Cell Biochem Biophys (2007) 47:199–208
DOI 10.1007/s12013-007-0006-9
ORIGINAL PAPER
The antiproliferative role of ERG K+ channels in rat
osteoblastic cells
Leonardo Hernandez Æ Ki Ho Park Æ Shi-Qing Cai Æ
Ling Qin Æ Nicola Partridge Æ Federico Sesti
Published online: 10 May 2007

Springer Science+Business Media B.V. 2007
Abstract We report on the role of K+ currents in the
mechanisms regulating the proliferation of UMR 106-01
osteoblastic osteosarcoma cells. Electrophysiological
analysis showed that UMR 106-01 cells produce robust
K+ currents that can be pharmacologically separated into
two major components: a E-4031-susceptible current,
IE-4031, and a tetraethylammonium (TEA)-susceptible
component, ITEA. Western blot and RT-PCR analysis
showed that IE-4031 is produced by ether a go-go (eag)-
related channels (ERG). Incubation of the cells with E-
4031 enhanced their proliferation by 80%. Application of
E-4031 in the bath solution did not induce instantaneous
changes in the membrane resting potential or in the level of
cytosolic calcium; however, the cells were slightly depo-
larized and the calcium content was significantly increased
upon prolonged incubation with the compound. Taken
together these findings indicate that ERG channels can
impair cell proliferation. This is a novel finding that
underscores new modes of regulation of mitosis by voltage-
gated K+ channels and provides an unexpected insight into
the current view of the mechanisms governing bone tissue
proliferation.
Keywords Potassium channel
Bone
Proliferation
L. Hernandez
K. H. Park
S.-Q. Cai
L. Qin

N. Partridge
F. Sesti (&)
Department of Physiology and Biophysics, UMDNJ-Robert
Wood Johnson Medical School, 683 Hoes Lane, Piscataway
NJ 08854, USA
e-mail: sestife@umdnj.edu
Present Address:
L. Hernandez
Department of Physiology, University of Wisconsin-Madison,
601 Science Dr., Madison, WI 53711, USA
Abbreviations
TEA Tetraethylammonium
4-AP 4-Aminopyridine
Introduction
Potassium (K+) channels constitute a diverse family of
integral membrane proteins that generate cellular excit-
ability by allowing diffusion of potassium ions across the
plasma membrane. K+ channels are implicated in a
variety of biological functions that range from sensory
transduction to control of processes as varied as blood
volume homeostasis and hormone secretion and when
defective, they can cause congenital and acquired chan-
nelopathies [1, 8, 20, 30, 39, 40, 49]. In non-excitable
cells, K+ channels play important roles in differentiation
and growth. The observation that K+ channel blockers
are antiproliferative in several cell types and that the
expression and/or activity of K+ channels is augmented
by mitogens [32, 36, 52] has produced a consensus
model that predicts that K+ channels—through the con-
trol of the membrane potential—promote cellular prolif-
eration by regulating calcium influx [7, 12–14, 16, 17,
21, 23, 28, 31, 33, 37, 48, 50]. In addition to this pri-
mary mechanism, K+ channels have also been shown to
be components of mitogen-activated protein kinase
(MAPK) pathways [53] and to assist proliferation by
controlling cell volume [36]. Among the various types of
K+ channels, those related to the eag sub-family (ERG)
appear to be prominent players in the regulatory mech-
anisms of neoplastic cell proliferation and survival. ERG
channels have been shown to support proliferation of
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200 Cell Biochem Biophys (2007) 47:199–208

tumor cell lines of different species (human and murine) fc = 1 kHz and sampled at 2.5 kHz. About 5–10 MW
and distinct histogenesis (neuroblastoma, rhabdomyosar- patch pipettes were pulled using a Flaming-Brown puller
coma, adenocarcinoma, lung microcytoma, pituitary (Sutter, CA). Bath solution was (in mM) 3 KCl, 140
tumors, insulinoma beta-cells, and monoblastic leukemia) NaCl, 10 Hepes (adjusted to pH 7.6 with NaOH), 2.5
[5, 6, 10, 25, 43, 46, 51]. Even though potassium channels CaCl2, and 1.0 MgCl2. Pipette solution was (in mM) 140
are abundant in bone cells, their role in determining bone KCl, 10 Hepes (adjusted to pH 7.3 with KOH), 1.0
development—a complex process which is regulated by MgCl2, 1.0 CaCl2, and 11 EGTA (adjusted to pH 7.5
multiple factors including homeobox transcription factors, with KOH). Whole-cell currents were evoked by voltage
growth factors (FGF, VEGF, BMPs, IGF), transcription jumps of variable amplitude and duration (as specified in
factors (Runx2, Sox), hormones (GH, PTH) matrix me- the text) from a holding potential of –80 mV with 1 s
talloproteinases, protein kinases, vitamin D, sex steroids interpulse intervals if not otherwise stated.
and calcium [11]—is poorly understood. To elucidate the
role of potassium channels in the mechanisms that deter-
mine the proliferation of bone tissue we characterized Data analysis and statistics
potassium currents expressed in UMR 106-01 osteoblastic
osteosarcoma cells. Here we show that inhibition of Data are indicated as means ± SEM. The number of
endogenous ERG channels by the class III antiarrhythmic determinations, n, is indicated in parentheses. Students t-
compound, E-4031, leads to a significant increase in the test was used to evaluate the statistical significance of
proliferation of the cells. We further show that the mito- unpaired averages. * and ** indicate a P < 0.05 and
genic effect of E-4031 involves the activation of calcium- P < 0.01, respectively.
dependent pathways through the inhibition of the ERG The dependence of the reversal potential Vrev. on the
channels. Taken together these findings underscore new inside/outside concentrations of K+ in the test solutions
roles of ERG K+ channels in bone tissue cells and provide ([K+]O and [K+]i, respectively) was fit to the Nernst
an unexpected insight into the current view of the mech- equation:
anisms governing proliferation of bone.
 þ 
kT ½K O
Vrev: ¼ ln ð1Þ
ze ½Kþ i
Methods
where k is the Boltzmann constant, T the absolute tem-
Cell culture perature, z the valence, and e the electronic charge.
The dependence of the normalized macroscopic con-
Cells were maintained in Eagle’s minimal essential ductance, G/Gmax, on the test voltage was fit to the
medium (UMR 106-01) or minimum essential medium Boltzmann equation:
alpha (CHO) supplemented with 10% (v/v) fetal bovine
1
serum plus 50 units/ml penicillin, and 50 lg/ml strepto- G=Gmax ¼ 
 ð2Þ
1 þ exp V1=2  V =VS
mycin (Sigma). The cells were seeded in 35-mm dishes
at 10,000 cell/cm2 in the same medium or the same
where
medium with test drugs. E-4031 dihydrochloride was
purchased from Tocris Cookson Inc., TEA-Cl was from I
Sigma and ionomycin from Alexis Biochemicals. If not G¼ ð3Þ
V  Vrev:
otherwise stated media (with or without drugs) was re-
placed every 24 h. After digestion with trypsin cells and V1/2 is the voltage at which G/Gmax = 0.5.
were resuspended in MEM and then were counted using The dependence of the fractional current, I/Imax, on the
a bright line counting chamber. For electrophysiology, concentration of drug in the test solution was fit to the Hill
cells were seeded onto poly-L-lysine-treated glass cov- equation:
erslips at a density of 10,000 cells/cm2.
I KinH
¼ ð4Þ
Electrophysiology Imax KinH þ ½drugnH

Data were recorded with an Axopatch 200B (Axon) a PC where Ki is the concentration at which the fractional
(Dell) and Clampex software (Axon), filtered at current is 0.5 and nH is the Hill coefficient.

123
Cell Biochem Biophys (2007) 47:199–208
Biochemistry
UMR 106-01 and CHO cells were washed with 10 ml
ice-cold PBS and lysed with ~2 ml ice-cold RIPA buffer
(50 mM Tris pH 7.4, 150 mM NaCl, 1 mM EDTA, 1%
IGEPAL CA-630, 0.5% (w/v) deoxycholate, 0.1% (w/v)
SDS, freshly added 10 mM iodoacetamide, phosphatase
and protease inhibitors for 30 min at 4C. Cell lysates
were centrifuged for 60 min at 4C and the supernatant
was incubated in SDS sample buffer at ~90–95C for
15 min, blotted and stained with rabbit polyclonal anti-
HERG antibody (Sigma) diluted (1:500) for 1.5 h.
Membranes were carefully washed and stained with
secondary antibodies for Western visualization.
RT-PCR
mRNA was extracted from UMR 106-01 cells using
Tri reagent (Sigma) followed by RT-PCR as described
previously [35]. The PCR product was amplified using
primers (5¢-CGTCTTCACACCGTACTCGGC-3¢ and 5¢-
CCAGAGCCAAAGATGAGCAGG-3¢) flanking a stretch
of 261 bp between the second and fourth transmembrane
domains of rat ERG. The PCR product was cloned in the
TOPO vector (Invitrogen) and confirmed by automated
DNA sequencing.
Determination of calcium and protein content
UMR 106-01 cells were plated on 60 mm tissue culture
plates at a density of 8,000 cells/cm2 and cultured under
control conditions or with the addition of 1 mM TEA
or 20 lM E-4031 in the media. Cells were incubated
for 5 days, then they were washed three times with
Dulbecco’s phosphate buffered saline (PBS) containing
10 mM EGTA (to chelate external calcium) and then
three times with normal PBS to remove the remaining
EGTA. Cells were lysed mechanically by adding water
and harvested with a scraper. This suspension was
divided into two samples: one for the determination of
calcium and one for protein content. The calcium content
was measured using a calcium assay kit (QuantiChromTM
Bioassay Systems, Hayward, CA) and the protein content
was determined using the Bradford Protein Assay after
adding 1 M NaOH to the sample.
Fura-2-microfluometry
Cells were washed with bath solution twice and then were
soaked in bath solution containing Fura-2 AM (5 mM) for
1 h at 37C and 15 min at room temperature. Dishes were
201
mounted on the stage of an inverted microscope (Nikon TE
200), and fluorescence measurements were performed at
37C using a dual-wavelength spectrofluorometer (Photon
Technology International, Inc.) with excitation wave-
lengths at 340 and 380 nm and emission at 520 nm.
Results
UMR 106-01 cells express robust K+ currents
We characterized native UMR 106-01 ionic currents using
the whole-cell configuration of the patch-clamp. Figure 1
A shows that depolarizing voltages (from an holding
voltage of –80 mV) evoked robust outward currents, that
reversed at –52.9 ± 1.1 mV (n = 11), a potential close to
the equilibrium potential for potassium. The average peak
current density, which gives a measure of the whole-cell
current normalized for cell size, was 46.7 ± 4.7 pA/pF at
+80 mV (Fig. 1B). This outward current could be con-
ducted only by chloride, potassium or both. Progressive
increase of K+ in the bath solution caused a rightward
shift in the reversal potential of the macroscopic current
by kT/e = 25.3 ± 4.0 mV when fit to the Nernst equation
(Eq. 1, Fig. 1C) under conditions predicting a value of
25.6 mV for ideal potassium selectivity. This indicated
that the voltage-dependent current was carried mainly by
potassium ions. The voltage-dependence of the endoge-
nous potassium current, IK, is shown in Fig. 1D. Fit of the
data to the Boltzmann equation (Eq. 2) gave
5.9 ± 0.9 mV and Vs = 16.7 ± 3.3 mV. IK was also
characterized by a moderate time-dependent decline at
large depolarizing potentials (Fig. 1A). The time course
of this decline, s, calculated by fitting the current at
+80 mV to a single exponential function was 1.2 ± 0.2 s
(n = 50, not shown).
A E-4031-sensitive conductance contribute to IK
in UMR 106-01 cells
E-4031, is a methanesulfonanilide class III antiarrhythmic
drug that specifically targets ERG channels [44]. When we
applied E-4031 in the bath solution the molecule partially
blocked IK, suggesting that these cells express functional
ERG channels (Fig. 2A), consistent with previous findings
based on DNA microarray analysis [35]. Blockade by
E-4031 needed repetitive activating pulses to achieve
equilibrium (Fig. 2B) and was irreversible (not shown).
Further, E-4031 blockade did not exhibit voltage-depen-
dence (Fig. 2C) suggesting that the mechanism of
inhibition was state-dependent. Normalized macroscopic
conductances were fitted to the Boltzmann equation with a
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202
Fig. 1 Native UMR 106-01 macroscopic currents. (A) Native UMR
106-01 macroscopic currents, evoked by voltage jumps from –80 to
+80 mV in +20 mV increments (inset: voltage protocol). (B) Mean
peak current density–voltage relationship. Data were calculated from
current envelopes as shown in (A) and are from 33 cells.
(C) Dependence of the membrane potential on external K+
concentration. KCl was isotonically replaced with NaCl. Data were
fitted to Eq. 1 with kT/e = 25.3 ± 4.0 mV. (D) Steady-state depen-
dence of activation. Macroscopic conductance curves were calculated
according to Eq. 3, normalized to the maximum value and fitted to Eq.
2 (Boltzmann equation) with V 1 / 2 = 5.9 ± 0.9 mV and
Vs = 16.7 ± 3.3 mV. Data are from 62 cells
midpoint for voltage activation and a slope factor, V1/2 = –
8.4 ± 1.1 mV and Vs = 7.2 ± 2.4 mV, respectively
(Fig. 2D), typical values for ERG channels. ERG channels
are activated upon depolarization but inactivate almost
instantaneously so that little outward current is passed. On
repolarization, these channels recover from inactivation
and generate large tail inward currents. Tail currents were
elicited at –120 mV under symmetrical K+ (140 mM)
solutions. Figure 2E shows the effect of applying 100 and
300 nM E-4031 in the bath solution: the amplitude of the
repolarizing tail current was reduced by roughly 20 and
40%, respectively. Increasing the concentration of E-4031
did not augment the block proportionally, however, prob-
ably reflecting the fact that other channels contributed to
the current. Current–dose relationships in the presence of
increasing amounts of E-4031 (Fig. 2F) could be fitted to
the Hill equation (Eq. 4), with equilibrium inhibition
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Cell Biochem Biophys (2007) 47:199–208
constant, Ki = 0.44 ± 0.12 lM (n = 10) and Hill coeffi-
cient, nH, = 0.9 ± 0.1. Leak-subtracting the residual tail
current in the presence of 1 lM E-4031 gave dose–re-
sponse curves that could be fitted with Ki = 76 ± 9 nM,
which is a typical value for ERG channels (data not
shown). IK was significantly blocked (~75%) by 1 mM
tetraethylammonium (TEA), which is perhaps the most
non-specific blocker of K+ channels (data not shown).
When TEA and E-4031 were simultaneously added to the
bath solution IK was not completely suppressed. A residual
current roughly 15% or less of the total current remained,
revealing the presence of one or more K+ channel types
that were insensitive to both E-4031 and TEA (at the
concentrations used, data not shown).
ERG is expressed in UMR106-01 cells
We next employed biochemistry to detect ERG expression
in the UMR 106-01 cells. Chinese Hamster ovary (CHO)
cells, which do not express endogenous ERG channels
were used as a control [27]. Figure 3A shows a Western
blot of CHO (lane 1) and UMR106-01 cells (lane 2) lysates
stained with polyclonal anti-HERG antibodies. A
~120 kDa band, which corresponds to the molecular
weight of ERG, was detected in UMR 106-01 cells lysates
and was absent in CHO cells lysates. Treatment with
10 lM E-4031 did not change the level of ERG expression
in UMR106-01 cells (Fig. 3B). RT-PCR was also em-
ployed to detect ERG expression. mRNA was extracted as
described previously [35], and a stretch of 261 bp encoding
the region comprised between the second and fourth
transmembrane domain of ERG could be amplified
(Fig. 3C, lane 2). Taken together these data suggest that
UMR106-01 cells express robust K+ currents that are car-
ried by at least two major distinct channels: a ERG chan-
nel, susceptible to E-4031 and a presently unidentified
voltage-gated K+ channel that is inhibited by the generic
blocker TEA.
Mitogenic and antimitogenic effects of K+ channel
blockers
We incubated the UMR 106-01 cells with E-4031 to
evaluate the physiological role of ERG channels. CHO
cells, which do not express endogenous K+ currents were
used as a control [27]. We found that the presence of
E-4031 in the media enhanced cellular proliferation
(Fig. 4A). E-4031 neither did affect the appearance or
morphology of the cells nor its mitogenic effect could be
ascribed to reduced apoptosis, which is negligible (less
than 1%) in this cell line (data not shown). Since UMR
106-01 cells express a K+ current which is insensitive
to E-4031 but it is blocked by TEA we also evaluated the
Cell Biochem Biophys (2007) 47:199–208
Fig. 2 Biophysical characteristics of IE-4031 currents. (A) UMR
106-01 whole-cell currents in the absence and in the presence of
20 lM E-4031 in the bath solution. The E4031-sensitive component
is obtained by subtracting the total current from the unblocked current
(right). (B) Representative whole-UMR 106-01 cell current evoked by
repetitive voltage jumps from –80 to +80 mV. The arrow marks the
start of 10 lM E-4031 perfusion. (C) Voltage-dependence of E-4031
blockade. Fractional currents were normalized to the value at 20 mV.
Data are from 20 cells. (D) Steady-state dependence of activation of
contribution of ITEA to the proliferation of the cells.
Figure 4B shows that progressive increase of the con-
centration of TEA in the media proportionally impaired
the proliferation of the cells. In contrast neither 20 lM
E-4031 nor 1 mM TEA had any effect on the prolifera-
tion of the CHO cells (data not shown). Other broad-
spectrum K+ channel blockers such as 4-aminopyridine
(4-AP) also impaired the proliferation of UMR 106-01
cells (Fig. 4C). However 4-AP inhibited the proliferation
of CHO cells indicating that its effects could not be as-
cribed to specific inhibition of K+ channels as in the case
of E-4031 and TEA. The effects of TEA and E-4031 were
additive (Fig. 4D). Thus, the decrease in proliferation
caused by 0.3 mM TEA could be rescued by 20 lM
E-4031 and cells proliferated slightly better than untreated
203
the E-4031-sensitive conductance. Macroscopic conductance was
calculated according to Eq. 3 and fitted to the Boltzmann equation
with V1/2 = –8.4 ± 1.1 mV and Vs = 7.2 ± 2.4 mV (n = 10). (E)
UMR 106-01 tail currents in the absence and in the presence of the
indicated concentrations of E-4031 in lM (inset). Cells were
depolarized to 40 mV for 1 s from an holding potential of –80 mV
and then to –120 mV for 3 s. (F) Dose–response relationships for E-
4031. Data were fitted to Eq. 4 with Ki = 0.44 ± 0.12 lM and
nH = 0.9 ± 0.1. Data are from 12 cells
cells; further increase in the TEA concentration inhibited
proliferation even in the presence of E-4031. These
observations suggest first, that a TEA-sensitive and a
E-4031-sensitive channel present in UMR 106-01 cells
can affect proliferation in opposite ways and second,
that the mechanisms underlying their mitogenic and
antimitogenic action are distinct.
Inhibition of IE-4031 is associated with cell
depolarization and altered calcium homeostasis
In many cell types, K+ channels promote proliferation by
regulating calcium influxes through the control of the
membrane potential, therefore we investigated whether this
general mechanism was responsible for the IE-4031-mediated
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204 Cell Biochem Biophys (2007) 47:199–208

Fig. 3 ERG is detected in UMR 106-01 cells. (A) Western blot

visualization with anti-HERG antibodies of CHO cells (lane 1) and
UMR 106-01 cells (lane 2) lysates. The antibodies detected a
~120 kDa band which corresponds to the molecular weight of ERG
only in UMR 106-01 lysates. The marker was BenchMark (Invitro-
gen). (B) As in (A) for CHO cells (lane 1) or UMR106-01 cells
incubated 5 days with (lane 2) or without (lane 3) 10 lM E-4031. To
account for the E-4031-dependent proliferation the cells were counted
and an approximately equal number of cells was used. (C) RT-PCR
reveals the presence of ERG in UMR 106-01 cells. mRNA was
extracted from UMR 106-01 cells using Tri reagent (Sigma) followed
by RT-PCR. The PCR product shown in lane 1 (~280 bp band)
encodes the second, third and fourth transmembrane domains of rat
ERG. Lane 2 represents a PCR amplification of the extracted mRNA
without the RT step
Fig. 4 Effects of 4-AP, TEA and E-4031 on the proliferation of
UMR 106-01 cells. UMR 106-01 and CHO cells were plated at
10,000 cells/cm2 density at day 0 and were counted at 24-h
effects on the proliferation of UMR 106-01 cells. The intervals. Data are normalized to the maximum value in control
opening of K+ channels produces a current that moves the (normalized proliferation). (A) Proliferation of UMR 106-01 in
membrane potential toward the equilibrium potential control conditions (circles) or with the addition of the following
of potassium according to Eq. 3. If E-4031 inhibit IK, the concentrations of E-4031 in the media (in lM): 10 (triangles), 20
(squares). Inset: maximum proliferation versus E-4031 concentra-
conductance for potassium, GK, changes according to: tion. Data are from 8 experiments. (B) UMR 106-01 cells in the
absence/presence of the following concentrations of TEA in the
GK max media (in mM): 0 (circles), 0.1 (squares), 0.3 (triangles), 1.0
GK ¼ ð5Þ
1 þ ð½drug=Ki ÞnH (diamonds). Data are from 4 experiments. (C) Proliferation of UMR
106-01 (circles) and CHO cells (triangles) in control conditions or
with the addition of 10 mM 4-AP (hollow symbols) in the culture
where GKmax is a constant and Ki and nH have the usual media. Data are from 4 experiments. (D) Proliferation of UMR 106-
meanings. The membrane potential, Vm, changes by DV: 01 cells at day 7 in the absence (diamonds) or presence (squares) of
20 lM E-4031 and of the indicated TEA concentrations. Data are
IK GK from 4 experiments
DV ¼ ¼ ðVK  Vm Þ ð6Þ
Gm GK þ G

where the membrane conductance Gm = GK + G is
expressed as the sum of GK and of all other ions, G, and VK
is the equilibrium potential for potassium. To investigate
whether the presence of E-4031 in the bath solution altered
the membrane resting potential (MRP) and/or the concen-
tration of cytosolic calcium, we employed the patch-
clamp technique in the current clamp configuration and
fura-2 microfluorimetry. Figure 5A shows that application
of 20 lM E-4031 (a level sufficient to inhibit the
corresponding sensitive conductance) did not depolarize
the cells when the external concentration of K+ was
physiological (3 mM) and the compound also failed to
further depolarize the membrane in symmetrical solutions
(140 mM, data not shown). In contrast, application of
5 mM TEA caused significant depolarization (~10 mV,
data not shown). Changing the concentration of K+ from
low (3 mM) to high (140 mM) and vice versa did not
evoke calcium-mediated fluorescence either in the absence

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Cell Biochem Biophys (2007) 47:199–208
or in the presence of E-4031 (Fig. 5B, n = 4 experiments).
The failure of E-4031 to elicit changes in the MRP or
calcium intake was probably a consequence of the fact that
E-4031 blocking is state-dependent. On the other hand cells
are expected to exhibit oscillations in the magnitude of the
MRP during particular phases of their cycle. For instance,
it is thought that membrane hyperpolarization induced by
the opening of K+ channels helps the transition toward the
G1 phase in several cell types [52]. It is possible that the
inhibitory action of E-4031 takes place during one of these
phases and since blocking is irreversible, this should result
in permanent depolarization. To address this point we
measured the resting potential in cells incubated with
E-4031 over 6 days. Cells treated with E-4031 were more
depolarized that untreated cells (Fig. 6A, E-4031-A).
Consistent with the notion that prolonged voltage stimu-
lation in the presence of E-4031 induce cell depolarization,
the MRP was measured before and after evoking ERG
currents by a train of 30 depolarizing steps to +60 mV. As
expected, the MRP was ~12% more positive (n = 5) after
voltage-stimulation (Fig. 6A, E-4031-I). To evaluate
the concentration of calcium in control cells or in cells
Fig. 5 The effect of inhibiting IE-4031 on the MRP and calcium
intake. (A) Representative recording of the membrane potential of a
UMR 106-01 cell in the whole-cell current clamp configuration.
Application of 20 lM E-4031 in the bath solution and wash out are
indicated by arrows. Test solution was physiological (3 mM K+).
(B) Representative microfluorimetric recording of a UMR 106-01 cell
loaded with fura-2. Arrows indicate the addition/wash out of 20 lM
E-4031 in both physiological (3 mM) or high (140 mM) K+ test
solutions. Triton-X-100 was added at the end of the experiment to test
the viability of the cell
205
incubated with E-4031 for six consecutive days we used a
kit that employs a mix of calcium-sensitive dyes (Quanti-
ChromTM, Bioassay Systems, Hayward, CA). Fluorescence
measurements were normalized for the protein content of
the total lysates, which we assumed to be proportional to
the number of cells. Consistent with our previous results,
the calcium content in cells incubated with E-4031 was
significantly increased (Fig. 6B). If ERG channels regulate
calcium influx, the presence of a calcium ionophore, which
makes calcium-selective pores in the plasma membrane,
should interfere with the proliferative effect of E-4031. As
expected, 100 ng/ml ionomycin, a dose that could be
tolerated, interfered with the proliferative action of E-4031:
cells co-incubated with ionomycin and 20 lM E-4031
proliferated less than control cells (Fig. 6C). Taken
together these data suggest that calcium-dependent
pathways enhance the proliferation of the UMR 106-01
cells and endogenous ERG channels inhibit these
processes.
Discussion
Electrophysiological, molecular and biochemical evidence
support the notion that eag-related K+ channels, are
expressed in and exert an antiproliferative effect on UMR
106-01 cells. These cells produce robust voltage-dependent
K+ currents that can be pharmacologically separated into
two major components: a E-4031-susceptible current which
is produced by ERG and which contributes to roughly 25%
to IK, and a TEA-susceptible component and whose
molecular identity is at present unknown. While ITEA is
proliferative, consistent with the well-established role of
K+ channels in stimulating mitosis [2, 29, 32, 38, 52],
IE-4031 is antiproliferative. This is to our knowledge a novel
observation—eag channels have been shown to exert a
mitogenic effect in a variety of tumor cell lines of different
species and distinct histogenesis [3, 4, 6, 34, 51]—but
never before they had been found to impair cell prolifera-
tion. Recently, Yangling et al. [54] showed that
down-regulation of KCNE2, suppresses the proliferation of
gastric cancer cells. Since KCNE2 is a well-recognized
partner of HERG in cardiomyocytes, the findings of
Yangling might imply that a eag-related channel might be
expressed in gastric cancer cells and its upregulation
(KCNE2 decreases the HERG current) exert antiproliferative
action.
More importantly, our findings suggest that ERG
channels can control the proliferation of UMR 106-01 cells
through novel mechanisms. The consensus model predicts
that eag-related channels maintain a constantly depolarized
value of the resting potential which is required for dereg-
ulated tumor growth. This mechanism seems to assist also
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Fig. 6 Inhibition of I E-4031 alters calcium homeostasis. (A)
Membrane resting potential of control cells or of cells incubated for
6 days with 20 lM E-4031 (E-4031-A) or in the presence of the same
concentration of E-4031 before and after voltage-stimulation by a
train of 30 consecutive voltage jumps to +60 mV (from an holding
voltage of –80 mV) with 1 s interpulse interval (E-4031-I). Statisti-
cally significant differences from control are indicated with
** (P < 0.01). Data are from n = 13, n = 21 and n = 5 cells,
respectively. (B) Calcium content of cells incubated for 5 days with
20 lM E-4031. The cells were thoroughly washed with PBS
containing EGTA to chelate external calcium, re-washed with normal
the proliferation of UMR 106-01 cells but with one major
difference: that the depolarization-mediated increase in
proliferation is achieved at the expenses of a diminished
ERG activity. A second major difference relates to the
mechanisms regulating calcium influx which is key to cell
proliferation. In human fibroblasts or T-lymphocytes for
example, calcium oscillations occur in synchrony with
membrane currents [33, 48]. In contrast, in UMR106-01
cells ERG channels decrease the proliferation rate by
inhibiting cellular pathways that lead to increased intra-
cellular calcium. This might be due to the increase in the
membrane depolarization associated with inhibition of
ERG, however other possibilities should also be consid-
ered. Recently, Hegle et al. [19] have shown that HERG
channels can regulate intracellular signaling pathways
independently of ion flux but through a mechanism that is
controlled by the voltage sensor. Alternatively, ERG might
keep ‘‘calcium-gates’’ closed during specific phases of the
cell cycle or it might control pathways involving some
proliferation factor—there is evidence of interactions of K+
channels with TNF-alpha receptor, 14-3-3, b-integrins,
p56lck and Src [9, 18, 24, 26, 47, 51]. In conclusion ERG
exerts an antiproliferative action by inactivating calcium-
dependent pathways whose molecular identity is at present
elusive.
123
Cell Biochem Biophys (2007) 47:199–208
PBS to remove residual EGTA, lysed and incubated with a mix of
calcium-sensitive dyes. Total calcium content was measured with a
spectrophotometer set at the 612 nm wavelength and normalized for
the protein content that was evaluated at the 595 nm wavelength.
Statistically significant differences from control are indicated with
** (P < 0.01). Data are from n = 10, n = 10 experiments, respec-
tively. (C) Cells incubated in the presence of 20 lM E-4031
(squares), 100 ng/ml ionomycin (triangles), 100 ng/ml ionomycin
plus 20 lM E-4031 (diamonds) and control conditions (circles). Data
are from 4 experiments
It is interesting to observe that genes and signaling
molecules that play a primary role in the nervous system,
such as neurotransmitters, receptors and transporters, are
expressed in, and can have profound effects on, bone cells
[15, 22, 41, 42, 45]. The most classic example is glutamate
which has been shown to have functions in bone that share
striking similarities with its role in synaptic neurotrans-
mission [42]. The presence of a eag-related K+ channel in
UMR 106-01 osteoblastic cells strengthens the emerging
notion that tissues that are so different in function and
properties share common molecular pathways and opens the
possibility to novel therapeutic approaches to bone diseases.
In conclusion, these findings emphasize a significant
heterogeneity in the mechanisms by which K+ channels
control the proliferation of biological cells. These data
are even more significant considering the crucial role
played by calcium in bone tissue histogenesis and prompt
several questions. What are the pathways involving dif-
ferent K+ channel types? In which phases of the cell
cycle is the activity of K+ channels crucial for prolifer-
ation? These lay ahead and will be the matter of future
investigations.
Acknowledgements We thank Drs. Zui Pan, Yutaka Hurata and
Jianjie Ma for help with microfluorimetry, Dr. John Lenard for critical
Cell Biochem Biophys (2007) 47:199–208
reading of the manuscript and Cinzia Sesti for help with the graphics.
This work was supported by an NIH Grant R01GM68581 to F.S. L.H.
was supported in part by a University of Colima fellowship.
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