is also supported by experiments in which
the patch-clamp technique was used to test the effect of herbimycin A
on ROMK1 channel activity. Figure 3 is a
channel recording demonstrating that the inhibition of c-Src increased the activity of ROMK1 channels. From an inspection of Fig. 3, it is
apparent that there was no K channel activity before addition of
herbimycin A. The inhibition of c-Src caused a sharp increase in the
number of ROMK1 channels. This increase was most likely the result of
fusion of a vesicle containing ROMK1 into the cell membrane because we
often observed that more than two channels were simultaneously open. In
10 experiments, blocking c-Src increased NPo
from 0.5 ± 0.1 to 3.0 ± 0.3. Because herbimycin A did not increase the channel open probability (data not shown), the effect of
herbimycin A must result from an increase in channel number. This
finding is consistent with a previous report that herbimycin A
increased the activity of ROMK1-like channels in the CCD obtained from
rats on a K-deficient diet from 0.7 ± 0.1 to 3.1 ± 0.3 (29). The effect of herbimycin A results from the
inhibition of c-Src because herbimycin A had no effect on channel
activity in cells transfected with ROMK1 alone (data not shown). Also,
herbimycin A significantly decreased the tyrosine-phosphorylated ROMK1
channel population. Figure 4 is a Western
blot analysis illustrating the effect of herbimycin A on the tyrosine
phosphorylation of ROMK1. The cells were treated with herbimycin A or
vehicle for 15 min. The ROMK1 channels were harvested by
immunoprecipitation of the cell lysate with GFP antibody;
phosphorylated protein was detected with py20 antibody (Fig.
4A), and total ROMK1 was recognized by ROMK antibody (Fig.
4B). Protein phosphorylation was normalized compared with
total ROMK1 proteins. Inhibition of c-Src reduced the
tyrosine-phosphorylated ROMK1 by 48 ± 5% (n = 5).
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We have previously demonstrated that tyrosine residue 337 is a major
site for PTK-induced phosphorylation (7). To examine the
role of tyrosine residue 337 in mediating the effect of inhibiting PTK
on the ROMK1, we used confocal microscopy to examine the effect of
herbimycin A on ROMK1 mutant R1Y337A. Figure
5 is a confocal image from nine
experiments showing the effect of herbimycin A on the distribution of
R1Y337A in cells cotransfected with c-Src. Clearly, inhibition of c-Src
did not have a significant effect on R1Y337A distribution because
intracellular R1Y337A location was not altered by 15-min treatment with
herbimycin A. This suggests that the effect of herbimycin A on ROMK1
results from the stimulation of tyrosine dephosphorylation of ROMK1.
Because R1Y337A cannot be phosphorylated by PTK, the population of the
tyrosine-phosphorylated ROMK1 channels was absent. Accordingly,
inhibition of PTK did not have an effect on ROMK1 membrane insertion.
This hypothesis is also supported by experiments in which the effect of
herbimycin A on ROMK1 surface number was investigated with the
biotin-labeling technique.
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Figure 6 is a Western blot analysis
showing the effect of herbimycin A on the surface localization of
R1Y337A in HEK-293 cells transfected with GFP-R1Y337A and c-Src. The
ROMK1 channels were harvested by immunoprecipitation of the cell lysate
with GFP antibody and detected with ROMK antibody (Fig. 6B),
and the surface-localized ROMK1 was identified by biotin labeling (Fig.
6A). The results clearly demonstrated that inhibition of
c-Src did not increase the intensity of the avidin-recognized 71-kDa
band (94 ± 11% of the control value, n = 5).
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After establishing that the inhibition of c-Src stimulates the
insertion of ROMK1, we investigated whether exocytosis induced by
inhibiting c-Src depended on SNARE proteins, using confocal microscopy
and biotin-labeling techniques. HEK cells were transfected with
GFP-ROMK1 and c-Src and incubated in media containing either tetanus
toxin (30 nM) or boiled tetanus toxin. Figure
7, A-C, is a
confocal image from nine experiments showing the effect of herbimycin A
on the distribution of ROMK1 channels in cells incubated with tetanus
toxin (30 nM). It is apparent that treatment of cells with tetanus
toxin abolished the effect of herbimycin A on ROMK1 trafficking because
fluorescence intensity in the intracellular compartment, an indication
of intracellularly located ROMK1, was not significantly decreased by
herbimycin A. Also, the fluorescence intensity in the cell membrane did
not increase. In contrast, we observed that addition of herbimycin A
significantly decreased the ROMK1 number in the intracellular
compartment in the cells treated with boiled toxin (Fig. 7,
D-F). The notion that the inhibition of
SNARE proteins abolishes the effect of inhibition of c-Src is also
supported by results obtained from biotin-labeling experiments. Figure
8 is a typical Western blot demonstrating
the effect of herbimycin A on biotin-labeled surface ROMK1 intensity in
the presence of boiled toxin (Fig. 8A) and unboiled toxin
(Fig. 8B). As expected, inhibition of c-Src can still
significantly increase the intensity of the avidin-recognized 71-kDa
band corresponding to the surface-located ROMK1 (herbimycin A
treatment, 175 ± 20% of the control value) in cells treated with
boiled toxin. In contrast, unboiled toxin completely abolished the
effect of herbimycin A because it did not increase the surface-located
ROMK1 channels (92 ± 11% of the control value, n = 7). This suggests that the inhibition of c-Src-induced exocytosis
depends on SNARE proteins.
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DISCUSSION |
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In the present study, we have demonstrated that the inhibition of PTK decreases the level of tyrosine-phosphorylated ROMK1 and stimulates membrane insertion of ROMK1. Because it is possible that other potential subunits such as the CFTR or sulfonylurea receptor may be required to form native SK channels in the CCD, the term ROMK1 means ROMK1 subunit in the present study. Two lines of evidence suggest that a decrease in tyrosine phosphorylated ROMK1 proteins is required for initiating the insertion of ROMK1 channels into the cell membrane. First, in vitro phosphorylation experiments have demonstrated that ROMK1 is a substrate of PTK. Second, an increase in ROMK1 insertion induced by inhibiting PTK was absent in cells transfected with c-Src and R1Y337A.
In a previous study, we have shown that tyrosine residue 337 of ROMK1 is the main site for the PTK-induced phosphorylation, and mutation of tyrosine residue 337 to alanine almost completely abolished the c-Src-induced tyrosine phosphorylation of ROMK1 (7). We used herbimycin A as a tool to reduce the level of the tyrosine phosphorylation of ROMK1. Three pieces of evidence strongly suggest that the effect of herbimycin A results from the inhibition of PTK: 1) tyrosine-phosphorylated ROMK1 was diminished when cells were treated with herbimycin A; 2) mutation of tyrosine residue 337 to alanine abolished the effect of herbimycin A on ROMK1 redistribution; and 3) a previous study showed that the effect of herbimycin A on ROMK1 channel activity was absent in the presence of the PTP inhibitor (30). This suggests that the effect of herbimycin A on ROMK1 results from an indirect potentiation of PTP function.
In the present study, we employed three different techniques, biotin-labeling, confocal microscopy, and a patch clamp, to demonstrate that inhibition of PTK in cells transfected with ROMK1 and c-Src stimulates the insertion of ROMK1 in the cell membrane. First, confocal microscopy showed that inhibition of PTK dramatically diminished ROMK1 located in the intracellular compartment. Second, application of the PTK inhibitor caused a sharp increase in ROMK1 channel activity. This observation is consistent with the previous finding that herbimycin A increased K current in oocytes injected with ROMK1 and c-Src. Finally, biotin labeling revealed that inhibition of PTK significantly increased the number of surface ROMK1 channels. Therefore, the data have unambiguously indicated that a decrease in the tyrosine phosphorylation level of ROMK1 enhances the insertion of ROMK1 channels. This notion is also supported by the observation that inhibition of microtubule assembly abolished the effect of inhibition of PTK (32) because the microtubule is critically involved in mediating vesicle transportation and fusion.
In contrast, several lines of evidence indicate that the stimulation of tyrosine phosphorylation of ROMK1 caused the internalization of ROMK1 (24). First, inhibition of PTP augmented the level of the tyrosine-phosphorylated ROMK1 in HEK-293 cells transfected with ROMK1 and c-Src and reduced the biotin-labeled surface ROMK1. Second, confocal microscopic imaging demonstrated that GFP-tagged ROMK1 was accumulated in the intracellular compartment. Third, the patch-clamp experiments showed that inhibition of PTP diminished the activity of ROMK1. The effect of inhibition of PTP on ROMK1 channels has also been observed in native isolated CCD, in which inhibition of PTP decreased the ROMK-like SK channels and the effect of the PTP inhibitor was blocked by a hypertonic bath solution (30).
Two lines of evidence strongly suggest that the interaction between target membrane (t)-SNAREs and vesicle (v)-SNAREs is involved in mediating the effect of the inhibition of PTK on ROMK1 insertion: 1) inhibition of PTK did not decrease ROMK1 content in the intracellular compartment in cells incubated with tetanus toxin; and 2) toxin treatment abolished the herbimycin A-induced increase in biotin-labeled ROMK1 channels. The effect of tetanus toxin was specific because inhibition of c-Src could still increase the number of surface ROMK1 in cells treated with boiled tetanus toxin. Tetanus toxin has also been shown to inhibit exocytosis of the proton pump in the male reproductive tract (1).
A large body of evidence indicates that SNARE proteins involved in synaptic vesicle translocation, docking, and membrane fusion (18) are also required for the regulation of membrane fusion in epithelial tissues (1, 6, 12, 17, 22, 23). Relevant to this conclusion is the finding that insulin-induced insertion of the glucose transporter GLUT4 depends on SNARE proteins (25). According to the present concept regarding the role of SNARE proteins in the regulation of vesicle fusion process, there are two types of SNAREs: t-SNAREs, which serve as the docking points for vesicles at the plasma membrane, and v-SNAREs, which are responsible for the interaction with t-SNAREs. The interaction between t-SNAREs and v-SNAREs causes vesicle docking at the plasma membrane. The mechanism by which tetanus toxin blocks vesicle docking and fusion is that the toxin cleaves v-SNAREs and abolishes the interaction between t-SNAREs and v-SNAREs (16).
SNARE proteins are involved in the regulation of membrane transporter protein trafficking (6, 12, 17, 22, 23). It has been shown that syntaxin 1A stoichometrically binds to the NH2 terminus of CFTR and regulates Cl currents of CFTR (12). Moreover, syntaxin 1A and other members of the SNARE family are involved in the control of cell surface number of epithelial Na channels (17, 22). In the CCD, SNAP-23 has been demonstrated to be colocalized with aquaporin-2 and play an important role in mediating the effect of vasopressin on aquaporin-2 insertion (23). Although the observation that tetanus toxin abolished the effect of herbimycin A on ROMK1 trafficking strongly suggests that SNARE proteins are involved in the regulation of ROMK1 trafficking, it is not known which syntaxin and SNAP-25 homologues are specifically responsible for ROMK1-containing vesicle fusion. However, several investigations have reported that SNAP homologues and syntaxin are expressed in the CCD (6, 23). We need further experiments to determine which member of the SNARE family is involved in the regulation of ROMK1 insertion.
ROMK1 is responsible for K secretion in the CCD under physiological conditions (4). We and others have demonstrated that a high-K intake increases the number of ROMK-like SK channels in the CCD (13, 29). Although channel activity in the CCD from rats on a low-K diet is not significantly lower than those on a normal-K diet (15, 31), it is possible that channel activity in the CCDs obtained from rats on a normal-K diet is underestimated. This is because the patch-clamp experiments were performed in split-open tubules, a preparation that is different from in vivo conditions. For instance, an increase in Na delivery can stimulate the basolateral Na-K-ATPase, and activity of Na-K-ATPase is closely coupled to apical ROMK-like SK channel activity (11, 26). Because the activity of Na-K-ATPase in the split-open CCD may be lower than that in in vivo conditions, the SK channel activity observed in our experimental conditions may not represent "true" channel activity in vivo. Therefore, it is conceivable that the activity of SK channels in CCD from rats on a normal-K diet is higher than that from rats on a low-K diet. Moreover, it is possible that there are fewer "silent" ROMK1 channels in the apical membrane of CCD from K-depleted rats than those of rats on a normal-K diet. This speculation is supported by the observation that ROMK expression in the cell membrane fraction was significantly lower in K-depleted rats than that in rats on a normal-K diet (8). We hypothesize that there are two populations of ROMK1 in the cell membrane of the CCD: active and inactive ROMK1 channels. Inactive ROMK1 channels serve as a reserved K channel pool, which can become active in response to a variety of stimuli such as hormones and other local factors. However, the pool size of inactive ROMK1 diminishes when dietary K intake is low and PTK activity is stimulated. Accordingly, ROMK1 channels are internalized and the response of ROMK1 channels to a given hormone is abolished. This can function as a K-saving mechanism. When dietary K intake is normal or high, the activity of PTK is suppressed. Consequently, ROMK1 channels are tyrosine dephosphorylated, reinserted into the apical membrane, and ready to respond to a particular factor that stimulates K secretion. This notion is supported by the observation that the tyrosine-phosphorylated ROMK1 channel level is significantly less in kidneys from rats on a high-K diet than those from animals on a K-deficient diet (7).
We conclude that inhibition of PTK stimulates the tyrosine-dephosphorylated ROMK1 channels and increases the insertion of ROMK1 in the cell membrane by a SNARE protein-dependent mechanism.
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ACKNOWLEDGEMENTS |
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This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-47402 and DK-54983.
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FOOTNOTES |
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Address for reprint requests and other correspondence: W.-H. Wang, Dept. of Pharmacology, BSB Rm. 537, 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.
First published December 30, 2002;10.1152/ajprenal.00309.2002
Received 28 August 2002; accepted in final form 5 November 2002.
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