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Am J Physiol Cell Physiol 273: C1657-C1665, 1997;
0363-6143/97 $5.00
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Vol. 273, Issue 5, C1657-C1665, November 1997

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

    ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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

    INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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.

    MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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 MOmega 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(beta -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.

    RESULTS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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 (black-square) or with 0 mM K+ in the extracellular solution (bullet ). 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.

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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  		Table 1.   Effect of cAMP-dependent protein kinase on K + channel activity in ML-1 cells


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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.

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 (black-square) and 5 ng/ml epidermal growth factor (EGF; bullet ) treatments. Intracellular cGMP level (black-triangle) 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.

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.

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).

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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  		Table 2.   Effect of altered culture conditions for 24 h on viability of ML-1 cells


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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.

    DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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-gamma 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-alpha (TNF-alpha ), a cytokine associated with activated macrophages (19, 25). Treatment of oligodendrocytes with TNF-alpha 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-alpha 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.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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AJP Cell Physiol 273(5):C1657-C1665
0363-6143/97 $5.00 Copyright © 1997 the American Physiological Society


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