Role of the cytoskeleton in mediating effect of vasopressin and herbimycin A on secretory K channels in CCD

Yuan Wei and Wen-Hui Wang

Department of Pharmacology, New York Medical College, Valhalla, New York 10595


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

We have previously demonstrated that inhibiting protein tyrosine kinase (PTK) and stimulating protein kinase A (PKA) increase the activity of the small-conductance K (SK) channel in the cortical collecting duct (CCD) of rat kidneys (Cassola AC, Giebisch G, and Wang WH. Am J Physiol Renal Fluid Electrolyte Physiol 264: F502-F509, 1993; Wang WH, Lerea KM, Chan M, and Giebisch G. Am J Physiol Renal Physiol 278: F165-F171, 2000). In the present study, we used the patch-clamp technique to study the role of the cytoskeleton in mediating the effect of herbimycin A, an inhibitor of PTK, and vasopressin on the SK channels in the CCD. The addition of colchicine, an inhibitor of microtubule assembly, or taxol, an agent that blocks microtubule reconstruction, had no significant effect on channel activity. However, colchicine and taxol treatment completely abolished the stimulatory effect of herbimycin A on the SK channels in the CCD. Removal of the microtubule inhibitors restored the stimulatory effect of herbimycin A. In contrast, treatment of the tubules with either taxol or colchicine did not block the stimulatory effect of vasopressin on the SK channels. Moreover, the effect of herbimycin A on the SK channels was also absent in the CCDs treated with either cytochalasin D or phalloidin. In contrast, the stimulatory effect of vasopressin was still observed in the tubules treated with phalloidin. However, cytochalasin D treatment abolished the effect of vasopressin on the SK channels. Finally, the effects of vasopressin and herbimycin A are additive because inhibiting PTK can still increase the channel activity in CCD that has been challenged by vasopressin. We conclude that an intact cytoskeleton is required for the effect on the SK channels of inhibiting PTK and that the SK channels that are activated by inhibiting PTK were differently regulated from those stimulated by vasopressin.

ROMK1; microtubule; exocytosis; protein tyrosine phosphatase; vasopressin; cortical collecting duct


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

THE APICAL SMALL-CONDUCTANCE K (SK) channel contributes significantly to apical K conductance and is responsible for K secretion in the cortical collecting duct (CCD) (6, 7, 19). A large body of evidence has demonstrated that dietary K intake plays an important role in regulating K secretion: high K intake increases, whereas low K intake decreases, renal K secretion (6). Moreover, protein tyrosine kinase (PTK) mediates the effect of low dietary K intake on the SK channels in the CCD (20, 22). We have previously demonstrated that inhibiting PTK increased the number of the SK channels in the CCD from rats on a K-deficient (KD) diet, whereas inhibiting protein tyrosine phosphatase (PTP) decreased the number of the SK channels in the CCD from rats on a high-K (HK) diet (20, 21). Also, we have observed that the effect on channel activity of inhibiting PTP could be blocked by either concanavalin A or hyperosmolarity, a maneuver that inhibits the endocytosis of membrane proteins (21). This suggests that inhibition of the SK channels induced by blocking PTP is the result of increasing internalization of the SK channels, whereas stimulation of the SK channels induced by blocking PTK is mediated by enhancing exocytosis. It has been well established that microtubules and actin filaments are involved in regulating exocytosis (9). Therefore, we examined the role of the microtubules and actin filaments in mediating the effect on channel activity of inhibiting PTK in the rat CCD.

In addition to dietary K intake, vasopressin has been shown to stimulate the SK channels in the CCD via a cAMP-dependent pathway (3, 6). There are at least two mechanisms by which vasopressin stimulates the SK channels: vasopressin could increase the channel activity by opening the previously silent K channels in the cell membrane; or alternatively, the hormone may increase the insertion of the SK channels into the cell membrane by a mechanism similar to the inhibition of PTK. Therefore, we extended our study to examine the role of the cytoskeleton in mediating the effect of vasopressin to determine whether the effect of vasopressin also depends on microtubules and actin filaments.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Preparation of CCD. Pathogen-free Sprague-Dawley rats (Taconic Farms, Germantown, NY) were used in the experiments. Because the effect of herbimycin A on the SK channels is more robust in CCD from rats on a KD diet than from those on a normal diet (22), we carried out the experiments in the rats on a KD diet for 5-7 days. The method for preparation of CCD has been previously described (3). To immobilize the CCDs, we placed the tubules on a 5 × 5-mm cover glass coated with Cell-Tak (Collaborative Research, Bedford, MA). The cover glass was transferred to a chamber mounted on an inverted microscope (Nikon, Melville, NY), and tubules were superfused with a bath solution containing (in mM) 140 NaCl, 5 KCl, 1.8 MgCl2, 1.8 CaCl2, 5 glucose and 10 HEPES (pH = 7.4). We used a sharpened pipette to open the CCDs to gain access to the apical membranes. In the present study, only principal cells were patched.

Patch-clamp technique. Electrodes were pulled with a Narishige model PP83 vertical pipette puller and had resistances of 4-6 MOmega when filled with 140 mM NaCl. The channel current recorded by an Axon 200A patch-clamp amplifier was low-pass filtered at 1 kHz using an eight-pole Bessel filter (902LPF, Frequency Devices, Haverhill, MA). The current was digitized at a sampling rate of 44 kHz using an Axon interface (Digidata 1200) and was transferred to an IBM-compatible Pentium computer (Gateway 2000) at a rate of 4 kHz and analyzed using the pCLAMP software system 7.0 (Axon Instruments, Burlingame, CA). Channel activity was defined as NPo, a product of channel open probability (Po) and channel number (N). NPo was calculated from data samples of 60-s duration in the steady state as follows
NP<SUB>O</SUB><IT>=&Sgr;</IT>(1<IT>t</IT><SUB>1</SUB><IT>+</IT>2<IT>t</IT><SUB>2</SUB><IT>+… </IT>i<IT>t</IT><SUB>i</SUB>) (1)
where ti is the fractional open time spent at each of the observed current levels. We did not make an effort to distinguish whether stimulation of channel activity resulted from increasing the channel number or channel open probability.

Solution and statistics. The pipette solution was composed of (in mM) 140 KCl, 1.8 Mg2Cl, and 5 HEPES (pH = 7.4). Herbimycin A was purchased from Biomol and dissolved in DMSO. Vasopressin, taxol, colchicine, cytochalasin D, and phalloidin were obtained from Sigma (St. Louis, MO). Taxol and cytochalasin D were dissolved in methanol and ethanol, respectively. The final concentrations of ethanol, methanol, or DMSO were <0.1%, and they had no effect on channel activity.

Experiments were carried out in cell-attached patches, at 37°C. We recorded the channel activity for at least 1 min under control conditions, and the bath solution was then switched to a solution containing a given cytoskeleton inhibitor such as colchicine or cytochalasin D from 30-60 min while the channel activity was continuously monitored. For studying the effect of herbimycin A and vasopressin in the tubules treated with cytoskeleton inhibitors, the CCDs were incubated in media containing a given cytoskeleton inhibitor for 30 min. We then carried out the experiments in the continuous presence of the cytoskeleton inhibitor. To determine whether the effect of herbimycin A can be reversed in the tubules treated with cytoskeleton inhibitors, the CCDs were incubated in a control bath medium for 30 min after removal of taxol, colchicine, cytochalasin D, or phalloidin.

Data are presented as means ± SE. We used paired and unpaired Student's t-tests to determine the statistical significance.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

We first examined the effect of taxol, an agent which freezes the microtubule, or colchicine, a microtubule inhibitor, on the activity of the SK channel in the CCD. We confirmed the previous observation that addition of 20 µM taxol or colchicine did not affect the channel activity within 60 min (21). After establishing that taxol and colchicine had no direct effect on channel activity, we investigated, in rats on a KD diet for 5-7 days, the effect of herbimycin A, an inhibitor of PTK, on the activity of the SK channels in the CCDs treated with either 20 µM taxol or colchicine for 30 min. We have previously demonstrated that herbimycin A increased the number of SK channels in CCDs harvested from rats on a KD diet (22). Figure 1 summarizes the results of 14 experiments in which the effect of 1 µM herbimycin A on channel activity was examined in the presence of colchicine or taxol. It is apparent that the stimulatory effect (NPo) of the PTK inhibitor was completely absent in the CCDs treated with either colchicine or taxol (control, 0.42 ± 0.03; herbimycin A + colchicine, 0.42 ± 0.03; herbimycin A + taxol, 0.41 ± 0.03). The lack of an effect of herbimycin A resulted from inhibiting microtubules because washout of colchicine or taxol restored the stimulatory effect of herbimycin A. Figure 1 demonstrates that addition of herbimycin A stimulated the SK channels and increased NPo from 0.42 ± 0.03 to 1.2 ± 0.1 (n = 22) after removal of colchicine and to 1.12 ± 0.15 (n = 14) after removal of taxol, respectively. This strongly suggested that an intact microtubule is essential for the effect of herbimycin A. Figure 2 is a representative recording showing the effect of herbimycin A on the SK channel after washout of taxol. It is clear that inhibiting PTK stimulates the SK channels in CCDs from rats on a KD diet. We observed that herbimycin A increased two SK channels at the same time. This suggests that inhibiting PTK-induced increase in channel activity may be mediated by insertion of new SK channels.


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Fig. 1.   Effect of 1 µM herbimycin A on the activity of the small-conductance K (SK) channels in the presence of 20 µM colchicine or taxol in cortical collecting ducts (CCDs) from rats on a K-deficient (KD) diet. Channel activity was measured in the control tubule (-colchicine/-taxol, -herbimycin A), in tubules treated with either colchicine or taxol, in the CCDs treated with herbimycin A and colchicine/taxol, and in those treated with herbimycin A 30 min after removal of colchicine/taxol. NPo, product of channel open probability and channel number. *Significant difference between control and experimental group (P < 0.05).



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Fig. 2.   A channel recording shows the effect of 1 µM herbimycin A on the SK channel 30 min after removal of 20 µM taxol in CCDs from rats on a KD diet. The experiment was performed in a cell-attached patch, and the pipette holding potential was +30 mV (hyperpolarization). The channel conductance is 38-40 pS. Top trace: time course of the experiment. Bottom traces: 2 parts of the top trace extended at a fast time resolution. c···, Channel closed.

To determine whether the effect of vasopressin on the SK channel also depends on an intact microtubule, we examined the effect of vasopressin (200 pM) on the SK channels in CCDs from rats on a KD diet for 5-7 days. Figure 3 is a typical recording showing the effect of vasopressin on channel activity in a cell-attached patch in CCDs treated with taxol. Clearly, vasopressin stimulated the SK channels in the presence of taxol and increased NPo from 0.55 ± 0.1 to 1.70 ± 0.2 (n = 17). Figure 4 also summarizes the effect of vasopressin in CCDs treated with colchicine, showing that, in the presence of colchicines, vasopressin increased NPo to 1.75 ± 0.2 (n = 10) in CCDs from rats on a KD diet for 5-7 days.


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Fig. 3.   A channel recording demonstrates the effect of 200 pM vasopressin on the SK (38 pS) channels in CCDs treated with 20 µM taxol. The experiment was performed in a cell-attached patch, and the holding potential was 0 mV. Bottom traces: 2 parts of the top trace extended at a fast time resolution.



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Fig. 4.   Effect of vasopressin (200 pM) on the activity of the SK channels in the presence of either colchicine or taxol (20 µM). *Significant difference between control (-vasopressin and -colchicine/-taxol) and experimental group (P < 0.05).

After establishing that inhibition of the microtubule abolished the effect of herbimycin A but not the effect of vasopressin, we explored the role of actin filaments in mediating the effect of herbimycin A on channel activity. We confirmed the previous observation (18) that addition of 10 µM cytochalasin D, an agent that inhibits F-actin polymerization (11), completely blocked channel activity (Fig. 5). Moreover, Fig. 5 also shows that the effect of herbimycin A on the SK channels was absent in the CCDs treated with cytochalasin D (n = 13). Also, removal of cytochalasin D restored the effect of inhibiting PTK, and the addition of herbimycin A (1 µM) increased channel activity from 0.5 ± 0.05 to 1.2 ± 0.15 (n = 13). In contrast to cytochalasin D, addition of 10 µM phalloidin, which stabilizes F-actin against depolymerization (11), did not significantly change the channel activity within 60 min. However, the stimulatory effect of herbimycin A is completely abolished in the presence of phalloidin (n = 13) (Fig. 5). The inhibitory effect of phalloidin on herbimycin A-induced stimulation of channel activity was reversible because removal of phalloidin restored the effect of herbimycin A and NPo increased from 0.52 ± 0.06 to 1.3 ± 0.16 (n = 13). This suggests that actin filaments are also involved in mediating the effect of inhibiting PTK.


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Fig. 5.   Effect of 1 µM herbimycin A on the activity of the SK channels in the presence of 10 µM cytochalasin D or 10 µM phalloidin in CCDs from rats on a KD diet. Channel activity was measured in the control tubule (-cytochalasin D/-phalloidin, -herbimycin A), in tubules treated with either cytochalasin D or phalloidin, in the CCDs treated with herbimycin A and cytochalasin D/phalloidin, and in those treated with herbimycin A 30 min after removal of cytochalasin D or phalloidin. *Significant difference between control and experimental group (P < 0.05).

Also, we examined the effect of vasopressin on the SK channels in the CCD treated with phalloidin or cytochalasin D. Figure 6 is a recording showing the effect of vasopressin on the SK channels in CCDs treated with 10 µM phalloidin. It is apparent that vasopressin increased the activity of the SK channels in CCDs treated with phalloidin. In 10 experiments, we observed that vasopressin increased NPo from 0.5 ± 0.06 to 1.5 ± 0.17 (Fig. 7). However, the effect of vasopressin was absent in CCDs treated with cytochalasin D (n = 9) (Fig. 7). Moreover, the notion that actin filaments are required for the effect of vasopressin is also supported by experiments in which washout of cytochalasin D restored the effect of vasopressin, which increased NPo from 0.5 ± 0.06 to 1.6 ± 0.2 (n = 5) (data not shown).


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Fig. 6.   A channel recording demonstrates the effect of 200 pM vasopressin on the SK (38-40 pS) channels in CCDs treated with 10 µM phalloidin. The experiment was performed in a cell-attached patch, and the holding potential was +30 mV (hyperpolarization). Bottom traces: 2 parts of the top trace extended at a fast time resolution. *Vasopressin-induced increase in channel activity.



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Fig. 7.   Effect of vasopressin (200 pM) on the activity of the SK channels in the presence of either 10 µM phalloidin or 10 µM cytochalasin D. *Significant difference between control (-vasopressin and -phalloidin/-cytochalasin D) and experimental group (P < 0.05).

To determine whether the effects of vasopressin and herbimycin A are additive, we examined the effect of herbimycin A on channel activity in CCDs that had been challenged by vasopressin. Figure 8 is a typical channel recording showing the effect of 1 µM herbimycin A in the presence of vasopressin. Addition of vasopressin (200 pM) stimulated channel activity and increased NPo from 0.6 ± 0.05 to 1.7 ± 0.2 within 5 min (n = 7). In the presence of vasopressin, application of herbimycin A increased NPo to 3.4 ± 0.4 within 15 min (n = 7). In contrast, the second addition of vasopressin did not further stimulate channel activity (data not shown).


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Fig. 8.   A channel recording showing the effects of herbimycin A (1 µM) and vasopressin (200pM). The experiment was carried out in a cell-attached patch, and the pipette holding potential was 30 mV. Top trace: the time course of the experiment. Bottom traces: 3 parts of the trace (1-3) extended to demonstrate the fast time resolution. The channel conductance was 40 pS.


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

In the present study, we have demonstrated that treatment of cells with cytochalasin D, phalloidin, taxol, or colchicine completely abolished the stimulatory effect of herbimycin A. This indicates that microtubules and actin filaments are involved in mediating the effect of herbimycin A on the SK channels in the CCD. In contrast, the effect of vasopressin on the apical SK channels was still observed in the presence of the inhibitors of microtubules and phalloidin, suggesting that the effect of vasopressin does not depend on the microtubule. Although cytochalasin D also abolished the effect of vasopressin on the SK channels, the finding that phalloidin treatment abolished the effect of inhibiting PTK and had no effect on the vasopressin-induced stimulation suggests that actin filaments may have a different role in mediating the effect of vasopressin and herbimycin A, respectively.

We have previously shown that the stimulatory effect of herbimycin A on SK channels results specifically from inhibiting PTK, which facilitates the tyrosine phosphorylation of SK channels (20, 21). This conclusion is supported by several lines of evidence. First, the effect of herbimycin A was observed only in CCDs harvested from rats on a KD diet but was absent in the tubules from rats on an HK diet (20). The different response of the channels to herbimycin A is due to the fact that the expression of PTKs such as cSrc and cYes was significantly lower in CCDs from rats on an HK diet than in those from rats on a KD diet (22). Second, herbimycin A stimulates ROMK1, which is closely related to the native SK channel in the CCD, only in oocytes injected with cSrc and ROMK1 but not in oocytes injected with ROMK1 alone (15). Third, treatment of oocytes injected with cSrc and ROMK1 with PTP inhibitor abolished the effect of herbimycin A. This indicates that the effect of herbimycin A is induced by favoring the tyrosine dephosphorylation by PTP after inhibition of PTK. It is possible that stimulation of tyrosine phosphorylation decreases, whereas stimulating tyrosine dephosphorylation increases, the activity of SK channels in the CCDs (15, 22). Although the mechanism by which PTK increases the number of SK channels is not completely understood, it is unlikely that tyrosine phosphorylation directly inhibits SK channels because adding exogenous cSrc did not decrease channel activity in inside-out patches (20).

Using confocal microscopy, our laboratory has demonstrated that stimulating tyrosine phosphorylation by blocking tyrosine phosphatase decreased the surface location of ROMK1, whereas enhancing tyrosine dephosphorylation with herbimycin A increased the density of ROMK1 in oocytes injected with ROMK1 and cSrc (15). Therefore, it is possible that the effect of herbimycin A on the SK channels is mediated by increasing the exocytosis of the SK channels in the CCD. Moreover, insertion of the SK channels depended on the intact cytoskeleton because inhibiting microtubules or disrupting actin filaments abolished the effect of herbimycin A. On the other hand, our laboratory has shown that inhibition of microtubules did not influence the effect of inhibiting tyrosine phosphatase on ROMK1 (15). The same observation has been confirmed in the CCD. We have observed that inhibition of protein tyrosine phosphatase reduced the channel activity in the CCDs treated with microtubule inhibitors (Wei Y and Wang W-H, unpublished observations). This suggests that endocytosis may not depend on the microtubule. Also, the finding that application of microtubule inhibitors and phalloidin has no significant effect on channel activity suggests that tyrosine phosphorylation-induced endocytosis and tyrosine dephosphorylation-induced exocytosis are highly regulated processes. Only if the balance between tyrosine phosphorylation and dephosphorylation is disturbed by either dietary K intakes or pharmacological approaches can endocytosis or exocytosis take place. In addition to tyrosine phosphorylation-regulated endocytosis, it is possible that ROMK1 channels are internalized by a constitutive endocytosis mechanism. However, the constitutive pathway of endocytosis may be a slow process because addition of microtubule inhibitors had no effect on channel activity within 30-60 min. The cytoskeleton, such as microtubules and actin filaments, has been shown to be involved in mediating exocytosis of membrane proteins (9). For instance, inhibiting actin filament polymerization has been reported to block GLUT-4 exocytosis (8, 16). In addition, the cytoskeleton has been shown to be a substrate for PTK (10), suggesting the role of PTK in mediating endocytosis or exocytosis.

In addition to insertion and internalization of the SK channels, regulation of SK channel activity could also be achieved by closing or opening K channels in the cell membrane. We have previously speculated that there are at least three populations of SK channels in the CCD. The first group of SK channels is located in the cell membrane and is active. The second population of the K channel is phosphorylated by PTK and retrieved from the cell membrane. The third pool of the K channel is silent but may be located in the cell membrane. This hypothesis was supported by our laboratory's previous observation that the effect of herbimycin A on SK channel activity was progressively increased by prolonged low K intake and absent in tubules from rats on an HK diet (22). Moreover, prolonged low K intake increased, whereas high K intake decreased, the expression of cSrc and cYes PTK in the renal cortex (22). It is possible that low K intake increases, whereas high K intake diminishes, the pool size of the SK channels responding to inhibition of PTK. The hypothesis that there are different SK channel pools responding to a variety of stimuli is further supported by the present findings. First, the effect of vasopressin on channel activity was not affected by colchicine, taxol, or phalloidin, whereas these agents completely abolished the effect of herbimycin A. Second, addition of herbimycin A increased channel activity in CCDs in which vasopressin had first been used to stimulate channel activity. In contrast, the second exposure of CCDs to vasopressin had no additional stimulatory effect. This strongly indicates that vasopressin activates SK channels from a pool different from that containing channels responding to an inhibition of PTK. Alternatively, it is also possible that vasopressin and PTK inhibitor may stimulate the same set of SK channels by two different pathways. It is conceivable that herbimycin A-induced SK channels are from the microtubule-dependent membrane fusion. In contrast, vasopressin activated the previously silent SK channels that were already present in the cell membrane because the effect of vasopressin did not depend on the microtubule.

The effect of vasopressin is the result of stimulating cAMP, because the effect of vasopressin can be mimicked by a membrane-permeable cAMP analog (3). It is well established that PKA-induced phosphorylation has an important role in regulating the activity of the SK channels. It was reported that mutating one PKA phosphorylation site significantly diminished the K current in oocytes injected with ROMK1 (13, 14, 23). A large body of evidence has indicated that the specificity of PKA may partially be determined by the A kinase anchoring protein (AKAP), which binds the regulatory subunit of PKA and brings PKA to the close vicinity of effector proteins such as ion channels (2, 5, 17). Our laboratory has previously shown that AKAP is involved in mediating the effect of PKA on ROMK1 (1, 2). Because AKAP is associated with a membrane by the cytoskeleton, it is possible that the lack of vasopressin's effect in CCDs treated with cytochalasin D may result from disrupting the membrane targeting of the associate proteins such as AKAP.

Increasing insertion of the SK channels induced by herbimycin A can result from either stimulation of SK channel recycling or from enhancing export of the de novo synthesized SK channels from the endoplasmic reticulum. However, our unpublished observation (Wei Y and Wang W-H) that herbimycin A can still increase the number of the SK channels in CCDs treated with brefeldin A strongly suggests that the newly inserted SK channels were, at least in part, mediated by stimulating the insertion from a recycling pool. However, further experiments are required to explore the source of the newly appeared SK channel in response to inhibition of PTK.

We conclude that an intact cytoskeleton is required for mediating the effect of inhibiting PTK on the SK channels, whereas microtubules are not directly required for mediating a vasopressin-induced increase in channel activity. Therefore, it is possible that an increase in SK channels induced by herbimycin A and by vasopressin results from two different channel populations or is regulated by two separate pathways.


    ACKNOWLEDGEMENTS

The authors thank Melody Steinberg for help in preparing the manuscript.


    FOOTNOTES

This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-47042 and DK-54983.

Address for reprint requests and other correspondence: W.-H. Wang, Dept. of Pharmacology, New York Medical College, Valhalla, NY 10595 (E-mail: wenhui_wang{at}nymc.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

10.1152/ajprenal.00229.2001

Received 20 July 2001; accepted in final form 1 November 2001.


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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Am J Physiol Renal Fluid Electrolyte Physiol 282(4):F680-F686
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