Department of Neurobiology, University of Alabama School of Medicine, Birmingham, Alabama 35294
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ABSTRACT |
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Ransom, Christopher B. and
Harald Sontheimer.
BK Channels in Human Glioma Cells.
J. Neurophysiol. 85: 790-803, 2001.
Ion channels in inexcitable
cells are involved in proliferation and volume regulation. Glioma cells
robustly proliferate and undergo shape and volume changes during
invasive migration. We investigated ion channel expression in two human
glioma cell lines (D54MG and STTG-1). With low
[Ca2+]i, both cell types
displayed voltage-dependent currents that activated at positive
voltages (more than +50 mV). Current density was sensitive to
intracellular cation replacement with the following rank order;
K+ > Cs+ Li+ > Na+. Currents were
>80% inhibited by iberiotoxin (33 nM), charybdotoxin (50 nM), quinine
(1 mM), tetrandrine (30 µM), and tetraethylammonium ion (TEA; 1 mM).
Extracellular phloretin (100 µM), an activator of
BK(Ca2+) channels, and elevated intracellular
Ca2+ negatively shifted the I-V curve
of whole cell currents. With 0, 0.1, and 1 µM
[Ca2+]i, the half-maximal
voltages, V0.5, for whole cell current
activation were +150, +65, and +12 mV, respectively. Elevating
[K+]o potentiated whole
cell currents in a fashion proportional to the square-root of
[K+]o. Recording from
cell-attached patches revealed large conductance channels (150-200 pS)
with similar voltage dependence and activation kinetics as whole cell
currents. These data indicate that human glioma cells express
large-conductance, Ca2+-activated
K+ (BK) channels. In amphotericin-perforated
patches bradykinin (1 µM) activated TEA-sensitive currents that were
abolished by preincubation with
bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid-AM (BAPTA-AM). The BK channels described here may influence the
responses of glioma cells to stimuli that increase
[Ca2+]i.
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INTRODUCTION |
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The vast majority of primary
brain tumors in adult humans arise from glial cells. These neoplasms
carry a very poor prognosis due to their invasive migration that
renders surgical treatment untenable (Cotran et al.
1994). Ion channels may contribute to this invasive behavior by
influencing salt and water movements between intracellular and
extracellular compartments during shape and volume changes associated
with migration through the tortuous extracellular space of brain tissue
(Soroceanu et al. 1999
). In addition, ion channels in
glia and other inexcitable cell types have been shown by many
laboratories to be functionally involved in proliferation
(Bringmann et al. 2000
; Chin et al. 1997
;
DeCoursey et al. 1984
; Dubois and Rouzaire-Dubois
1992
; Nilius and Wohlrab 1992
; Pappas et
al. 1994
; Puro et al. 1989
;
Rouzaire-Dubois and Dubois 1990
, 1998
; Schlichter
et al. 1996
; Wiecha et al. 1998
; Wilson
and Chiu 1993
). Thus there is good reason to believe that ion
channels in glioma cells could contribute to the malignant behavior of
these cells (i.e., invasive migration and uncontrolled proliferation).
Moreover, ion channels expressed by glioma cells may represent novel
therapeutic targets in the treatment of this deadly disease.
Human glioma cells express a variety of ion channels. These include
voltage-gated K+ currents (Chin et al.
1997), voltage-gated Na+ currents
(Brismar and Collins 1989
),
Ca2+-activated K+ currents
(Brismar and Collins 1989
; Pallotta et al.
1987
), voltage-gated Cl
currents (Ullrich and Sontheimer 1996
), and
volume-regulated Cl
currents (Bakhramov
et al. 1995
) (for review, see Brismar 1995
). The
expression of large-conductance, Ca2+-activated
K+ channels (BK) by glioma cells is of particular
interest because these channels are related to the degree of
differentiation and proliferative state of retinal glial (Muller) cells
(Bringmann et al. 2000
). Specifically, Muller cells lose
their BK channels during development and regain them when cells
proliferate in response to injury or disease. These results suggest
dedifferentiated/proliferating glial cells, glioma cells representing
the most extreme case, revert to a developmental biophysical phenotype
that includes BK channel expression (Bordey and Sontheimer
1997
; Bringmann et al. 2000
; MacFarlane
and Sontheimer 1997
). We examined ion channel expression in two
human glioma cell lines and found that both cell types highly express
large-conductance, Ca2+-activated
K+ channels (BK channels). Our study confirms
that BK channel expression is a common feature of human glioma cells
(Brismar 1995
). In addition, we provide additional
descriptions of the pharmacology, Ca2+
dependence, and [K+]o
dependence of these currents in glioma cells. This detailed biophysical
thumbprint is necessary for critical evaluation of the functions of BK
channels in human glioma cells.
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METHODS |
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Cell culture
All experiments were performed on the glioma cell lines STTG-1
(anaplastic astrocytoma, WHO grade III) and D54-MG (glioblastoma multiforme, WHO grade IV). STTG-1 cells were obtained from American Type Tissue Collection (Rockville, MD), and D54-MG cells were a gift
from Dr. D. Bigner (Duke University). We received D54MG cells at
passage 515 and STTG1 cells at passage 14. Vials of cells arrived
frozen and were thawed and resuspended in culture medium (see following
text). These cells were plated on four large culture flasks (Becton
Dickinson, Lincoln Park, NJ) and grown to confluency. Cells were
detached from the flasks with a 1- to 2-min exposure to culture media
supplemented with trypsin (1.5 mg/ml). This suspension was added to an
equal volume of culture medium and spun at 1,200 g for 5 min
in a centrifuge (Lab-Line Instruments Inc., Melrose Park, IL). We
aspirated the supernatant and resuspended the pellet from three flasks
in 90 ml of a freezing solution (culture medium with 5% DMSO). This
suspension was divided into 180 0.5-ml aliquots and stored in liquid
nitrogen for later use. Some cells were plated directly onto glass
coverslips in 24-well plates (Becton Dickinson) for experiments and
into a culture flask (Becton Dickinson) for future passage. Data in
this paper were obtained from cells passed 100 times. However, no
appreciable difference in membrane currents were observed in cells
passed
300 times.
Our culture medium was Dulbecco's modified essential medium (Life Technologies, Grand Island, NY) with 10% fetal calf serum (Hyclone, Logan, UT). Cells were kept in an incubator (Lab-Line Instruments) at 37°C in a 90% O2-10% CO2 humidified environment. This resulted in a pHo of 7.4.
Electrophysiology
Standard patch-clamp techniques were used to record whole cell
and single-channel membrane currents (Hamill et al.
1981). Patch pipettes were pulled on an upright puller (PP-83,
Narishige Instruments, Tokyo) from thin-walled, glass capillary tubing
with filament (MTW150F-4, WPI, Sarasota, FL) and had resistances of 3-5 M
. For experiments with amphotericin B (Sigma, St. Louis, MO)
perforated patches, we closely followed the procedures of Rae et
al. (1991)
. Briefly, amphotericin was dissolved and thoroughly triturated in DMSO (final concentration of 65 µM). This stock was
added to our standard pipette solution (final amphotericin concentration of 0.3 µM). Pipettes used for amphotericin
perforated-patch recording were flame-polished on a microforge (MF-83,
Narishige Instruments) and had resistances of 1-3 M
. Inclusion of
Lucifer yellow (Sigma) in our pipette solutions for
amphotericin-perforated patch recordings allowed us to distinguish
perforated-patch recordings from whole cell recordings (fluorescence
rapidly appeared in cells following breakthrough). We used an Axopatch
200B amplifier (Axon Instruments, Redwood City, CA) controlled by a
PC-compatible microcomputer (Dell Computers, Dallas, TX) running Axon
instruments software (pClamp7). Data were stored directly to disk using
a Digidata 1200 A-D interface (Axon Instruments). Data were acquired at
10 kHz and filtered at 1 and 2 kHz for patch and whole cell recordings, respectively. Capacitance and series resistance,
Rs, compensation was performed with
the Axopatch amplifier. Rs was
compensated
80%. No post hoc correction of
Rs was performed; because the currents
under study were quite large, we simply note that the voltage error
associated with Rs will underestimate
the steepness of the I-V curve. Experiments were not
performed on cells with a Rs >10 M
(except with amphotericin-perforated patches). Cells were visualized
with an inverted microscope (Nikon, Melville, NY). A three-axis
micromanipulator (Newport, Irvine, CA; mounted onto a custom frame
fitted to the microscope) held the preamplifier headstage and pipette
holder. The recording chamber had a volume of
300 µl and was
constantly superfused with control extracellular solution at a rate of
0.5 ml/min. A triple-barreled microperfusion device with a stepper
motor (SF-77B perfusion fast-step, Warner Instruments, Hamden, CT) was
used to apply test solutions directly to cells. Two barrels were fed by
2-to-1 manifolds, and one barrel was fed by a 4-to-1 manifold. Control
solutions were continuously flowing in each barrel between applications
of the five test solutions. The microperfusion flow pipes and stepper
motor were mounted on a manual micromanipulator (MX-110, Soma
Scientific Instruments, Irvine, CA) attached to our isolation table
(Micro-g, Peabody, MA) with a magnetic base. Grounding the recording
chamber via an agar salt bridge (4% agar, 1 M KCl) minimized liquid
junction potentials produced by test solutions.
Solutions
Our standard bath solution contained the following (in mM): 5 KCl, 135 NaCl, 1.6 Na2HPO4,
0.4 NaH2PO4, 1 MgSO4, 10 glucose, and 32.5 HEPES (acid). pH was
adjusted to 7.4 with NaOH. The osmolarity was 300 mOsm. In
experiments with elevated
[K+]o, KCl was
substituted with an equimolar amount of NaCl. Drugs were added directly
to this solution. Our standard pipette solution contained (in mM): 145 KCl, 1 MgCl2, 10 HEPES (acid), and 10 EGTA. pH
was adjusted to 7.25 with Tris-base, and Ca2+ was
added from a stock solution to achieve a target free
Ca2+ concentration of 20 nM. We calculated the
calcium to add to our pipette solution in experiments with elevated
free calcium concentrations with a software program based on equations
provided in Marks and Maxfield (1991)
. This program
takes into account ionic strength and pH. We corrected for EGTA purity.
For target free Ca2+ concentrations of 0.1 and 1 µM, we added 4.3 and 8.6 mM Ca2+, respectively.
To inhibit rises of
[Ca2+]i, we loaded cells
with the acetoxymethyl ester form of 1,2-bis(2-aminophenoxy) ethaneN,N,N',N'-tetraacetic acid (BAPTA-AM; Molecular
Probes, Eugene, OR). BAPTA-AM was dissolved in DMSO and added to our
culture media at a final concentration of 100 µM. Cells were
incubated for 20-30 min before recording. All chemicals were purchased
from Sigma unless otherwise noted. Scorpion toxins (charybdotoxin and iberiotoxin) were purchased from Alomone Labs (Jerusalem, Israel).
Analysis
Data were analyzed off-line with the software package Origin
(v0.5.0, MicroCal Software, Northhampton, MA). All curve-fitting was
performed using a least-squares curve-fitting routine provided by the
software. Inhibition curves were fit with the following equation
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RESULTS |
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Whole cell recordings from glioma cells
Under typical whole cell recording conditions (i.e., low
intracellular Ca2+), the human glioma cells
studied (D54MG and STTG-1) had stereotypical I-V
relationships. Voltage-dependent currents were seen that activated only
at positive potentials (more than +50 mV; see Fig.
1). Due to the voltage dependence and
rapid deactivation of this current (see Single-channel
recordings), tail-current analysis of the reversal potential was
not a feasible approach to determine the charge-carrying species. We
therefore opted to examine the effect of intracellular cation
replacement on whole cell current density (pA/pF). Figure 1 shows whole
cell currents in a representative STTG-1 cell that was serially patched
with pipettes containing KCl- or CsCl-based pipette solutions. Current
density (pA/pF) was reversibly reduced by intracellular cations other
than K+ with the following rank order:
K+ > Cs+ Li+ > Na+ (see Fig.
1C). These data suggest the currents are largely carried by
K+ ions. Data from singly and serially patched
cells are included in Fig. 1C. These data were obtained from
a single passage of STTG-1 cells. Unless otherwise stated, the voltage
protocol we used to elicit currents was to step the membrane potential
from
120 to +180 mV for 40-80 ms in 20-mV increments from a holding potential of
40 mV (see Fig. 1A, inset). With KCl-based
pipette solutions, the average resting membrane potential (measured as the 0 current potential) of 16 STTG-1 astrocytoma cells was
43 ± 13 mV (mean ± SD for all subsequent values, range =
9
to
64 mV), much smaller than typically seen in "normal" rodent
glia (Bordey and Sontheimer 1997
).
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Pharmacology of the voltage-dependent currents
The voltage-dependent currents in glioma cells were sensitive to
several well-known K+ channel blockers. These
included the organic compounds tetraethylammonium ion (TEA) and quinine
and the scorpion venom peptides charybdotoxin and iberiotoxin. Because
we wanted to quantitatively assess the effects of these drugs on the
voltage-dependent currents, on-line leak-subtraction was performed for
these experiments. Charybdotoxin was effective at 50 nM and inhibition
was voltage-dependent; block was reduced as the membrane potential was
made more positive (at 50 nM,
Idrug/Icontrol
was 0.05 at +80 mV and
0.5 at +180 mV; data not shown).
Iberiotoxin, a selective inhibitor of BK channels (Galvez et al.
1990
), inhibited currents in a voltage-independent fashion with
an apparent half-maximal concentration (IC50) of
10 nM (see METHODS for details and Fig.
2). In a subset of experiments, we tested
iberiotoxin at 10 nM and found it was effective at this concentration
(I/Icontrol = 0.88 at +140
mV). TEA inhibition of voltage-dependent currents had an
IC50 of
250 µM (see Fig.
3). In a subset of experiments, we tested
10 µM TEA on currents at +140 mV and found no effect.
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Tetrandrine is an inhibitor of BK currents that has different effects
on channels with and without auxiliary subunits. At 3 µM,
tetrandrine has negligible effects on channels composed of
subunits
alone but causes >50% inhibition of channels associated with
subunits (Dworetzky et al. 1996
). On average, the
voltage-dependent currents in glioma cells were inhibited by 63 and
91% by 3 and 30 µM tetrandrine, respectively (n = 5, see Fig. 4). These results are consistent
with the presence of a
subunit. The illustrated currents are
"TEA-sensitive" currents obtained by subtracting the average
current evoked with a voltage step to +120 mV in the presence of 10 mM
TEA from each trace. No time-dependent current component remained in
the presence of 10 mM TEA. The effects of all of these drugs were
completely reversible.
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The pharmacological profile [inhibition by low concentrations of TEA
(IC50 < 0.5 mM), charybdotoxin, and
iberiotoxin] of the voltage-dependent currents is consistent with them
representing the activity of BK channels. To pursue this further, we
extracellularly applied phloretin, a plant molecule that activates BK
channels (Gribkoff et al. 1997), during recordings to
observe its effects on whole cell currents. Extracellular phloretin
(0.1 mM) increased current amplitude and negatively shifted the
activation potential (see Fig. 5). The
apparent half-maximal voltages, V0.5,
determined from Boltzmann fits to the mean currents elicited with ramp
voltages (normalized to the peak value seen with phloretin), were +177 and +65 mV under control conditions and in the presence of phloretin, respectively (n = 4, Fig. 5B). Off-line leak
subtraction was performed on the ramp currents comprising the data
illustrated in Fig. 5B (using the linear portion of these
data between
80 and
20 mV). Phloretin-induced currents were reduced
by >50% with low concentrations of TEA (0.5 mM) as would be predicted
if they represent the activity of the same channel population
underlying the voltage-dependent current (Fig. 5C).
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The pharmacology of the voltage-dependent currents in glioma cells strongly suggests that they are mediated by BK channels.
[Ca2+]i dependence
If the voltage-dependent currents in human glioma cells
are mediated by BK channels, elevation of the intracellular
Ca2+ concentration,
[Ca2+]i, would be
predicted to shift the I-V curve toward more negative potentials. To evaluate this, we made whole cell recordings from STTG-1
cells with pipette solutions with 10 EGTA/zero-added
Ca2+ and free Ca2+
concentrations of 0.1 and 1 µM (see Fig.
6). Increasing
[Ca2+]i resulted in
modest increases in current density at negative potentials (likely due
to linear Cl currents) but dramatically
increased current densities across the range of 0 to +120 mV (see Fig.
6B). The half-maximal voltages, V0.5, determined from Boltzmann fits
to the summary data from these experiments were +150 mV (range = +141 to +166 mV, n = 6), +65 mV (range = +49 to
+102 mV, n = 8), and +12 mV (range =
7 to +28
mV, n = 6), with approximate free
Ca2+ concentrations of 0, 0.1, and 1 µM,
respectively. The effects of Ca2+ were often
maximal within 30 s following breakthrough but sometimes continued
to develop over
20 min. For consistency, all current measurements
for the data in Fig. 6B were made 8-12 min after obtaining
a whole cell recording. We observed wide variability in
V0.5 (25-50 mV), even between cells
on the same coverslip with the same
Ca2+/EGTA-buffered pipette solutions. We suggest
some of this variability is due to incomplete equilibration between
pipette and cell and/or different biochemical states of the channels.
Currents evoked with ramp voltages in experiments with elevated
[Ca2+]i were reduced by
>50% with 0.5 mM TEA and 50 nM iberiotoxin (data not shown). The
illustrated currents in Fig. 6 were leak-subtracted by subtracting the
current remaining in 10 mM TEA from control currents (TEA-sensitive
currents).
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Single-channel recordings
To identify the ion channels underlying the Ca2+-activated K+ currents, we made cell-attached recordings from these cells. Of primary interest were the single-channel conductance, voltage dependence, and the activation kinetics of these channels in cell-attached patches. The latter two parameters could be compared with whole cell currents to identify channels as those underlying the whole cell currents.
The activation kinetics of large-conductance channels in cell-attached
patches resembled those of whole cell currents. We constructed
ensemble-average currents (average of 200-300 voltage steps to an
activating test potential) and compared the kinetics of the ensemble
average to whole cell currents at or near the same membrane potential
(see Fig. 7). The accuracy of these
comparisons was limited by our ability to measure the resting membrane
potential of a cell after obtaining a whole cell recording because this value was used to determine the transmembrane potential during the
cell-attached phase of the experiment. We measured the resting potential as the zero-current potential seen immediately following breakthrough. Figure 7A illustrates one such experiment in
which the transmembrane potential produced by the test potential was judged to be +101 mV (Vpip,test was
120 mV and the measured Vm was
19
mV). It was clear by inspection that the ensemble average strongly
resembled the whole cell current (Fig. 7C), and the time constants of activation (
, determined from single exponential fits
of the data; Fig. 7B, dashed line) were also very similar (
ensemble = 10 ms and
whole-cell = 8 ms). In four ensemble/whole cell pairs at
+120 mV,
ensemble was 4 ± 1 ms and
whole-cell was 5 ± 2 ms
(Fig. 7D). The slightly faster
of ensemble-average currents is expected because these channels were exposed to 135 mM
[K+]o (concentration of
K+ in our pipette solution) while whole cell
currents were recorded with 5 mM
[K+]o and the
of
whole cell currents was slightly reduced by elevated [K+]o (see following
text). Inward currents through these channels were observed
infrequently following an activating voltage step (see Fig.
7A). The tail current in the ensemble average in Fig. 7B has completely deactivated in <2.0 ms. These last
findings support our initial approach to study the ionic selectivity of these currents (intracellular cation replacement). Under conditions of
low [Ca2+]i, the voltage
dependence and rapid deactivation of these BK channels limits the
ability to measure reversal potentials with a tail-current protocol.
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Activation of channels in cell-attached patches required positive
potentials, similar to whole cell currents with low
[Ca2+]i.
Channels in the cell-attached patch illustrated in Fig. 7A were only seen at transmembrane potentials greater than +40 mV (see
Fig. 7E). The unitary current of channels was determined from Gaussian fits of amplitude frequency histograms (see Fig. 7E, inset). With standard pipette solution (KCl-based), the
slope of the unitary current-V plot for channels activating
at positive potentials suggested an average single-channel conductance
of 150 pS. However, a wide range of single-channel conductances was
observed (range = 120-220 pS, n = 32), similar to
previous studies on BK channels (Reinhart et al. 1989
).
The unitary conductance for the channels illustrated in Fig.
7A was 194 pS (see Fig. 7E). Consistent with the
high current density of whole cell currents (80 pA/pF at +120 mV), only
2/32 cell-attached patches did not display multi-channel activity. In
light of this and the fact that
[Ca2+]i (an
unknown value during cell-attached recordings) modulates the voltage
dependence of the channel, we elected not to undertake a more detailed
analysis of the voltage dependence of channels in cell-attached patches.
Channels in outside-out patches displayed all the properties of whole cell currents, including activation at positive potentials, block by low concentrations of TEA and iberiotoxin, and dependence on intracellular K+. It was difficult to measure unitary current in outside-out patches due to the large number of channels in our patches. In outside-out patches with sufficiently low numbers of channels to resolve single-channel currents over a range of potentials, the unitary-current slope conductance was 92 pS (n = 4) in standard bath solution (low K+, high Na+). The lower unitary conductance in outside-out patches compared with cell-attached patches is likely due to the higher [K+]o experienced by channels during cell-attached recording (see following text, Fig. 9D). The unitary current-V curve of channels in outside-out patches developed a pronounced negative slope at potentials greater than +60 mV (20 nM [Ca2+]i).
[K+]o-dependence of whole cell currents
Our ability to measure the reversal potential of the
voltage-dependent currents was previously limited by their high voltage dependence in the absence of intracellular Ca2+.
With elevated [Ca2+]i (1 µM), clear shifts in reversal potential were observed when [K+]o was raised from 5 to 135 mM (see Fig. 8). With 135 mM
[K+]o, currents reversed
near EK (2 mV under these
conditions). These experiments confirm the results of our intracellular
cation replacements. Despite the 84 mV decrease in driving force for K+ ions, current amplitudes above +30 mV were
potentiated by elevating [K+]o to 135 mM. For the
D54-MG cell illustrated in Fig. 8, this potentiation translated into a
three- to six-fold increase in whole cell conductance across a range of
potentials (see Fig. 8C). Elevated
[K+]o had insignificant
effects on voltage dependence, using
V0.5 as an index, in experiments with
or without [Ca2+]i (see
Fig. 8C, inset).
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The potentiation by [K+]o
allowed us to detect BK current activation by 1 µM
[Ca2+]i near typical
resting membrane potentials (40 mV; see Fig. 8D). We made
rapid applications of 135 mM
[K+]o to cells. High
[K+]o resulted in large
inward currents at
40 mV in cells with elevated [Ca2+]i. The TEA
sensitivity of
[K+]o-induced currents
was consistent with BK currents. The current remaining in 10 mM TEA is
likely a K+-dependent leak current (note the lack
of increased noise in Fig. 8D). The latency from application
of high [K+]o solution to
the response may reflect the speed of our stepper motor and the wash-on
and -off times of the high
[K+]o solution.
Because glioma cells in the interior of a tumor mass are likely to
experience elevated
[K+]o, especially near
the necrotic center, we wished to determine the range of
[K+]o over which glioma
BK currents are potentiated. In 6/6 cells tested, current amplitudes
were increased by 25 mM
[K+]o (see Fig.
9, A and B). In
Fig. 9C, the mean specific K+
conductance (nS/pF) of six cells was plotted as a function of [K+]o. Conductance
increased proportional to the
[K+]o as has been
described for [K+]o
potentiation of the conductance of inwardly rectifying
K+ currents (Newman 1993
;
Ransom and Sontheimer 1995
), with an
5-fold increase
in specific conductance going from 5 to 135 mM
[K+]o. Specific
conductances were 0.44 ± 0.1, 0.86 ± 0.3, 1.71 ± 0.4, and 2.12 ± 0.7 nS/pF (n = 6) in 5, 25, 83, and
135 mM [K+]o,
respectively. Elevated
[K+]o slightly decreased
the time constant of activation (at +120 mV,
m = 4.8 ± 0.5 ms in control and
m = 3.9 ± 0.4 ms in 135 mM K+). The
potentiation of currents by K+ was not due to
reduction of [Na+]o per
se because Na+ substitution with
choline+ or
N-methyl-D-glucamine+
(NMDG, data not shown) did not increase glioma BK currents.
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Consistent with previous studies (Hurley et al. 1999;
Lerche et al. 1995
; Wann and Richards
1994
), the unitary current was increased by elevating
[K+]o. Figure
9D illustrates the effect of increasing
[K+]o on unitary current
amplitude in a representative outside-out patch. At a constant voltage
(+70 mV), the unitary current amplitude was increased from +6.7 pA in 5 mM [K+]o to +13.6 pA in
135 mM [K+]o (see Fig.
9E).
Endogenous activation of BK channels
Large-conductance channels in cell-attached patches were generally
only observed at positive potentials. However, there were examples of
patches in which we detected large-conductance channels at the resting
potential (Vpip = 0 mV) during
cell-attached recording (see Fig. 10).
In Fig. 10A, the current response of a cell-attached patch
to ramping the pipette potential from +100 to 100 mV from a holding
potential of 0 mV is illustrated (STTG-1 cell). The currents show a
strong voltage dependence. Single-channel currents could be resolved
across a range of potentials and the unitary slope conductance of the
channels illustrated in Fig. 10B was 226 pS (see Fig.
10C). The large conductance and voltage dependence is
consistent with these channels being BK channels.
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Bradykinin activation of glioma BK currents
The activation of glioma BK channels at negative potentials
requires a rise in
[Ca2+]i. Cellular signals
increasing [Ca2+]i are
expected to activate these currents. Bradykinin is one such mediator
that increases [Ca2+]i in
many cell types, including human glioma cells (T. Manning and H. Sontheimer, unpublished observations). We used amphotericin perforated-patch recordings to demonstrate activation of glioma BK
currents by bradykinin (see Fig. 11).
In our amphotericin-perforated patch experiments, we gained electrical
access within 3 min after establishing a giga seal with series
resistances of 12-35 M (see Fig. 11A). Because we were
interested in bradykinin effects on the voltage dependence of glioma BK
currents, we applied voltage ramps from
120 to +120 mV from a holding
potential of
40 mV every 5 s (rate of change was 1 mV/ms). In
9/11 D54MG cells and 5/5 STTG-1 cells, application of bradykinin (1 µM) resulted in a rapid (
5 s) and transient activation of currents
with a strong voltage dependence (see Fig. 11B). This
activation was manifested as both an increase in current amplitude and,
most importantly, a negative shift in the activation potential. The
data in Fig. 11B are displayed on an expanded scale in the
inset to demonstrate that bradykinin resulted in current
activation at negative potentials. This shift was much larger in some
cells (down to
50 mV). The negative shift in current activation can
also be appreciated with the time course of current change at +40 mV
illustrated in Fig. 11C. Because bradykinin effects were
transient and we acquired data at 5-s intervals, it is possible that we
missed the peak response to bradykinin (and therefore the maximum
negative shift in activation). The bradykinin-induced currents were
sensitive to 1 mM TEA as expected for BK currents, although these
experiments were complicated by the transient nature of bradykinin
effects and the fact that we could only elicit a single response in
cells (even
15 min after the initial application). To evaluate
whether the activation of currents by bradykinin was due to elevation of [Ca2+]i, we incubated
cells with the membrane-permeant, fast calcium-chelator BAPTA-AM (100 µM) for 20-30 min. No responses to bradykinin were seen in 4/4 D54MG
cells and 2/2 STTG1 cells incubated with BAPTA-AM during
perforated-patch recording (see Fig. 11, D and
E). In addition, no response to bradykinin was seen during
conventional whole cell recording. These data indicate that bradykinin
elevates [Ca2+]i of human
glioma cells and leads to a transient activation of BK currents at
typical resting potentials.
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DISCUSSION |
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Our study demonstrates that two human glioma cell lines
(STTG-1 and D54MG) express BK channels. We have obtained data from another glioma cell line frequently used by others (U373MG) and confirmed BK expression by these cells as well. The identification of
these currents as BK currents is based on the
[K+]i-dependence,
pharmacological profile, Ca2+ sensitivity of
whole cell currents, and the similar voltage dependence and kinetics of
large-conductance channels in cell-attached patches to whole cell
currents. In addition to confirming that BK channel expression is a
common feature of many human glioma cells (Brismar 1995), our study provides additional descriptions of the
pharmacology, Ca2+ sensitivity,
[K+]o dependence of the
BK channels in these cells, and one example of an endogenous ligand
(bradykinin) that activates them. The expression of BK channels by
human gliomas is intriguing because, as pointed out by
Tseng-Crank et al. (1994)
, the single gene for these
channels is located on chromosome 10, which is affected in many tumors
including >60% of glioblastomas (Kleihues et al. 1995
). Our laboratory has recently PCR cloned a novel BK
-subunit from glioma cells using specific primers for BK channels,
confirming BK expression in these cells (X. Liu and H. Sontheimer, unpublished observations).
The glioma cells studied here expressed high levels of BK channels. We
routinely recorded very large (>10 nA) currents from these cells. The
mean current density of five STTG-1 cells at +60 mV with 0.1 µM
[Ca2+]i was 70 pA/pF.
Using this value and a single-channel current of
9 pA at +60 mV
(from 4 outside-out patches with normal bath solution, i.e., high
Na+, low K+), one can
calculate a lower limit for channel density of seven to eight
channels/µm2 [using a specific membrane
capacitance value of 1 pF/µm2 (Hille
1992
)]. Given this density, it is not surprising that our
outside-out patches had large numbers of channels. Our channel density
estimate is a lower limit because open probability is unlikely to be
unity under these conditions and the single-channel conductance
(determined with 0 [Ca2+]i) if anything
would be reduced by elevations of
[Ca2+]i (Marty
1981
).
Previous studies on these cell lines showed that a substantial
proportion of the voltage-gated outward currents were inhibited by
[Cl]o removal
(Ullrich and Sontheimer 1996
). However, under our
experimental conditions we found that the majority of the
voltage-dependent outward currents were sensitive to intracellular
cation replacement and iberiotoxin. We suspect these differences may
relate to the variations in the relative expression of
K+ and Cl
currents in
these cells (unpublished observations).
In STTG-1 astrocytoma cells, we found a half-maximal voltage for whole
cell current activation, V0.5, of +12
mV with 1 µM [Ca2+]i.
The V0.5 for BK channels in human
smooth muscle was +35 mV with 1 µM
[Ca2+]i (Hurley et
al. 1998) and in human skeletal muscle the
V0.5 was +50 mV with 0.5 µM
[Ca2+]i (extrapolated
V0.5 of +35 mV at 1 µM
[Ca2+]i) (Lerche
et al. 1995
). Two BK channels cloned from human brain had
V0.5s of +18 mV with 10 µM
[Ca2+]i (hbr5) and +9 mV
with 24 µM [Ca2+]i
(hbr3) (Tseng-Crank et al. 1994
). In native human
macrophages, BK channels had a V0.5 of
+22 mV with 3 µM
[Ca2+]i (Gallin
1984
). The Ca2+ sensitivity of BK
channels in a leukemic human macrophage cell line was the closest to
that reported here for BK channels in glioma cells;
V0.5 was
7.5 mV with 3 µM
Ca2+ (DeCoursey et al. 1996
).
Thus, glioma BK channels have an equal or greater
Ca2+ sensitivity than those described in many
other human preparations. An enhanced Ca2+
sensitivity would presumably allow BK channels in human glioma cells to
gate in response to smaller
[Ca2+]i rises than is
required in other cells. Clear activation of channels was seen near
typical resting potentials (
40 mV) with 1 µM
[Ca2+]i (see Fig.
8D). The tetrandrine sensitivity of glioma BK currents was
consistent with the presence of a
subunit, and this would contribute to the Ca2+ sensitivity
(Dworetzky et al. 1996
; McManus et al.
1995
). The possibility exists that our data (obtained from
whole cell recordings) may underestimate the true
Ca2+ sensitivity of these channels. If
[Ca2+]i regulatory
mechanisms are still intact during whole cell recordings, the
steady-state [Ca2+]i may
not equal [Ca2+]pipette,
particularly at distal parts of cells (Mathias et al. 1990
).
The high voltage dependence of BK currents in the absence of Ca2+ raises questions about their functional role. Gating at the resting potential is requisite for a meaningful role of BK channels in glioma biology. We were able to demonstrate endogenous activation of these channels in cell-attached patches and bradykinin stimulation of cells during amphotericin-perforated patch recording shifted activation of BK currents into the range of typical resting membrane potentials. We suggest that bradykinin is only one example of a mediator that could lead to BK activation via elevation of [Ca2+]i.
The [K+]o dependence of
the BK currents in glioma cells is a feature of these channels with
functional implications. The square-root relationship of conductance
and [K+]o makes the
relative modulation of BK channels by
[K+]o the greatest in the
physiologic range of
[K+]o. The potentiation
of BK currents by [K+]o
suggests that the functions of glioma BK channels would be augmented
under conditions of elevated
[K+]o, such as may exist
near the center of a rapidly-growing tumor. Inhibition of BK channels
reduced Muller cell proliferation only in the presence of 20 mM
[K+]o (Bringmann
et al. 2000). This was suggested to be due to effects on
membrane potential but could also relate to BK potentiation by
[K+]o. Our data suggest
that the whole cell BK conductance would be increased approximately
twofold under these conditions. BK current enhancement by elevated
[K+]o is due in part to
an increased single-channel conductance, in line with other studies
(Hurley et al. 1999
; Lerche et al. 1995
;
Wann and Richards 1994
). This may be a result of
allosteric actions of K+ ions themselves on the
channel or altered permeation properties with high
[K+]o. Our data are
suggestive of an allosteric action of K+ because
the observed potentiation was opposite to that predicted by driving
force considerations or single-file pore models of permeation. No
potentiation was seen during Na+ substitution
with choline+ or NMDG+,
indicating that the potentiation was not due to
Na+ removal per se. However, we cannot rule out
complicated ion-ion interactions between Na+ and
K+ within the pore of the channel as giving rise
to the increased unitary currents in high
[K+]o.
The electrophysiology of the glioma cells studied here is greatly
different from that of "normal" rodent glia (Bordey and Sontheimer 1997; Sontheimer 1994
). Specifically,
mature astrocytes have low input resistances, inwardly
rectifying K+ currents, and large resting
potentials. The glioma cells studied here had larger input resistances,
outwardly rectifying I-V curves, and small resting
potentials. In addition, there are no descriptions of
large-conductance, Ca2+-activated
K+ channels in astrocytes (but see Nowak
et al. 1987
; Quandt and MacVicar 1986
).
Furthermore, astrocytes express voltage-gated delayed-rectifier and
A-type K+ currents that were absent from the
glioma cells under investigation here (Bordey and Sontheimer
1997
). Thus the biophysics of glioma cells are distinct from
"normal" glia. In normal rodent glia, K+
channel expression is functionally involved in proliferation (Bordey and Sontheimer 1997
; MacFarlane and
Sontheimer 1997
, 2000a
,b
). In rat astrocytes, inwardly
rectifying K+ channels
(Kir) are associated with a quiescent
proliferative state and selective inhibition of
Kir increases proliferation (MacFarlane and Sontheimer 2000a
). Given the role of
K+ channels in the proliferation of normal glia,
it is reasonable to suggest that the altered biophysics of glioma
cells, in particular BK expression, could contribute to their malignant
behavior. The relevance of studies on rodent astrocytes to human glioma
cells is supported by the observations that only STTG-1 cells expressed Ba2+-sensitive inwardly rectifying
K+ currents, and these cells grew to confluency
approximately threefold slower than D54MG cells when plated at the same
density (unpublished observations).
There are several types of Ca2+-activated
K+ channels [K(Ca)]. In addition to the
voltage-dependent large-conductance
Ca2+-activated K+ channels
(the type described in this study), there are small- and intermediate-
conductance Ca2+-activated
K+ channels that are voltage independent. In
excitable cells, the varieties of Ca2+-activated
K+ channels likely perform similar roles, namely
to oppose depolarizations accompanied by rises in
[Ca2+]i. In inexcitable
cells, the different classes of Ca2+-activated
K+ channels may underlie different functions in
different cell types. Retinal glial (Muller) cells from diseased retina
(proliferative retinopathy) show increased expression of BK channels,
suggesting a correlation between BK channel expression and
proliferation (Bringmann et al. 2000). The link between
BK channels and a proliferative/undifferentiated state is substantiated
by the observations that normal Muller cells lose their BK channels
postnatally with a time course paralleling the maturation of these
cells and BK channel inhibition reduced proliferation of rabbit Muller
cells and human endothelial cells (Bringmann et al.
2000
; Wiecha et al. 1998
). Likewise,
intermediate-conductance Ca2+-activated
K+ channels are up-regulated in transformed
fibroblasts, suggestive of a role in proliferation (Pena and
Rane 1999
; Rane 1991
). In human THP-1 monocytes,
however, BK channels were only observed in PMA-differentiated cells
that are nondividing (DeCoursey et al. 1996
). Other
studies on astrocytoma cells and meningioma cells found no effects of
charybdotoxin on cell growth (Chin et al. 1997
;
Kraft et al. 2000
), suggesting K(Ca) channels do not
contribute to the proliferative response of these tumor cells, in
contrast to data from Muller cells, endothelial cells, and fibroblasts. We have performed [H3]-thymidine incorporation
assays on our D54MG and STTG1 cells and found no effect of TEA on
[H3]-thymidine incorporation (unpublished
observations), similar to the results in other astrocytoma cells and
meningioma cells (Chin et al. 1997
; Kraft et al.
2000
). Alternatively, K(Ca) channels have been shown to
participate in volume regulation and migration (Pasantes-Morales
et al. 1994
; Schwab et al. 1999
). Potassium channels are postulated to participate in migration by affecting net
salt fluxes, in conjunction with Cl
channels.
These salt fluxes, with their accompanying water, result in
volume/shape changes permissive to migration through narrow spaces and
promote the movement of cytosol into the expanding leading edge. In
human glioma cells, TEA (at concentrations that selectively inhibit BK
channels, i.e., <1 mM) reduced migration by
40% (Soroceanu
et al. 1999
). In contrast, other studies on human glioma cells
have suggested that BK activation completely stops migration
(Bordey et al. 2000
). The precise role of BK channels in
glioma cells remains to be determined.
In summary, we have shown that the human glioma cell lines we studied are well endowed with BK-type K+ channels. Taken together with the work of others, our study indicates that BK channels are a common feature of human glioma cells. The BK channels in glioma cells were active at typical resting potentials with [Ca2+]i near 1 µM. The [K+]o dependence of BK channels in glioma cells is an attribute of these channels with functional implications for glioma biology. We propose that these channels will contribute to the response of glioma cells to stimuli that increase [Ca2+]i, such as bradykinin.
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ACKNOWLEDGMENTS |
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The authors appreciate the discussions and comments of Drs. Robin Lester, David Weiss, and Zucheng Ye.
This work was supported by National Institute of Neurological Disorders and Stroke Grant NS-36692 and American Cancer Society Grant RPG-97-083-01CDD to H. Sontheimer and by a Medical Scientist Training Program scholarship to C. B. Ransom.
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FOOTNOTES |
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Address for reprint requests: H. Sontheimer, Dept. of Neurobiology, University of Alabama, 1719 6th Ave. S. CIRC 545, Birmingham, AL 35294 (E-mail: hws{at}nrc.uab.edu).
Received 19 June 2000; accepted in final form 13 October 2000.
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REFERENCES |
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