Am J Physiol Renal Physiol 282: F680–F686, 2002;
10.1152/ajprenal.00229.2001.
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
Received 20 July 2001; accepted in final form 1 November 2001
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.
ROMK1; microtubule; exocytosis; protein tyrosine phosphatase; vasopressin; cortical collecting duct
METHODS
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).
Address for reprint requests and other correspondence: W.-H.
Wang, Dept. of Pharmacology, New York Medical College, Valhalla,
NY 10595 (E-mail: [email protected]).
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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
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.
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Wei, Yuan, and Wen-Hui Wang. Role of the cytoskeleton
in mediating effect of vasopressin and herbimycin A on secretory K channels in CCD. Am J Physiol Renal Physiol 282:
F680–F686, 2002; 10.1152/ajpcell.00229.2001.—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 patchclamp 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.
ROLE OF THE CYTOSKELETON IN MEDIATING EXOCYTOSIS
NP O ⫽ ¥共1t1 ⫹ 2t2 ⫹ . . . iti兲
(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
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 ␮M 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 ␮M 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 ␮M
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
Fig. 1. Effect of 1 ␮M herbimycin A on the
activity of the small-conductance K (SK)
channels in the presence of 20 ␮M 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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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 M⍀ 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
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ROLE OF THE CYTOSKELETON IN MEDIATING EXOCYTOSIS
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.
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.
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 ␮M 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 ⫽
AJP-Renal Physiol • VOL
13). Also, removal of cytochalasin D restored the effect
of inhibiting PTK, and the addition of herbimycin A (1
␮M) increased channel activity from 0.5 ⫾ 0.05 to 1.2 ⫾
0.15 (n ⫽ 13). In contrast to cytochalasin D, addition of
10 ␮M 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.
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 ␮M 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. 2. A channel recording shows the effect of 1 ␮M herbimycin A on the SK
channel 30 min after removal of 20 ␮M
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䡠䡠䡠, Channel closed.
ROLE OF THE CYTOSKELETON IN MEDIATING EXOCYTOSIS
F683
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 ␮M herbimycin A in the presence of vasopressin. Addition of vaso-
Fig. 4. Effect of vasopressin (200 pM) on the activity of the SK
channels in the presence of either colchicine or taxol (20 ␮M).
*Significant difference between control (⫺vasopressin and ⫺colchicine/⫺taxol) and experimental group (P ⬍ 0.05).
AJP-Renal Physiol • VOL
pressin (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).
Fig. 5. Effect of 1 ␮M herbimycin A on the activity of the SK
channels in the presence of 10 ␮M cytochalasin D or 10 ␮M 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).
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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 ␮M 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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ROLE OF THE CYTOSKELETON IN MEDIATING EXOCYTOSIS
DISCUSSION
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
Fig. 7. Effect of vasopressin (200 pM) on the activity of the SK
channels in the presence of either 10 ␮M phalloidin or 10 ␮M
cytochalasin D. *Significant difference between control (⫺vasopressin and ⫺phalloidin/⫺cytochalasin D) and experimental group (P ⬍
0.05).
AJP-Renal Physiol • VOL
Fig. 8. A channel recording showing the effects of herbimycin A (1
␮M) 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.
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 vasopressininduced 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
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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
␮M 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.
ROLE OF THE CYTOSKELETON IN MEDIATING EXOCYTOSIS
AJP-Renal Physiol • VOL
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
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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
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ROLE OF THE CYTOSKELETON IN MEDIATING EXOCYTOSIS
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.
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The authors thank Melody Steinberg for help in preparing the
manuscript.
This work was supported by National Institute of Diabetes and
Digestive and Kidney Diseases Grants DK-47042 and DK-54983.
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