Growth factor-mediated K+ channel activity
associated with human myeloblastic ML-1 cell
proliferation
Ling
Wang,
Bo
Xu,
Richard E.
White, and
Luo
Lu
Department of Physiology and Biophysics, School of Medicine,
Wright State University, Dayton, Ohio 45435
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ABSTRACT |
ML-1 cell proliferation is dependent on the presence of serum
growth factors. Removing serum from the culture medium results in
growth arrest and promotes differentiation. In this study, we found
that a 4-aminopyridine-sensitive
K+ channel was highly expressed in
proliferating ML-1 cells and significantly diminished in
G1-arrested ML-1 cells induced by serum deprivation but was restored within 30 min in these cells with
addition of 10% fetal bovine serum (FBS) or 5 ng/ml epidermal growth
factor (EGF). Intracellular adenosine 3',5'-cyclic
monophosphate (cAMP) levels, but not guanosine 3',5'-cyclic
monophosphate, were significantly increased in serum-deprived cells
stimulated by FBS or EGF, and the effects of FBS and EGF on the channel
activation were mimicked by exogenous cAMP. In inside-out patches,
K+ channel activity was
significantly increased by the cAMP-dependent protein kinase catalytic
subunit, whereas the effect of EGF on K+ channel activation was blocked
by Rp-8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphothioate. Together, our results
demonstrate that serum growth factors stimulate
K+ channel activity in
proliferation of ML-1 cells through protein kinase-induced
phosphorylation and suggest an important molecular mechanism for serum
growth factor-stimulated mitogenesis in ML-1 cells.
patch clamp; adenosine 3',5'-cyclic monophosphate; protein kinase A; epidermal growth factor; DNA synthesis
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INTRODUCTION |
HUMAN MYELOBLASTIC leukemia (ML-1) cells proliferate in
tissue culture as immature myeloblasts, and this proliferation is stimulated by various growth factors present in the culture serum. ML-1
cells can be programmed to differentiate into granulocytes or
macrophages when specifically stimulated (4), and these differentiated
cells play significant roles in the immune defense system and require
membrane-mediated transduction of cell-cell and cell-environment
signals. Our previous studies demonstrated that a voltage-gated
K+ current in ML-1 cells was
altered during the entire process of differentiation induced by
12-O-tetradecanoylphorbol 13-acetate (TPA) and revealed that K+ channel
activity varied depending on the stage of ML-1 cell proliferation and
differentiation (18). Channel activity was dramatically diminished in
the early stages of TPA-induced ML-1 cell differentiation and was
completely suppressed in differentiated cells. However, it is still
unclear how voltage-gated K+
channel activity is regulated and what is the precise role of this
channel in ML-1 cell proliferation and differentiation.
Ion channels located at the cell membrane sense chemical and physical
changes in the cell growth environment and mediate functional adaptation of the cell to environmental changes. In excitable tissues,
such as nerves and muscle and some hormone-releasing cells,
voltage-dependent K+ channels play
important roles in regulation of cell electrical activities in response
to various stimulations. Voltage-dependent K+ channels also play crucial
roles in cell development, volume regulation, membrane potential
stabilization, and proliferation (5, 6, 8, 13, 20). A variety of
studies have suggested that the voltage-gated
K+ channel plays a functional role
in the onset of cellular events associated with both T and B lymphocyte
activation (1, 17, 21). It has been found that enhanced
K+ channel gene expression or
increased K+ channel activity is
associated with mitogenesis in several cell types (21). Application of
different K+ channel blockers to
cultured cells significantly inhibits various types of cell
proliferation (1, 7, 10). Recently, the important role of the
voltage-gated K+ channel in
mitogenesis has been suggested to be a key determinant for cell
progression through G1 phase
before the G1 checkpoint in ML-1
and other cells (10, 16, 32). In
K+ channel activity-suppressed
ML-1 cells, retinoblastoma protein (pRB) is dephosphorylated and
effectively inhibits the cell from progressing through the
G1/S transition (32).
To investigate the precise role the voltage-gated
K+ channel plays in growth
factor-mediated ML-1 cell proliferation, we have designed a series of
experiments to study mechanisms that underlie the correlation of
channel activity to ML-1 cell growth control and the effect of growth
factors on K+ channel activity
during ML-1 cell proliferation. We found that the growth-related
K+ channel activity was markedly
diminished in serum-deprived cells, and channel activity could be
restored to its full activity within 30 min after physiological
concentrations of serum or epidermal growth factor (EGF) were applied
to the patch chamber. EGF-stimulated K+ channel activity was mediated
through elevation of intracellular adenosine 3',5'-cyclic
monophosphate (cAMP) levels and activation of cAMP-dependent protein
kinase (PKA)-induced phosphorylation. Our results suggest that the
K+ channel in ML-1 cells is
regulated by growth factor-mediated intracellular signaling pathways
and plays an important role in controlling cell proliferation,
specifically in the G1/S
transition of the cell cycle.
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MATERIALS AND METHODS |
Cell culture.
ML-1 cells were originally isolated from an acute myeloblastic leukemia
patient and were received as a generous gift from Dr. R. W. Craig,
Dartmouth Medical School (Hanover, NH). Cells were maintained in
suspension culture as described previously (18). Briefly, culture
medium RPMI 1640 containing 25 mM
N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) buffer was supplemented with 7.5% heat-inactivated fetal
bovine serum (FBS; GIBCO, Grand Island, NY). For the
high-K+ culture medium, NaCl in
the RPMI 1640 medium was isotonically replaced by 135 mM KCl. Cells
were grown in a humidified incubator with 5%
CO2 at 37°C and passed at a
seeding density of 3 × 105
cells/ml. Cells were washed twice with phosphate buffer solution (PBS) before they were transferred for patch-clamp experiments.
Cell growth assays.
Proliferation of ML-1 cells was determined by counting cell numbers and
measuring
[3H]thymidine
incorporation into the host DNA. ML-1 cells from suspension cultures
were plated in triplicate into 35-mm culture dishes at a density of 3 × 105 cells/ml. The effect
of K+ channel blockers on cell
growth was tested by dilution of concentrated stock solution directly
into the plating medium. After incubation for 4 or 24 h, all cultures
were pulsed with 1 µCi/ml
[3H]thymidine for 2 h.
Cells were then harvested and washed twice with PBS. Nucleic acids were
precipitated with 10% trichloroacetic acid, and radioactivity of
samples was quantified by liquid scintillation counting. Growth arrest
induced by serum deprivation was achieved by culturing cells in RPMI
1640 medium containing 0.3% FBS at 37°C for 24 h.
Intracellular cAMP and guanosine 3',5'-cyclic
monophosphate assays.
ML-1 cells were synchronized in the
G1 phase of the cell cycle by
serum deprivation for 24 h. Cells were then aliquoted into 35-mm
culture dishes at a final concentration of 1 × 106 cells/ml and were stimulated
with either 10% FBS or 5 ng/ml EGF. At the times indicated, cells were
collected and washed twice with ice-cold PBS and then resuspended in 1 ml of 65% (vol/vol) ice-cold ethanol. After they had settled for 60 min at 22°C, supernatants were drawn into new test tubes and
remaining precipitates were washed with ice-cold 65% ethanol. Washing
solutions were added into the appropriate tubes. Cell extracts were
centrifuged at 2,000 g for 15 min at
4°C, and supernatants were transferred into fresh tubes. The
extracts were then dried overnight by a vacuum lyophilizer.
Intracellular cAMP and guanosine 3',5'-cyclic monophosphate (cGMP) levels were assayed with the use of the enzyme immunoassay system (EIA, nonacetylation protocol) provided by Amersham Life Sciences (Buckinghamshire, UK).
Patch-clamp studies.
Both cell-attached and inside-out patch-clamp techniques were used in
the present study. Detailed methods for the patch pipette preparation,
data acquisition, and single-channel analysis were described previously
(31). Briefly, pipettes were manufactured with a two-stage puller
(PP-83, Narishige) with a resistance of 3-4 M
when filled with
150 mM KCl solution. The solutions used in these experiments were
1) KCl bath solution containing (in mM) 140 KCl, 2 MgCl2, 0.5 CaCl2, 1 ethylene
glycol-bis(
-aminoethyl ether)-N,N,N',N'-tetraacetic
acid, and 10 HEPES (pH 7.4) and 2) pipette solution containing (in mM) 140 KCl, 2 MgCl2, 1 CaCl2, and 10 HEPES (pH 7.4).
Single-channel currents were recorded with an Axonpatch 200A amplifier
(Axon Instruments, Foster City, CA) and filtered with a four-pole
low-pass filter at 1 kHz and digitalized at 22 kHz by a pulse-code
modulator (A. R. Vetter, Rebersburg, PA). The pCLAMP program (Axon
Instruments) was used to analyze the single-channel data. The channel
activity was determined as NPo, where
N represents number of channel
openings in the patch and
Po represents the
channel open probability. All experiments were performed at room
temperature (21-23°C). Data are presented as original values
or as means ± SE, when indicated. Significant differences were
determined by using the paired t-test
at the confidence interval indicated.
Reagents.
The catalytic subunit of PKA, MgATP, dithiothreitol (DTT),
8-(4-chlorophenylthio)-cAMP (CPT-cAMP), and 4-aminopyridine (4-AP) were
purchased from Sigma Chemical (St. Louis, MO).
Rp-8-(4-chlorophentlthio)adenosine 3',5'-monophosphothioate
(Rp-CPT-cAMPS) and EGF were obtained from Biolog Life Science (La Jolla, CA) and Calbiochem (La Jolla, CA),
respectively. The catalytic subunit of PKA was diluted in KCl bath
solution, and 1 mg/ml DTT was added to the mixture. The mixture was
then allowed to stand for 10 min at room temperature (22°C) before
use or before storage at
80°C.
K+ channel blockers were prepared
as stock solutions with concentrations of 500 mM to 1 M in sterile
water.
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RESULTS |
A voltage-gated K+ channel in proliferating
ML-1 cells.
A voltage-gated and 4-AP-sensitive
K+ channel at the whole cell level
in ML-1 cells has been shown previously (18). To investigate the role
of this channel in growth factor-mediated ML-1 cell
proliferation, single K+ channel
currents were measured (Fig.
1A).
The microscopic current-voltage (I-V)
relationship was linear with a single-channel conductance of 31 ± 0.7 pS (measured from the slope of
I-V
curves; n = 6) in symmetric 140/140 mM
KCl solution (Fig. 1B). When
extracellular KCl was isotonically replaced with NaCl, the inward
current was abolished (Fig. 1B),
suggesting selectivity of this channel for K+. The
K+-selective channel was also
confirmed pharmacologically by demonstrating sensitivity of the channel
to extracellular 4-AP. Channel activity was inhibited 63 and 97% by
extracellular application of 50 or 100 µM 4-AP, respectively (Fig.
1C). These results indicate that the
single-channel current is carried by
K+ through a 4-AP-sensitive
K+ channel.

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Fig. 1.
Single-channel recordings of
K+ channel in proliferating
ML-1 cells. Single-channel currents were recorded from cell-attached patches at various membrane potentials.
A: outward current recorded as
an upward deflection and inward current as a downward deflection. Arrows, closed states of channel. Extracellular solution in the pipette
contained 140 mM K+.
B: current-voltage
(I-V)
relationships obtained from patches with symmetrical 140 mM
K+ in the extracellular solution
( ) or with 0 mM K+ in the
extracellular solution ( ). Data were plotted as means with SE bars
and fit with a linear curve (n = 6).
C: blocking effect of 4-aminopyridine
(4-AP) on the K+ channel activity
(NPo, where
N represents number of channel
openings in the patch and
Po represents
channel open probability). In the cell-attached patches, 50 or 100 µM
4-AP was added in the extracellular solution at a membrane potential of
60 mV. Data were presented as means ± SE
(n = 6). * Significant
difference (P < 0.05), determined by
the paired t-test.
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Serum growth factors activate the K+
channel.
Proliferation of ML-1 cells is dependent on various serum growth
factors in the culture medium. If the
K+ channel plays a role in growth
factor-mediated ML-1 cell proliferation, then channel activity should
be regulated by serum growth factors. To test this hypothesis, the
precise role of this channel in growth factor-mediated ML-1 cell
proliferation was determined by patch-clamp experiments. Possible
regulatory effects of serum growth factors on
K+ channel function were examined
with the cell-attached patch clamp. Channel activity was observed in
ML-1 cells cultured with serum-rich medium (with 7.5% FBS), and the
channel was frequently open with an average activity
(NPo) of 21 ± 5.5% (at
60 mV, n = 9)
in cell-attached patches (Table 1). In
contrast, channel activity was significantly diminished to an
NPo of 0.4 ± 0.2% (at
60 mV, n = 16, P < 0.001) in ML-1 cells that were
cultured in serum-free medium (with 0.3% FBS) for at least 12 h (Fig.
2A). The
specific effect of growth factors on channel activity was studied by
addition of 10% FBS onto serum-starved cells. When 10% FBS was
applied in the patch chamber, channel activity was restored in
cell-attached patches in a few minutes and reached full activity within
30 min (Fig. 2B). Channel activity
was significantly increased, from 0.4 ± 0.2% to 40 ± 15%
within 30 min and to 57 ± 5% after 30 min
(n = 6, P < 0.001) (Fig.
2C). In some patches lasting for 150 min or longer, high channel activity was observed continuously. Thus these findings suggest that K+
channel function in proliferating ML-1 cells is regulated by serum
growth factors.

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Fig. 2.
Effect of serum growth factors on
K+ channel activity. Cell-attached
patch-clamp experiments were performed in a symmetrical 140 mM
K+ condition at a membrane
potential of 60 mV. A:
comparison of K+ channel activity
in proliferating (top trace) and
serum-deprived (bottom trace) ML-1
cells. Histogram bars represent channel activities from traces shown at
top.
B: activation of the
K+ channel by 10% fetal bovine
serum (FBS) in serum-deprived ML-1 cells. Channel activities were
observed in the same cell before and after addition of 10% FBS in the
patch chamber. Histogram bars represent channel activities from traces
shown at top.
C: statistics of
K+ channel activity stimulated by
10% FBS in serum-deprived ML-1 cells. Bars (means with SE bars)
represent K+ channel activity
stimulated by 10% FBS at 0, <30, and >30 min. Maximal activity
(Maxi) was selected from the entire patch period after the FBS
stimulation. * Significant difference
(P < 0.001); data were collected
from 6 independent experiments.
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Intracellular signaling pathway stimulated by serum growth factors.
To investigate the possible intracellular signaling pathway involved in
regulating K+ channel activity,
intracellular cAMP and cGMP concentrations were measured before and
after stimulation with 10% FBS in growth-arrested ML-1 cells. The
intracellular cAMP level was significantly increased within 5 min
(P < 0.001) after FBS
treatment and continued to rise for 55 min (Fig.
3). Because it has been shown that EGF
stimulates cAMP production in cardiac myocytes (18, 19) and hepatoma cells (20), we examined the effect of EGF on the cAMP levels in ML-1
cells. When 5 ng/ml EGF was applied to growth-arrested ML-1 cells in
culture, the intracellular cAMP concentration was significantly
increased within 5 min and reached a plateau level at 30 min (Fig. 3).
On the other hand, application of FBS or EGF did not significantly
increase intracellular cGMP levels.

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Fig. 3.
Stimulation of intracellular cAMP levels by serum growth factors in
serum-deprived ML-1 cells. Intracellular cAMP level was measured with
the enzyme immunoassay system, following the time course of 10% FBS
( ) and 5 ng/ml epidermal growth factor (EGF; ) treatments.
Intracellular cGMP level ( ) was also measured before and after
stimulation with 5 ng/ml EGF, following the indicated time course. Data
were plotted as means with SE bars and collected from 5 independent
experiments.
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The effects of EGF on K+ channel
activity were further characterized by applying EGF to the bath
solution of cell-attached patches in serum-deprived ML-1 cells. Channel
activity was markedly increased by 5 ng/ml EGF in a few minutes and
reached maximal activity within 30 min (Fig.
4A). The
channel activity measured at
60 mV was significantly increased,
from 0.4 ± 0.2% to 47 ± 15% within 30 min and to 48 ± 16% after 30 min (n = 4, P < 0.001). The time course showed
that the increase of EGF-induced
K+ channel activity was parallel
to the increase of intracellular cAMP level (Fig.
4B). It was notable that there was a
lag phase (a few minutes) in the increase of the channel activity.
Increasing the concentration of EGF to 25 ng/ml stimulated the
K+ channel activity in
serum-deprived ML-1 cells, but there was no further increase after 30 min (data not shown). Effects of FBS and EGF on
K+ channel activity were not a
voltage-dependent process in hematopoietic ML-1 cells when the membrane
potential was varied from
60 to +60 mV (Fig.
4C). To further confirm the effect
of cAMP on K+ channel regulation,
100 µM CPT-cAMP, a membrane-permeable cAMP analog, was added directly
to serum-deprived ML-1 cells in the patch chamber. Channel activity was
stimulated by CPT-cAMP within a few minutes (Fig.
4D) and significantly increased to
65 ± 12% (n = 8) within 30 min
(Table 1). Together, these results suggest that the intracellular
signaling pathway for the growth factor-mediated K+ channel activity involves
production of the second messenger cAMP and that EGF may be one of the
growth factors that stimulates this pathway.

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Fig. 4.
EGF-stimulated K+ channel
activity. Cell-attached patch-clamp experiments were performed in a
symmetrical 140 mM K+
condition at a membrane potential of 60 mV.
A: activation of the
K+ channel by 5 ng/ml EGF in
serum-deprived ML-1 cells. Channel activities were observed from the
same patch before and after addition of 5 ng/ml EGF in the patch
chamber. Histogram bars demonstrate channel activities from traces
shown at top.
B: time course
(inset) and statistics of
K+ channel activity stimulated by
5 ng/ml EGF in serum-deprived ML-1 cells. Bars (mean values ± SE)
represent K+ channel activity
stimulated by 5 ng/ml at 0, <30, and >30 min. Maximal activity was
selected from the entire patch period after the EGF stimulation.
* Significant difference (P < 0.001); data were collected from 6 independent experiments.
C: effect of membrane potential on
FBS- and EGF-induced K+ channel
activity. Data were collected from 4-6 independent experiments and
plotted as mean values with SE bars.
D: activation of
K+ channel by 100 µM
8-(4-chlorophenylthio)-cAMP (8-CPT-cAMP) in serum-deprived ML-1 cells.
Channel activities were observed from the same patch before
(top trace) and after
(bottom trace) addition of 100 µM
8-CPT-cAMP in the patch chamber. Histogram bars demonstrate channel
activities from traces shown at top.
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Verification of PKA involvement.
To further characterize the intracellular signaling pathway and the
potential involvement of PKA-induced phosphorylation in growth
factor-mediated K+ channel
activation, inside-out patches from ML-1 cells cultured in normal
medium were exposed to a phosphorylation mixture solution containing
MgATP and the catalytic subunit of PKA. Excised patches were held at
60 mV for 2 min to measure control channel activity, and then 1 mM MgATP was added to the patch bath solution.
K+ channel activity was not
significantly changed during a waiting period of 7-10 min. Then,
50 nM PKA catalytic subunit was added in the bath, resulting in a
significant increase in
NPo from 15 ± 3.2% to 38 ± 5.1% (n = 4, P < 0.05) in 5-10 min (Fig.
5A and Table 1). In inside-out patches that were not exposed to the phosphorylation mixture solution,
K+ activity faded away within
10-30 min after patch excision. Results from these experiments
suggest that phosphorylation of the channel protein induced by PKA is
required to maintain the normal activity of the
K+ channel in these cells. This
conclusion was further supported by experiments that demonstrated that
Rp-CPT-cAMPS, an antagonist of PKA,
blocked channel activity induced by EGF in serum-deprived ML-1 cells
(Fig. 5B). Cell-attached patches
were held at
60 mV, and 5 ng/ml EGF was then added to stimulate
channel activity. Rp-CPT-cAMPS (100 µM) was added to the patched cell after a dramatic increase of
channel activity was observed (5 min). The
K+ channel activity induced by EGF
was significantly diminished, from 47 ± 15% to 10 ± 3%
(n = 4, P < 0.01), within 30-50 min
after application of 100 µM
Rp-CPT-cAMPS (Fig.
5C).

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Fig. 5.
Increased K+ channel activity by
cAMP-dependent protein kinase (PKA)-induced phosphorylation.
A: activation of the
K+ channel by PKA-induced
phosphorylation. Activities of the
K+ channel in the excised
inside-out patch were compared before and after a cocktail containing
PKA catalytic subunit and MgATP was applied in the bath solution at
60 mV. Bottom: channel activity from the traces shown at top.
B: blockade of EGF effect on the K+ channel activity by
Rp-8-(4-chlorophenylthio)adenosine
3',5'-cyclic monophosphothioate
(Rp-CPT-cAMPS). In the cell-attached
patch from a serum-deprived cell,
K+ channel was activated 15 min
after 5 ng/ml EGF was applied at 60 mV. Channel activity was
then inhibited by application of 100 µM
Rp-CPT-cAMPS in the same cell.
Bottom: channel activities from traces
shown at top.
C: statistics of
Rp-CPT-cAMPS blockade. Bars represent
the blockade of EGF-stimulated K+
channel activity by 100 µM
Rp-CPT-cAMPS. Data were plotted as means with SE bars. * Significant difference
(P < 0.01, n = 4).
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Effect of suppressed K+ channel activity on
ML-1 cell proliferation.
The effect of inhibition of K+
channel on ML-1 cell proliferation was evaluated by adding
K+ channel blockers in the culture
medium individually or by replacing Na+ with
K+ in the medium and by monitoring
cell numbers and DNA synthesis measured by
[3H]thymidine
incorporation. ML-1 cells were growth arrested in the
G1 phase by culturing cells in
serum-deprived medium for 24 h. Growth-arrested cells were then
released into normal culture medium with 7.5% FBS (controls), in 135 mM K+ medium with 7.5% FBS (high
K+ medium), or in normal culture
medium containing 7.5% FBS plus 2 mM 4-AP, 2.5 mM
BaCl2, 10 mM tetraethylammonium,
or 30 µM quinine. After applications of different
K+ channel blockers at the
indicated concentrations or in the
high-K+ concentration culture
condition for 24 h, there were no significant changes in cell viability
measured with the trypan blue exclusion method. Viability measurements
of ML-1 cells in the absence and presence of different
K+ channel blockers and in
high-K+ concentration culture
condition are summarized in Table 2. The fractional inhibition of DNA synthesis was measured at 4 and 24 h after
release of growth-arrested cells. Rates of
[3H]thymidine
incorporation after exposure to different channel blockers or the
high-K+ concentration culture
condition were significantly inhibited as early as 4 h and reached a
much higher level at 24 h (Fig. 6). These
results suggest that blockade of the
K+ channel by
K+ channel blockers and growing
cells in the high-K+ concentration
medium inhibited DNA synthesis, preventing ML-1 cells from progressing
through the G1 phase to S phase of
the cell cycle.

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Fig. 6.
Effect of suppressed K+ channel
activity on ML-1 cell proliferation. Fractional inhibition of
[3H]thymidine
incorporation was measured at 4 h and at 24 h in
high-K+ culture condition (high
K+); in the presence of 2 mM
4-AP, 2.5 mM BaCl2, 10 mM
tetraethylammonium (TEA), or 30 µM quinine; or in the culture after
serum-deprived ML-1 cells were supplemented with 7.5% FBS
(starvation). Fractional inhibition was calculated by the equation (1 T)/T0,
where T represents rate of
[3H]thymidine
incorporation in the altered culture conditions or in the presence of
individual K+ channel blockers and
T0 represents
rate of [3H]thymidine
incorporation in the normal culture condition. Data were plotted as
means with SE bars and were collected from 6 independent experiments.
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DISCUSSION |
K+ channel activity has been found
to influence cell proliferation and differentiation in various systems
(2, 13, 18). In the present study, we demonstrate that
K+ channel activity is closely
correlated to serum growth factor-stimulated ML-1 cell proliferation.
In proliferating ML-1 cells, the
K+ channel activity was observed
in abundance (Table 1), but this activity was greatly suppressed in
serum-deprived ML-1 cells that were synchronized in the
G1 phase of the cell cycle (32).
The diminished K+ channel activity
in serum-deprived cells was then restored within 30 min by direct
exposure of ML-1 cells to physiological concentrations of FBS and EGF
(Figs. 2 and 4). Activation of K+
channels by serum growth factors has been found in other cell types.
For example, in PC-12 cells, nerve growth factor regulates the
abundance and distribution of delayed rectifier
K+ channels (24) and exposure of
resting microglial cells to interferon-
or granulocyte/macrophage
colony-stimulating factor results in an inhibition of outward
K+ current (11). These results
suggest that a shift of the resting membrane potential to more
hyperpolarized levels may be a prerequisite for intracellular
mechanisms involved in macrophage and microglial cell activity.
Generally, effects of growth factors and cytokines on
K+ channel activity can be divided
into long-term and short-term effects. The short-term effect is
characterized by altering channel gating, most likely through second
messenger-mediated modulation of channel protein. However, the
long-term effect corresponds to the maximal
K+ conductance affected by total
channel numbers resulting from altered
K+ channel gene expression and
insertion of channel proteins to the membrane. In human cultured
oligodendrocytes, inward rectifier K+ channels were modulated by
tumor necrosis factor-
(TNF-
), a cytokine associated with
activated macrophages (19, 25). Treatment of oligodendrocytes with
TNF-
for 24-48 h significantly decreases expression of the
K+ channel gene and diminishes the
mean open time of the K+ channel
relative to control value. These data suggest that TNF-
possesses
both short- and long-term effects on the inward rectifier K+ channel in human
oligodendrocytes. In the present study, activation of
K+ channels in ML-1 cells by 10%
FBS or 5 ng/ml EGF can be considered a short-term effect of serum
growth factors.
Growth factor/cytokine receptor-mediated second messenger systems, such
as the cAMP cascade, have been suggested to modulate K+ channel function, and a number
of different experimental approaches have been used to study the role
of the cAMP cascade in modulating K+ channel activity. Intracellular
levels of cAMP may be increased artificially by injection of cAMP
through microelectrodes, extracellular application of
membrane-permeable cAMP analogs, such as dibutyryl cAMP or
8-bromo-cAMP, use of phosphodiesterase inhibitors, or use of the
diterpene compound forskolin, a direct activator of adenylate cyclase
(20). Using EIA, we found that the effect of serum growth factors or
EGF on ML-1 cells was mediated through regulation of intracellular cAMP
levels but not intracellular cGMP levels (Fig. 3). The effect of
increased intracellular cAMP levels on
K+ channel activity was verified
by direct application of CPT-cAMP (Fig.
4D). Increased intracellular cAMP
can further activate PKA, leading to phosphorylation of
serine/threonine residues on a variety of substrate proteins, including
ion channels.
The present study demonstrates that serum growth factor-stimulated
K+ channel activity is mediated
via cAMP-dependent phosphorylation. Using the inside-out patch clamp,
we confirmed that serum growth factor-regulated
K+ channels in ML-1 cells can be
activated by direct phosphorylation by the PKA catalytic subunit in
vitro (Fig. 5A). Furthermore, we
found that the effect of EGF on K+
channel activity can be blocked by the PKA inhibitor
Rp-CPT-cAMPS (Fig.
5B). These results raise the
interesting possibility that PKA mediates the effects of growth factors
on ion channels and other proteins. A large body of evidence has shown
that protein phosphorylation by PKA is an important cellular mechanism
modulating K+ channel function.
The phosphorylation state of the channel subunits or associated
proteins can influence the amplitude or the time course of current
initiated by a change of membrane potential or ligand binding (7). For
example, the delayed rectifier K+
channel in Aplysia bag cell neurons,
the K+ channel in hippocampal
neurons, and Ca2+-activated
K+ channels in neuroendocrine
cells are also inhibited by cAMP analogs via activation of PKA (26,
29). However, it is important to point out that PKA-mediated
phosphorylation can induce the opposite effect in different
voltage-gated K+ channels. For
instance, the anomalous rectifier
K+ channel functions in
Aplysia neurons and cardiac cells are
upregulated by activation of PKA (2, 12, 27). PKA-mediated
phosphorylation affects the opening probability and the
Ca2+ or voltage sensitivity of rat
brain Ca2+-activated
K+ channels reconstituted into
artificial lipid bilayers (23). Correlation of PKA-mediated modulation
of intrinsic channel characteristics with the direct phosphorylation of
a K+ channel has been demonstrated
for three distinct voltage-gated K+ channels belonging to the
Shaker subfamily. Therefore, by
integrating electrophysiological and molecular biology techniques in a
Xenopus oocyte expression system, the
inactivation gating of the Shaker K+ channels and the opening time
that a single Kv1.2 channel spends in different conductance states were
shown to be regulated by PKA-induced phosphorylation at the
COOH-terminal region of the channels (9, 14). Direct phosphorylation of
channel proteins by PKA has been demonstrated biochemically for the
Shaker
K+ channels purified from rat
(Kv1.1) or bovine brain (Kv1.2) (15). The opening probabilities of
these channel on reconstituted lipid bilayers and the Kv1.3 channel
residing in T lymphocyte membrane can be increased by cAMP-dependent
phosphorylation (3, 22).
We have shown that K+ channel
activity was extremely low in growth-arrested ML-1 cells and that
channel activity can be restored by addition of 10% FBS (Fig. 2). To
study how the effect of growth factors on the channel activity might
influence cell proliferation, [3H]thymidine
incorporation was used to measure ML-1 cells entering the S phase and
proliferation (Fig. 6). Suppression of
K+ channel activity by different
K+ channel blockers effectively
prevented growth-arrested ML-1 cells from entering the S phase of the
cell cycle. It has been shown that pRB controls cell proliferation at
the G1 check point of the cell
cycle, whereas pRB dephosphorylation causes
G1 arrest in many cell types (28).
We have shown that increases in the dephosphorylated form of pRB is an
important event in the loss of proliferation in
K+ channel-suppressed ML-1 cells
(32). Our results have demonstrated that the effect of
K+ channel inhibition on ML-1 cell
proliferation is a phase-specific event.
In summary, the investigation of a functional role for a
growth-associated K+ channel in
human myeloblastic ML-1 cell proliferation has provided new evidence
that K+ channel activity involves
growth factor-mediated G1/S
transition of the cell cycle. Activity of this channel is closely
associated with this stage of cell growth and is controlled by serum
growth factors through the intracellular cAMP and cAMP-dependent kinase cascades. PKA-mediated phosphorylation of this
K+ channel resulted in increased
activity that paralleled serum growth factor-stimulated cell
proliferation. Our findings also provide an additional molecular
mechanism supporting a role for K+
channel activity in the G1/S
transition of the cell cycle, and this mechanism constitutes a novel
means of controlling ML-1 cell proliferation.
 |
ACKNOWLEDGEMENTS |
We thank Dr. R. W. Craig for giving us ML-1 cells as a generous gift.
 |
FOOTNOTES |
This study was supported by National Institute of General Medical
Sciences Grant GM-46834 (to L. Lu) and was partially supported by
National Heart, Lung, and Blood Institute Grant HL-54844 (to R. E. White) and by a grant from the American Foundation for Aging Research
(to R. E. White).
Address for reprint requests: L. Lu, Dept. of Physiology and
Biophysics, School of Medicine, Wright State Univ., Dayton, OH
45435.
Received 6 January 1997; accepted in final form 17 June 1997.
 |
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