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INTRODUCTION |
Unlike small conductance and big conductance
Ca2+-activated K+ channels (SK and BK,
respectively),1 intermediate
conductance Ca2+-activated K+ channels (IK) are
exclusively expressed in non-excitable cells, including fibroblasts,
endothelial cells, secretory epithelial cells, immature smooth muscle
cells, T-lymphocytes, and erythrocytes. The activity of IK channels has
been implicated in the regulation of secretion, in cellular migration,
and in the proliferation of mitogenically active cells (for review see
Refs. 1 and 2).
We recently demonstrated the expression of hIK1 (hSK4) mRNA in the
human keratinocyte cell line, HaCaT (3). In perforated-patch whole-cell
recordings, the extracellular mediator ATP produced a prominent and
long-lasting hyperpolarization of HaCaT cells through a signaling
pathway that involves IP3-mediated Ca2+ release
and subsequent activation of IK channels. Because ATP has been shown to
promote proliferation of keratinocytes (4), it is likely that the
mitogenic effect of ATP is at least partially mediated by the
activation of hIK1. In support of this notion, we found that the levels
of hIK1 mRNA declined as HaCaT cells began to differentiate
(3).
This finding is consistent with observations from other types of
non-excitable cells, which corroborate a link between IK channel
activity and cellular proliferation. For example, unstimulated T cells
express a low number of IK channels, whereas stimulation of T cells
with mitogens or specific antigens results in increased IK channel
density (5-8). In a myogenic fibroblast cell line, the mitogenic
action of basic fibroblast growth factor (bFGF) was linked to the
up-regulation of IK channels. Vice versa, the execution of
the myogenic program was associated with a decline in IK channel
transcripts (10, 11). Finally, IK conductance was much higher in
proliferating smooth muscle cells compared with differentiated cells
(12).
Given their apparent involvement in the regulation of cellular
proliferation, IK channels might emerge as a prime drug target to
manipulate the mitogenic behavior of non-excitable cells under various
conditions. Indeed, pharmacological suppression of IK channels by
charybdotoxin (ChbTx) or clotrimazole inhibited the mitogen-induced
conversion of resting T cells to activated, proliferating T cells (9,
13). In a myogenic fibroblast cell line, suppression by ChbTx of IK
channels abrogated the mitogenic effect of bFGF (10). Likewise, ChbTx
and clotrimazole inhibited the proliferative response of human
umbilical vein endothelial cells to the angiogenic factors bFGF and
vascular endothelial growth factor, both of which up-regulate IK1
transcripts in the same cells (14).
Although these studies examined the cellular consequences of blocking
IK channels, the recent advent of IK channel openers (reviewed in Ref.
1) now raises the question of how those compounds affect the pattern of
cellular proliferation and differentiation. Do positive modulators of
IK channels just act opposite to IK channel blockers, promoting
proliferation and delaying differentiation? We report here the
unexpected finding that prolonged exposure (3 days) of HaCaT
keratinocytes to the prototype IK channel opener 1-ethyl-2-benzimidazolinone (1-EBIO) (15) caused a dramatic down-regulation of the channels at the protein and mRNA level. This
novel feedback control of activity-dependent channel
expression led to a complete disappearance of functionally active IK
channels, as indicated by electrophysiological measurements.
Down-regulation of IK channels was associated with an almost entire
loss of mitogenic activity and a strong increase in cell size.
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EXPERIMENTAL PROCEDURES |
Cell Culture--
HaCaT keratinocytes (16) and C6 glioma cells
were grown in Dulbecco's modified Eagle's medium (DMEM) containing
10% fetal calf serum (FCS) and antibiotics (penicillin/streptomycin
100 units/ml). 1-2 × 105 cells were seeded into
3.5-cm culture dishes and incubated for 24 h in the plating medium
to allow for cell attachment and spreading on the dish. The plating
medium was subsequently replaced by DMEM/5% FCS. K+
channel openers dissolved in Me2SO or
Me2SO alone were added at this time. Cells were further
cultured with a daily medium change and analyzed at different time
points. 1-EBIO was purchased from Tocris (Cologne, Germany),
chlorzoxazone (CZ), and zoxazolamine (ZOX) from Sigma (Munich,
Germany). Medium, serum, and antibiotics were from Invitrogen
(Karlsruhe, Germany).
Proliferation Assay--
To determine the rate of HaCaT cell
proliferation, cells were seeded in 3.5-cm dishes as described above.
Growth medium was changed every day. At different time points after
seeding, cells were detached with trypsin (0.25%)/EDTA (0.5 mM) and counted in duplicate dishes using 0.04% trypan
blue to monitor cell viability.
Electrophysiology--
Electrophysiological recordings were
performed as previously described (3). Before recording, the culture
medium was always replaced with standard bath solution containing (in
mM): 130 NaCl, 3 KCl, 2 CaCl2, 2 MgCl2, 25 HEPES/NaHEPES, and 10 D-glucose, pH 7.4 (21-24 °C). Patch pipettes were fabricated from borosilicate glass using a two-stage pull protocol on a horizontal puller (DMZ, Zeitz, Germany) and filled with a solution containing (in
mM): 135 potassium gluconate, 10 KCl, 1.6 Na2HPO4, 0.4 NaH2PO4,
0.73 CaCl2, 1.03 MgCl2, 1 EGTA, 14 HEPES/NaHEPES, and 100 mg/liter nystatin, pH 7.2. After formation of a
seal in the G
range (typically 1.5-2 G
), the amplifier was
switched to current clamp mode and membrane potentials attained stable
values within 3-5 min. A remotely controlled, solenoid-operated Y tube
system was used for rapid application of test substances to the
keratinocytes under study. Electrophysiological signals were recorded,
amplified, and digitized with the use of an Axopatch 200 amplifier in
conjunction with a TL-1 Labmaster interface and AXOTAPE software (Axon
Instruments, Union City, CA). Data are expressed as mean ± S.E.
Statistical analysis (one-way analysis of variance) was done with the
use of Origin 4.1.
RNA Isolation and RNase Protection Assay--
Isolation of total
cellular RNA and RNase protection assay were performed as described
previously (17, 18). A fragment corresponding to nucleotides 740-1004
of the human hIK1/hSK4 cDNA (accession number AF000972) was used as
a probe. As a loading control, the same RNAs were simultaneously
hybridized with a probe corresponding to nucleotides 580-695
(accession number BC001601) of the human glyceraldehyde-3-phosphate
dehydrogenase (GAPDH) cDNA. Autoradiograms were scanned with a
Storm 820 PhosphorImager (Amersham Biosciences, Germany) and analyzed
by the Image 1.62b7 program (National Institutes of Health).
Cell Lysis and Immunoblotting--
Preparation of total cell
lysates was performed as described previously (19), and protein
concentrations were determined using the BCA kit (Pierce, Rockford,
IL). Proteins were separated by SDS-polyacrylamide gel electrophoresis
(10%) and transferred to nitrocellulose membranes. Membranes were
incubated with the primary antibodies followed by alkaline
phosphatase-conjugated secondary antibodies. Antibody-binding proteins
were detected with the nitro blue
tetrazolium/5-bromo-4-chloro-3-indolyl phosphate detection system
(Promega, Madison, WI). The following antibodies were used: mouse
monoclonal antibodies directed against keratin 10 (Dako, Glostrup,
Denmark), keratin 14 and involucrin (NeoMarkers, Fremont, CA), and
-actin (Sigma, Munich, Germany). Secondary antibodies were from
Promega.
Immunocytochemistry--
Cells were washed twice with ice-cold
PBS and fixed in acetone/methanol 1:1 for 20 min at
20 °C.
Endogenous peroxidase activity was blocked with 3%
H2O2 at room temperature. After blocking
unspecific binding sites with 3% bovine serum albumin (BSA) in PBS,
cells were incubated overnight at 4 °C with antibodies against
keratin 10 or keratin 14 (see above), with a mouse monoclonal antibody directed against Ki67 (NeoMarkers) or with a rabbit polyclonal antibody
directed against E-cadherin (kindly provided by Rolf Kemler, Freiburg,
Germany). Culture dishes were incubated at 4 °C overnight with the
primary antibodies and rinsed three times with PBS and once with
PBS/3% BSA. After a 2-h incubation with a peroxidase-coupled
anti-mouse IgG (Promega, Mannheim, Germany) at room temperature, cells
were washed three times with PBS, washed once with ddH2O,
and stained using the 3-amino-9-ethylcarbazole staining kit (Vector
Laboratories, Burlingame, CA).
Ca2+ Photometry--
HaCaT cells were loaded with
the fluorescent dyes via their membrane-permeant AM esters. Stock
solutions of 0.9 mM Calcium Green-1 AM and 1 mM
Fura Red AM (Molecular Probes, Eugene, OR) were prepared by dissolving
50 µg of each dye in 20 µl of Me2SO plus 20 µl of
Pluronic F-127 20% (Molecular Probes). Prior to loading, HaCaT cells
were rinsed three times with HEPES-buffered saline (HBS, see below).
The cell cultures were then incubated for 30 min at 37 °C in 3 ml of
HBS to which 20 µl of stock solution had been added (final
concentrations: Calcium Green-1, 6 µM; Fura Red, 7 µM) and were washed thoroughly after incubation. All
optical recordings were performed in HBS containing (in mM)
118 NaCl, 3 KCl, 1.5 CaCl2, 1 MgCl2, 20 sodium
gluconate, 6 Hepes, and 10 D-glucose (pH 7.4) at room temperature.
Culture dishes were mounted on an upright microscope (Olympus BX50WI,
objective: Olympus LUMPlanFl 40xW) to which a confocal laser-scanning
(Bio-Rad MRC 1024) and an independent custom-made photometric system
(20) were attached. The specimen was illuminated at 485 ± 30 nm
in intervals of 5 s. Two photodiodes collected fluorescent light
emitted from the cell layer at wavelengths 530 ± 30 nm (emission
Calcium Green) and 660 ± 50 nm (emission Fura Red) simultaneously
and produced voltage signals that were proportional to fluorescence
emissions. Signals were linearly amplified prior to PC-based data
digitization (DT2812, Data Translation, Marlboro, MA).
Data were monitored with a modified version of the program QTRAC (21).
Further analysis and ratio (r = F530/F660) calculations were performed using the scientific spreadsheet program Origin (Microcal, Northampton, MA). Ratio values are given as normalized r = R'/R0,
i.e. divided by reference value R0
obtained from the mean ratio value before drug administration. The time
course of Ca2+ transients was fitted with Origin 4.1.
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RESULTS |
Short-term and Long-term Effects of K+ Channel Openers
on Hyperpolarizing Response to ATP--
Whole-cell recordings in the
perforated-patch variation were performed on HaCaT cells, an
immortalized, non-tumorigenic human keratinocyte cell line that
maintains partial differentiation capacity in vitro and full
differentiation capacity in vivo (16). Fig.
1 compares the voltage response of
pre-confluent HaCaT cells to the physiological stimulus ATP, to its
non-hydrolyzable analogue, ATP
S, and to the three IK channel openers
1-EBIO, chlorzoxazone (CZ), and zoxazolamine (ZOX). As we have shown
before (3), ATP (10 µM, n = 28) produced
a biphasic voltage change, consisting of an initial, transient
depolarization due to the opening of Ca2+-activated
Cl
channels and non-selective cation channels, followed
by a strong hyperpolarization due to the opening of ChbTx-sensitive
hIK1 channels (Fig. 1A). ATP
S (10 µM,
n = 3) gave rise to virtually identical voltage
trajectories (Fig. 1B). 1-EBIO (1 mM,
n = 14), CZ (1 mM, n = 17)
and ZOX (1 mM, n = 8) all led to a marked
and ChbTx-sensitive hyperpolarization of the membrane potential (Fig.
1, C-E), demonstrating their pharmacological efficacy and
their functional coupling to IK channels in HaCaT cells.

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Fig. 1.
ATP and IK channel openers induce
ChbTx-sensitive hyperpolarization of the membrane potential.
Voltage responses were measured using the perforated-patch variation of
the whole-cell recording technique. A and B, the
extracellular signaling molecule ATP (10 µM) and its
non-hydrolyzable analogue ATP S (10 µM) produced
virtually identical, biphasic voltage changes, in which a brief
depolarization was followed by a pronounced hyperpolarization. The
latter was completely reversed by the IK channel inhibitor ChbTx (100 nM). C-E, the IK channel openers 1-EBIO (1 mM), CZ (1 mM), and ZOX (1 mM)
produced a rapid shift of the membrane potential in the hyperpolarizing
direction, which was sensitive to ChbTx (100 nM).
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Based on their strong effect on the resting membrane potential, all
three channel openers appeared as suitable tools to study how the
prolonged pharmacological activation of IK channels influences the
pattern of proliferation and differentiation in HaCaT cells. Before
doing so, however, we performed a series of control experiments to
prove the continued activation of IK channels during prolonged application of the channel openers. HaCaT cells were cultured in the
presence of 1 mM 1-EBIO, and the resting membrane potential and the electrophysiological response to ATP (10 µM) were
measured after drug incubation periods of variable duration (3 h to 3 days). To our surprise, incubation with 1-EBIO for just 3 h was
already sufficient to produce a dramatic desensitization of IK
channels, so that application of ATP (10 µM) failed to
give rise to the typical hyperpolarization (Fig.
2, A and B). As a
consequence, the depolarizing action of ATP, which is quickly reversed
by IK channel opening under control conditions, only gradually
declined. After 7-h incubation with 1-EBIO, activation of IK channels
by ATP was almost completely lost, as indicated by the plateau-like depolarization evoked by ATP (Fig. 2, A and
B).

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Fig. 2.
Loss of hyperpolarizing response
to ATP during prolonged application of IK channel openers.
A, representative voltage traces demonstrating the
gradual decline of the hyperpolarizing action of ATP (10 µM) at 3 h and at 7 h of 1-EBIO (1 mM) pretreatment. Note that, as the ATP-activated IK
conductance disappears, the depolarizing effect of ATP prevails, giving
rise to a plateau-like shift of the membrane potential in the
depolarizing direction. B, summary of the experiments on the
time dependence of decline of the ATP response. Voltage axis indicates
maximal voltage deviation from resting membrane potential (broken
line) that was induced by ATP (10 µM) at the various
time points of 1-EBIO (1 mM) treatment (control cells,
n = 12; 1-EBIO-treated cells, n = 13).
C, application of 10 µM ATP produced no
hyperpolarization in cells cultured for 3 days in the presence of
1-EBIO (1 mM, red trace), CZ (1 mM,
green trace) or ZOX (1 mM, blue
trace), whereas control cells showed the typical biphasic change
of membrane potential (black trace). Note that 3 days of
treatment with IK channel openers also caused a shift of resting
membrane potential in the depolarizing direction suggesting that the
activity of IK channels contributes to the resting membrane potential
under normal conditions. D, histogram summarizing the
experiments illustrated in C. All data were obtained after
3-day incubation with the different compounds or the solvent
(Me2SO) alone. Although the ATP-induced voltage shift in
the hyperpolarizing direction (given as  Vm) was
not affected by Me2SO or a low concentration of 1-EBIO (0.1 mM), 1 mM 1-EBIO, 1 mM CZ, and 1 mM ZOX almost completely abrogated this effect. *,
p < 0.05; **, p < 0.001.
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When keratinocytes were grown for 3 days in the presence of 1 mM 1-EBIO, they showed a markedly depolarized resting
membrane potential (
19 ± 2 mV, n = 9, Fig.
2C) compared with control cells (
53 ± 4 mV,
n = 9). These data are consistent with the depolarizing effect of ChbTx in normal HaCaT keratinocytes (3), suggesting that
tonic activity of ChbTx-sensitive IK channels contributes to their
resting membrane potential. Cells grown in the presence of 0.1 mM 1-EBIO showed only a slightly depolarized membrane
potential (
41 ± 5 mV, n = 8). The presence of
the solvent alone (Me2SO, 0.1%) did not influence resting
membrane potential (
52 ± 2 mV, n = 8).
Application of 10 µM ATP to control cells hyperpolarized the membrane potential by
22 ± 4 mV (n = 9),
which was not different from the effect in Me2SO
(0.1%)-treated cells (
Vm,
24 ± 2 mV,
n = 8, Fig. 2, C and D).
Incubation with 1 mM 1-EBIO virtually abrogated any effect
of ATP on IK channel activity (
Vm,
3 ± 1 mV, n = 8, Fig. 2, C and D). By
contrast, incubation with 1-EBIO at the lower concentration (0.1 mM) did not alter the hyperpolarizing response to ATP
(
Vm,
25 ± 3 mV, n = 8, Fig. 2D): This agrees with the observation that, during
acute application in control cells, 0.1 mM 1-EBIO displayed
only a weak electrophysiological effect (data not shown).
To determine whether the strong and unexpected down-regulation of the
channels is a common pharmacological feature of IK channel openers or
represents a peculiarity of 1-EBIO, we repeated the above experiments
using CZ (1 mM) and ZOX (1 mM), which were only recently identified as IK channel openers (22, 23). A 3-day incubation
with CZ or ZOX led to an almost complete disappearance of the
hyperpolarizing action of ATP:
Vm was
5 ± 1 mV (n = 4) in CZ-treated cells and
4 ± 2 mV
(n = 3) in ZOX-treated cells (Fig. 2, C and
D). In addition, both substances produced a depolarization
of the resting membrane potential (ZOX:
35 ± 2 mV,
n = 3; CZ:
42 ± 5 mV, n = 4).
This effect was weaker than that of 1 mM 1-EBIO (
19 ± 2 mV; n = 9), suggesting that 1-EBIO caused the most
complete down-regulation of IK channels.
Is the availability of functional IK channels in the membrane affected
in a similar way, when HaCaT cells are exposed for a longer time to a
natural stimulant of IK channel activity, such as ATP? This scenario
might occur, for example, after skin wounding when large amounts of ATP
are released into the extracellular space. To test this possibility,
HaCaT cells were incubated for 3 days with the stable ATP analogue,
ATP
S (10 µM), which, as shown above (Fig.
1B), fully reproduces the electrophysiological action of
ATP. In striking contrast to the down-regulation produced by the three
IK channel openers, preincubation with ATP
S did not diminish the
responsiveness of HaCaT cells to a single application of ATP, which
under this condition hyperpolarized the membrane potential by
33 ± 4 mV (n = 4, data not shown). This suggests that
prolonged channel activity will not per se initiate
subsequent down-regulation, unless induced by an IK channel opener that
binds to the channel or a closely associated protein of the channel complex.
Ca2+ Signaling and Down-regulation of IK
Channels--
In HaCaT cells, extracellular ATP binds to P2Y2
receptors, leading to inositol 1,4,5-trisphosphate formation and
subsequent release of intracellular Ca2+, which then
activates IK channels (3, 24). Is it conceivable that sustained
application of 1-EBIO somehow interferes with this pathway, so that the
fading voltage response to ATP would be secondary to changes in
receptor signaling, rather than reflect a down-regulation of the
channel itself? To examine whether P2Y2 receptor activation in
1-EBIO-treated keratinocytes still produces Ca2+
mobilization, we measured intracellular Ca2+ responses to
ATP (10 µM) in control cells, and in cells cultured for
3-4 days in 0.1 or 1 mM 1-EBIO, or in Me2SO
alone. Because we used ratiometric photometry with two fluorescent
Ca2+ indicators (Calcium Green/Fura Red), we cannot
calculate absolute intracellular Ca2+ concentrations from
changes in the fluorescence signal. Previous studies using the
indicators Fura-2 or Indo-1 to obtain quantitative measurements of
intracellular Ca2+ reported average resting levels of
78
145 nM in HaCaT cells, with ATP producing an
average increase of intracellular Ca2+ by 580 nM (25, 26). In our hands, ATP induced qualitatively very
similar cytosolic Ca2+ increases with rapid onset kinetics
in all four cell groups (Fig. 3A), indicating that P2Y2
signaling was not impaired by sustained 1-EBIO treatment. It is worth
noting, however, that the decline of the intracellular Ca2+
signal was dramatically accelerated after hIK1 down-regulation (Fig.
3A). In control cells, Me2SO-treated cells, and
low 1-EBIO-treated cells, the Ca2+ signal decreased slowly
with almost identical time constants of 2.34 ± 0.21 min
(n = 5), 2.38 ± 0.25 min (n = 2),
and 2.71 ± 0.25 min (n = 5), respectively (Fig.
3B). In high 1-EBI0-treated cells, however, Ca2+
signals declined much more rapidly (
of 0.52 ± 0.07 min,
n = 5). We did not further investigate the reason for
this discrepancy, but it is tempting to speculate that the slowly
falling phase of the Ca2+ signal in control cells reflects
Ca2+ influx from the extracellular side. The driving force
for the Ca2+ influx is provided by the hIK1-induced
hyperpolarization, which typically outlasts the period of ATP
application for several minutes or longer (Fig. 2, A and
C). After down-regulation of IK channels, however, an
increase in driving force is no longer generated, and the intracellular
Ca2+ signal reflects then solely intracellular
Ca2+ release and sequestration.

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Fig. 3.
Cytosolic Ca2+ response to ATP in
1-EBIO-treated HaCaT keratinocytes. Changes in cytosolic
Ca2+ were measured using ratiometric photometry.
A, representative traces showing the ATP-induced
Ca2+ rise in control cells (black trace) and in
cells treated with 1-EBIO (1 mM) for 3 days (gray
trace). B, histogram summarizes decay time constant of
Ca2+ signals after ATP (10 µM) application in
control cells, in cells treated with Me2SO alone, and in
cells treated with 1-EBIO (0.1 mM or 1 mM).
Duration of treatment was 3 days. **, p < 0.001.
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Down-regulation by 1-EBIO of IK Channels Occurs Also in C6 Glioma
Cells--
To determine whether down-regulation of IK channels is
unique to HaCaT cells or represents a more general mechanism of
negative feedback control, we performed a series of experiments in C6
glioma cells, which also express Ca2+-activated
K+ channels of intermediate conductance (25-35 pS) (27).
Because these cells do not respond to ATP, we used 1-EBIO to probe the presence of functional IK channels. As in HaCaT keratinocytes, application of 1 mM 1-EBIO to control or Me2SO
(0.1%)-treated C6 glioma cells had a strong hyperpolarizing effect
(control:
Vm
31 ± 5 mV, n = 5; Me2SO alone:
Vm
36 ± 5 mV, n = 3, Fig. 4,
A and B). Again, a 3-day incubation with 0.1 mM 1-EBIO was not sufficient to down-regulate IK channels (
Vm
33 ± 4 mV, n = 4, Fig. 4B), but after preincubation with 1 mM
1-EBIO, a single pulse of 1-EBIO completely failed to reveal functional
IK channels in C6 glioma cells (
Vm
2 ± 1 mV, n = 5, Fig. 4, A and B). This
suggests that prolonged application of 1-EBIO leads to a virtually
complete down-regulation of channel expression, irrespective of the
cell type under study.

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Fig. 4.
Down-regulation of IK channels by IK channel
openers in C6 glioma cells. A, voltage responses to
1-EBIO (1 mM) were recorded in control cells (black
trace) and in cells treated with 1-EBIO (1 mM) for 3 days (gray trace). B, quantitative comparison of
hyperpolarization of membrane potential ( Vm)
induced by acute application of 1 mM 1-EBIO in control
cells and in cells pre-treated for 3 days with 0.1% Me2SO,
0.1 mM 1-EBIO and 1 mM 1-EBIO. **,
p < 0.001.
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Loss of hIK1 Channel Activity Is Associated with Down-regulation of
hIK1 mRNA Levels--
The fading response to ATP and 1-EBIO in
cells incubated with IK channel openers might possibly involve
alterations of channel phosphorylation, channel internalization, and
degradation, and/or reduction of the mRNA levels. Because the
latter is arguably the most incisive mechanism of negative feed-back
control, we wondered whether the electrophysiologically determined loss
of functional IK channels is a result of reduced hIK1 mRNA levels.
We therefore used RNase protection assays to analyze hIK1 mRNA
levels under the various experimental conditions. Total RNA was
isolated after culturing HaCaT cells for 3 days under control
conditions, in Me2SO (0.1%) alone, or in 1-EBIO (0.1 or 1 mM). hIK1 mRNA levels were markedly reduced in cells
treated with 1 mM 1-EBIO for 3 days and slightly reduced in
cells grown in 0.1 mM 1-EBIO (Fig. 5A). In cells treated with
Me2SO alone, mRNA levels were not different from
control. The signal intensities of the bands corresponding to
transcripts of hIK1 and to transcripts of the housekeeping gene GAPDH
were determined by phosphorimaging. The signal intensity of hIK1 was
then normalized to that of GAPDH. The histogram of Fig. 5B
summarizes the relative change of normalized hIK1 mRNA under the
different experimental conditions, based on the results from eight
independent RNase protection assays. These data indicate that prolonged
application of IK channel openers leads to a pronounced reduction
of hIK1 mRNA levels.

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Fig. 5.
Expression of hIK1 mRNA is strongly
reduced in 1-EBIO-treated HaCaT keratinocytes. A, total
RNA was prepared from HaCaT keratinocytes cultured for 3 days in
control medium or in medium containing 0.1% Me2SO, 0.1 mM 1-EBIO or 1 mM 1-EBIO. Samples of 20 µg
were subjected to RNase protection analysis using an antisense probe to
hIK1. Hybridization of the same RNAs with a GAPDH antisense probe
served as a loading control. tRNA (20 µg) was used as a negative
control. 1000 cpm of the hybridization probes was loaded in the lanes
labeled "probe" and used as a size marker. B,
the signal intensities were determined by phosphorimaging and
normalized to the GAPDH signal intensity. The signal intensity of
non-treated cells was arbitrarily set at 100%. *, p < 0.05; **, p < 0.001.
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Down-regulation of hIK1 Channels Leads to Increased Cell Volume and
Inhibits Proliferation--
Because the expression of IK channels is
closely associated with, or even causally linked to cellular
proliferation (see the introduction), we wondered how the strong
down-regulation of the channel would alter the normal pattern of growth
and differentiation in HaCaT cells. To analyze proliferation of 1 mM 1-EBIO-treated HaCaT keratinocytes, cells were seeded in
culture dishes, and the number of cells was determined after 3 days in
the presence of 1-EBIO (0.1 and 1 mM), ATP
S (10 µM), or 0.1% Me2SO alone (Fig. 6A). 1-EBIO reduced the
proliferation rate in a concentration-dependent manner: 0.1 mM 1-EBIO decreased the rate by 31% (n = 12), whereas 1 mM 1-EBIO caused a 82% decrease
(n = 18). Me2SO alone (n = 18) had no effect on the proliferation of keratinocytes (control cells n = 18). Consistent with its lacking effect on the
expression of functional IK channels after 3 days of treatment, ATP
S
(10 µM, n = 6) did not reduce the
proliferation rate either. The results of the cell counts were
supported by immunocytochemical staining with an antibody against the
proliferation marker Ki67 in control cells and in cells treated with
1-EBIO (1 mM). As illustrated in Fig. 6B, most
control cells were stained with this antibody, whereas 1-EBIO treatment
for 3 days strongly reduced the number of Ki67-positive cells.

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Fig. 6.
1-EBIO inhibits keratinocyte
proliferation. A, HaCaT cells were seeded in 3.5-cm
dishes and cultured for 3 days in DMEM/5% FCS with or without
Me2SO, 1-EBIO, or ATP S as indicated (see "Experimental
Procedures"). They were subsequently trypsinized and counted in
duplicate dishes using 0.04% trypan blue to monitor cell viability.
The number of cells at day 3 after plating in relation to the number of
seeded cells is shown. B, HaCaT cells grown for 3 days in
medium with and without 1-EBIO (1 mM) were stained with an
antibody to Ki67. Note the reduced number of stained cells after
treatment with 1-EBIO. The scale bar in B
indicates 100 µm. **, p < 0.001.
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The most obvious effect of IK channel down-regulation already
visible under the light microscope was a massive increase in cell size.
This enlargement became particularly evident when the cellular borders
were visualized by immunostaining with an antibody against the cell
adhesion protein E-cadherin (Fig.
7B).

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Fig. 7.
Cellular effects of long-term 1-EBIO
treatment on HaCaT keratinocytes. A, samples of
10 µg of total protein from HaCaT keratinocytes cultured for 3 days
in the absence or presence of 1-EBIO were analyzed by Western blotting
for the presence of keratin 10 and keratin 14. 30 µg of protein were
used to determine expression of involucrin. Staining of the membrane
with an antibody to -actin served as a loading control. Control
cells and cells treated with 1 mM 1-EBIO for 3 days were
analyzed by immunocytochemistry for the presence of E-cadherin
(B), keratin 14 (C), and keratin10
(D). Antibody-bound E-cadherin was visualized by a
fluorescein isothiocyanate-labeled secondary antibody using a confocal
laser scanning microscope (filter, 530 ± 30 nm). Scale
bar indicates 50 µm.
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The expression of two keratins, keratin 10 (K10) and keratin 14 (K14)
served to determine the differentiation stage of the cells. K14, a
marker protein for non-differentiated keratinocytes (28), was uniformly
expressed in all control cells as well as in all 1 mM
1-EBIO-treated cells (n = 3; Fig. 7C).
Consistent with immunocytochemistry, results obtained by immunoblotting
did not reveal any difference in the amount of this protein between control and 1-EBIO-treated cells (n = 2, Fig.
7A). The differentiation-specific K10, which is induced in
HaCaT cells when grown at high density (29), was detectable in many
non-treated or Me2SO-treated cells within a confluent cell
layer. By contrast, it was largely absent from cells cultured in the
presence of 1 mM 1-EBIO (Fig. 7D,
n = 3). This down-regulation of K10 expression was
confirmed by immunoblotting (Fig. 7A, n = 3). Although this suggests that down-regulation of hIK1 interferes with
differentiation, it should be noted that the expression of involucrin,
a differentiation-specific protein deposited on the inner surface of
the plasma membrane, was not affected by 1-EBIO treatment (Fig.
7A).
Down-regulation of hIK1 and Associated Cellular Effects Are
Reversible--
To determine the reversibility of the cellular effects
of hIK1 down-regulation, 1-EBIO was withdrawn from the medium and the cells were cultured for 3 more days under standard conditions. In
electrophysiological measurements (Fig.
8, A and B), we did not observe a significant difference between the cell groups with respect to the ATP-induced maximal hyperpolarization (control:
73 ± 2 mV, n = 5; Me2SO:
73 ± 2 mV, n = 4; 0.1 mM 1-EBIO:
71 ± 2 mV, n = 6; 1 mM 1-EBIO:
74 ± 2 mV, n = 6). This finding agrees well with the results
of RNase protection assays (n = 4), which demonstrated
complete recovery of hIK1 mRNA levels in 1-EBIO-treated cells at 3 days post treatment (97 ± 16% of level in control cells). Finally, cell counts (n = 8) showed that, upon
withdrawal of the K+ channel opener from the medium and
subsequent re-expression of hIK1, mitogenic activity was re-ignited,
leading to a massive increase in the proliferation rate (Fig.
8C). Because the other cell groups (n = 8 per column) had already grown to confluency at this time point, they
showed only little further increase in cell number.

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Fig. 8.
Down-regulation of hIK1 channel by 1-EBIO and
concomitant inhibition of proliferation is reversible.
A, voltage responses to ATP (10 µM) were
virtually indistinguishable between control cells grown for a total of
6 days in control medium (black trace) and test cells grown
for 3 days in 1-EBIO (1 mM) followed by 3 days recovery in
control medium (gray trace). B, histogram
summarizes experiments of A for all cell groups examined.
C, recovery of hIK1 conductance was accompanied by strong
mitogenic activity in cells in which proliferation had been inhibited
by 1-EBIO (1 mM). Control cells were grown in control
solution for 6 days. Columns represent the ratio between the
number of cells after 3 days of drug application followed by 3 days of
recovery in control solution and the number of cells after 3 days of
drug application. **, p < 0.005.
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DISCUSSION |
We report here the novel and unexpected finding that, depending on
the duration of their application, the IK channel openers 1-EBIO, CZ,
and ZOX all exerted exactly opposite effects on hIK1 channels of HaCaT
keratinocytes: Although application of the drugs for minutes produced a
prominent and rapidly reversible hyperpolarization, as expected for a
K+ channel opener, exposure for hours and days led to a
complete disappearance of functional hIK1 channels, as indicated by the loss of the hyperpolarizing response to extracellular ATP. Our evidence
that the lacking effect of ATP did indeed result from a down-regulation
of hIK1 channels is the following: Optical measurements of
Ca2+ signals ruled out that the IK channel openers somehow
interfered with the P2Y2 receptor-activated pathway that triggers
channel opening. The second argument relies on the pharmacological
profile of the hyperpolarizing response: Although 1-EBIO, CZ, and ZOX also activate SK channels (30, 31), their antagonism by ChbTx, which
blocks IK and BK, but not SK channels (1), is only compatible with IK
channels being involved. Finally, we observed a close correlation
between channel activity and the levels of hIK1 mRNA. The loss of
the electrophysiological response was associated with a massive
down-regulation of hIK1 transcripts, and the recovery of the former was
associated with the recurrence of normal hIK1 mRNA levels.
The various mechanisms of short-term and long-term desensitization,
including down-regulation at the transcriptional level, have been
studied in detail for G protein-coupled receptors (32) and
ligand-activated ion channels (33-35). In addition, Levitan et
al. (36) showed that membrane depolarization by 50 mM
extracellular K+ causes rapid inhibition of the
transcription of the Kv1.5 channel gene, which was maximal at 3 h.
However, the complete disappearance of channel activity and the
concomitant decline of channel transcripts during prolonged application
of a direct channel opener is, to our knowledge, a novel observation.
Based on our RNase protection assay data, it seems likely that the
reduction in hIK1 mRNA levels as a result of reduced transcription and/or mRNA stability is responsible for the long-term
down-regulation of the channel. Turnover rates of
voltage-dependent Na+ and Ca2+
channels are relatively slow (t1/2, ~1 day) (37,
38), but the half-life of some K+ channels might be
substantially shorter, as demonstrated for Kv1.5 channels
(t1/2, ~4 h) (39). Although the turnover of hIK1
in HaCaT cells remains to be determined, it hence appears conceivable
that down-regulation of the mRNA upon continued drug application
might also be responsible for the lack of channel activity at earlier
time points. However, the fact that we already observed a reduced
response to ATP within 3 h suggests that 1-EBIO also induces a
conformational change in the channel protein that leads to more rapid
internalization and/or degradation.
Down-regulation of hIK1 channels had three distinct cellular
consequences in HaCaT keratinocytes when compared with cells grown
under standard culture conditions for the same period of time, namely
(i) inhibition of proliferation, (ii) increase in cell size, and (iii)
impaired expression of the differentiation marker K10. The association
of hIK1 down-regulation with inhibited cellular proliferation and the
parallel recovery of both parameters is consistent with a growing body
of evidence proposing a causal link between IK channel activity and
cellular proliferation (see the introduction). Because expression of
the early differentiation marker K10 is also diminished in
1-EBIO-treated cells, the reduced proliferation is obviously not
accompanied by a premature onset of differentiation in these cells.
Previous studies have implicated K+ channels and
Cl
channels of unknown molecular identity in the
proliferation and differentiation of keratinocytes (40-42). Our
findings suggest that hIK1 is a key player in these processes.
What is the relationship between the down-regulation of hIK1 channels
and the pronounced increase in cellular volume, and how could this
possibly be linked to the cessation of proliferation? A role of IK
channel in regulatory volume decrease in response to hypotonic cell
swelling is still disputed. Although IK channels are currently believed
not to contribute to regulatory volume decrease of the widely studied
Ehrlich cells (43, 44), this does not necessarily hold for other cell
types (reviewed in Ref. 1). In sickle cell anemia, for example, the
increased IK conductance of erythrocytes has been established as a
major cause of their salt loss and dehydration. This is in agreement
with studies from secretory epithelial cells, in which IK channels have
been implicated in the generation and maintenance of the ion gradients
required for secretion (see above). Although HaCaT cells do not have an appreciable secretory function, the down-regulation of functional IK
channels might impair extrusion of osmotically active substances and
thus lead to a substantial gain in cellular volume. We do not think,
however, that the large increase in cell size arises from uncontrolled,
pathological swelling. Such a process should inflict lasting damage on
the cell, but we found that 1-EBIO-treated cells resumed their normal
proliferation rate once the drug was withdrawn from the culture medium.
Is the gain in cell volume causally linked to the concomitant decline
in mitogenic activity? Rouzaire-Dubois et al. (45) have
recently proposed two mechanisms that might account for this highly
inverse relationship. First, cell volume changes may alter the
concentration of cellular components involved in the expression or
activity of cell cycle regulating proteins. Second, cytoskeleton
rearrangements due to cell volume changes may affect the protein
kinases or phosphatases responsible for the control of cell cycle progression.
Because IK channels are not only essential for cell proliferation but
play also a pivotal role in epithelial secretion (see the
introduction), IK channel openers are currently under consideration as
potentially beneficial agents in diseases such as cystic
fibrosis and chronic obstructive pulmonary disease (1). Given the
virtually identical down-regulation of IK channels in two independent
cell lines (HaCaT and C6 glioma), our data send a strong note of
caution regarding the clinical usefulness of IK channel openers as
secretion-stimulation compounds. Based on their inverse effect during
prolonged application, the compounds might actually worsen the symptoms
of cystic fibrosis and chronic obstructive pulmonary disease. In
striking contrast to their original purpose, K channel openers might
emerge as pharmacological alternatives to small molecule blockers of IK
channels such as clotrimazole or Tram-34. Owing to their inhibition of
IK channels at the mRNA level, 1-EBIO and related compounds could
even prove superior to small molecule blockers in the treatment of
disorders, in which long-term suppression of IK channel activity is
thought to counteract central pathophysiological processes. Examples
include epithelial and endothelial hyperproliferative disorders,
autoimmune diseases, and graft-versus-host reactions.