Regulation of ICl,swell in neuroblastoma
cells by G protein signaling pathways
Ana Y.
Estevez,
Tamara
Bond, and
Kevin
Strange
Departments of Anesthesiology and Pharmacology, Anesthesiology
Research Division, Laboratories of Cellular and Molecular
Physiology, Vanderbilt University Medical Center, Nashville,
Tennessee 37232
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ABSTRACT |
Guanosine
5'-O-(3-thiotriphosphate) (GTP
S) activated the
ICl,swell anion channel in N1E115 neuroblastoma
cells in a swelling-independent manner. GTP
S-induced current was
unaffected by ATP removal and broadly selective tyrosine kinase
inhibitors, demonstrating that phosphorylation events do not regulate G
protein-dependent channel activation. Pertussis toxin had no effect on
GTP
S-induced current. However, cholera toxin inhibited the current
~70%. Exposure of cells to 8-bromoadenosine 3',5'-cyclic
monophosphate did not mimic the effect of cholera toxin, and its
inhibitory action was not prevented by treatment of cells with an
inhibitor of adenylyl cyclase. These results demonstrate that GTP
S
does not act through G
i/o GTPases and that
G
s/G
G proteins inhibit the channel and/or channel
regulatory mechanisms through cAMP-independent mechanisms.
Swelling-induced activation of ICl,swell was
stimulated two- to threefold by GTP
S and inhibited by 10 mM
guanosine 5'-O-(2-thiodiphosphate). The Rho GTPase inhibitor
Clostridium difficile toxin B inhibited both GTP
S- and
swelling-induced activation of ICl,swell. Taken together, these findings indicate that Rho GTPase signaling pathways regulate the ICl,swell channel via
phosphorylation-independent mechanisms.
cell volume regulation; Rho GTPase; anion channel
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INTRODUCTION |
THE ABILITY TO SENSE AND
RESPOND to changes in volume is an essential and fundamental
property of all cells (32, 47). In response to cell
swelling, most vertebrate cells activate an outwardly rectifying anion
current termed ICl,swell. The
ICl,swell channel appears to play an important
role in regulating cell volume (reviewed in Refs. 43,
50, 57).
In addition to its role in volume homeostasis, the
ICl,swell channel may function in other
physiological processes and may contribute to organ system
pathophysiology. For example, it has been suggested that the
ICl,swell channel is a pathway for excitotoxic amino acid release in the central nervous system during cerebral ischemia and trauma (4, 29, 51).
ICl,swell is constitutively active in
ventricular myocytes isolated from dogs with tachycardia-induced congestive heart failure (CHF), suggesting that the channel contributes to electrophysiological and contractile abnormalities of CHF
(11). Swelling-induced taurine release via the
ICl,swell channel has been proposed to play a
role in controlling osmotic regulation of vasopressin secretion in
magnocellular neurons (13). The transformation of
microglia from an ameboid to a ramified shape is modulated by a
stretch-activated anion channel with biophysical characteristics
similar to ICl,swell (20). Changes
in cell volume are postulated to play important signaling roles in cell
metabolism, excitability, contraction, growth, proliferation, and
apoptosis (32, 38, 47). Volume-induced signaling
may be mediated in part by changes in ICl,swell activity.
Although volume-sensitive ion channels are expressed ubiquitously and
likely play important roles in cellular physiology and pathophysiology,
the molecular identity of the channel responsible for
ICl,swell is still unknown and the field is
fraught with controversy (21, 50, 56). In addition, the
signaling mechanisms by which cell swelling is transduced into channel
activation are incompletely understood and may vary between different
cell types. For example, some studies suggest a requirement for
serine/threonine or tyrosine kinase phosphorylation (9, 12,
64) in ICl,swell activation, whereas
others have demonstrated that ATP hydrolysis or phosphorylation events
are not required (6, 49, 59). In contrast, it has also
been suggested that dephosphorylation events mediate activation of the
ICl,swell channel (14, 61). The
apparent variation in signaling pathways that regulate the channel
suggests three possibilities: 1)
ICl,swell is due to the activity of more than a
single channel type, 2) channel regulation varies between
cell types, and/or 3) pharmacological and molecular
disruption of signaling pathways has indirect effects on channel activity.
The uncertainty that exists over the signaling mechanisms that regulate
ICl,swell and the molecular identity of the
channel(s) underscores the need for extensive additional
characterization of channel function and regulation. At present, it is
known that ICl,swell can be activated by cell
swelling (50, 57) and reduced intracellular ionic strength
(8, 22, 44). Doroshenko and colleagues (15,
16) demonstrated that guanosine
5'-O-(3-thiotriphosphate) (GTP
S) activates an outwardly
rectifying anion current with many of the properties of the
ICl,swell channel in bovine chromaffin cells.
More recently, Nilius and co-workers (46, 64) have shown
that activation of G proteins activates
ICl,swell in endothelial cells.
The purpose of the present study was to investigate the role of G
proteins in regulation of ICl,swell activation
in N1E115 neuroblastoma cells. Our results demonstrate that GTP
S
activates ICl,swell in the absence of swelling.
Current activation does not require phosphorylation events and is
insensitive to pertussis toxin. However, cholera toxin and
Clostridium difficile toxin B significantly inhibited
GTP
S-induced current activation. Swelling-induced activation of
ICl,swell was stimulated by GTP
S and
inhibited by guanosine 5'-O-(2-thiodiphosphate) (GDP
S)
and toxin B. Taken together, these results demonstrate that
G
s/G
and Rho G protein signaling pathways are
important regulators of the ICl,swell channel.
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MATERIALS AND METHODS |
Cell culture.
N1E115 mouse neuroblastoma cells (American Type Culture Collection,
Manassas, VA) were cultured in the presence of 5% CO2-95% air in high-glucose DMEM (GIBCO, Gaithersburg, MD) containing 25 mM
HEPES, 10% fetal bovine serum, 50 U/ml penicillin and 50 µg/ml
streptomycin. Cells were used between passages 13 and
34. The osmolality of the growth medium was 295-305
mosmol/kgH2O.
Patch-clamp recordings.
N1E115 cells were grown in 35-mm culture dishes and dissociated by
brief treatment with Ca2+- and Mg2+-free
modified Hanks' solution. Dissociated cells were allowed to reattach
to the poly-L-lysine-coated coverslip bottom of a bath
chamber (model R-26G; Warner Instrument, Hamden, CT) that was mounted
onto the stage of a Nikon TE300 inverted microscope. Patch electrodes
were pulled from 1.5-mm-outer diameter borosilicate glass
microhematocrit tubes (Fisher Scientific, St. Louis, MO) that had been
silanized with dimethyl-dichloro silane (Sigma Chemical, St. Louis,
MO). Electrodes were not fire polished before use.
The bath solution contained (in mM) 70 N-methyl-D-glucamine chloride, 5 MgSO4, 12 HEPES, 8 Tris, 5 glucose, 2 glutamine, 120 sucrose, and 0.4 or 1.3 CaCl2, (pH 7.4; osmolality = 300 mosmol/kgH2O). Bath osmolality was altered by
increasing or reducing sucrose concentration.
Patch clamping was carried out using a pipette solution that contained
(in mM) 125 CsCl, 10 HEPES, 10 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid
(BAPTA; tetracesium salt; Molecular Probes, Eugene, OR), 1 MgSO4, 5 CsOH, 2 ATP, and 0.5 GTP or GTP
S
(tetralithium salt; Sigma; pH 7.2). In some pilot studies, 1 mM EGTA
(Sigma) was used instead of BAPTA. To prevent spontaneous cell
swelling, the osmolality of the pipette solution was hypotonic (280 mosmol/kgH2O) with respect to the bath.
Experiments where the ATP requirement of channel activation was studied
utilized a pipette solution containing 125 mM CsCl, 10 mM HEPES, 10 mM
BAPTA, 1 mM EDTA (Sigma), 5 mM CsOH, 0.5 mM GTP
S, 40 µM
oligomycin, 5 µM iodoacetate, and 20 µM rotenone. ATP or
5'-adenylylimidodiphosphate (AMP-PNP; Boehringer Mannheim, Germany) were added as sodium and lithium salts, respectively. Metabolic inhibitors were added from concentrated stock solutions dissolved in DMSO. Final DMSO concentration in the pipette solutions was 0.2%.
Electrodes had direct current resistances of 3-5 M
.
Cells were used only if the series resistance was no greater than
~150% of the pipette resistance and the reversal potential was
within ±4 mV of the calculated value of +14.7 mV for a perfectly
anion-selective channel. Reversal potentials significantly below +14.7
mV were taken as an indication of loss of seal integrity.
An Axopatch 200A (Axon Instruments, Foster City, CA) patch-clamp
amplifier was used to voltage clamp N1E115 cells following gigaseal
formation and attainment of whole cell access. Command voltage
generation, data digitization, and data analysis were carried out on a
Pentium II computer using a DigiData 1200 AD/DA interface with pCLAMP 6 software (Axon Instruments). Data were digitized at 5 kHz and filtered
at 0.5 kHz using an eight-pole Bessel filter (model 902; Frequency
Devices, Haverhill, MA). Electrical connections to the amplifier were
made using Ag-AgCl pellets and 3 M KCl-agar bridges. Whole cell
currents were measured by varying membrane potential from
80 to +80
mV at 80 mV/s every 5 s.
Measurement of relative cell volume changes.
Whole cell currents and volume changes were measured simultaneously in
single patch-clamped cells. Cells attached to the coverslip bottom of
the patch-clamp bath chamber were visualized by video-enhanced differential interference contrast microscopy. Optical sectioning (58) demonstrated that the cells maintained a spherical
morphology for at least 60 min after attachment to the coverslip. Cells
were routinely removed from the bath chamber and replaced with fresh cells every 30-45 min. Given that the cells have a spherical
morphology, relative cell volume change was determined as
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(1)
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where CSA is the cell cross-sectional area measured at a single
focal plane. In all CSA measurements described in this paper, we imaged
cells at focal planes located at the point of maximum cell diameter.
Cell images were recorded continuously throughout a patch-clamp
experiment using a super VHS videocassette recorder (model SVO-2000;
Sony Electronics, San Jose, CA) and a Hamamatsu charge-coupled device camera (model C2400; Hamamatsu Photonics, Hamamatsu
City, Japan). CSAs of single cells were quantified by digitizing
recorded video images with an image-processing computer board (MV-1000; MuTech, Woburn, MA) with 512 × 480 × 8-bit resolution and a
Pentium II computer. Digitized images were displayed on the computer
monitor, and cell borders were traced using a mouse and a
computer-generated cursor. The CSA of a traced region was determined by
image analysis software (Optimas; Bioscan, Edmonds, WA). This image
acquisition and analysis system allows detection of changes in CSA with
an accuracy of ±2-3%.
Data analysis.
Whole cell currents were recorded within 15-20 s after membrane
rupture. The mean resting or baseline current is defined as current
measured before activation by GTP
S or cell swelling. Baseline
current was subtracted from all data points within a given record to
correct for variability in resting current levels between different
cells. Because of culture-to-culture variability in the response to
GTP
S, control measurements were performed in parallel with all
experimental treatments.
Rates of GTP
S current activation and inactivation and peak current
were measured. Current activation is defined as the point at which
there is a continuous increase in current amplitude above the baseline
current (6). Rates of current activation and inactivation were quantified by linear regression analysis.
Under control conditions, a small percentage (<10%) of cells treated
with GTP
S showed no current activation. To facilitate comparison
with experimental treatments that may have inhibited the GTP
S
response, nonactivating cells were included when calculating the
means ± SE rate of GTP
S-induced current activation.
Whole cell anion current was also activated by cell swelling. Rates of
current activation and cell swelling were determined by linear
regression analysis. For these studies, we also quantified the cell
volume set point of the channel. Cell volume set point is defined as
the relative cell volume at which current activation begins
(6).
Throughout the course of the experiments, a small percentage of cells
exhibited bleb formation during current recordings. These cells were
excluded from our analyses.
Statistical analysis.
Data are presented as means ± SE. Statistical significance was
determined using Student's two-tailed t-test for unpaired, independent means. When comparing three or more groups, statistical significance was determined by one-way analysis of variance. Values of
P < 0.05 were taken to indicate statistical significance.
 |
RESULTS |
GTP
S activates ICl,swell in the absence of cell
swelling.
Dialysis of N1E115 cells with 0.5 mM GTP
S activated an
outwardly rectifying whole cell anion current (Fig.
1A). Current
activation began within 0.85 ± 0.08 min after the whole cell
configuration was obtained and reached a plateau within 5.3 ± 0.8 min (n = 19; Fig. 1B). Activation was
transient, and current levels returned to baseline 11.5 ± 1.2 min
(n = 16; Fig. 1B) after the plateau was
reached. Cell swelling was not observed during GTP
S-induced current
activation (Fig. 1B). The mean relative cell volume at the
peak GTP
S-induced current was 0.99 ± 0.02 (n = 26).

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Fig. 1.
Guanosine 5'-O-(3-thiotriphosphate) (GTP S)
activates an outwardly rectifying anion current in N1E115 neuroblastoma
cells. A: steady-state current-voltage (I-V)
relationship of current activated by GTP S in a single neuroblastoma
cell. Calculated reversal potential for a perfectly anion-selective
channel is 14.7 mV. Measured reversal potential is 15.2 mV.
B: example of simultaneous current and volume measurements
performed on a single patch-clamped cell dialyzed with 0.5 mM GTP S.
Time 0 refers to the time at which recordings were
initiated.
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G proteins cycle between an active GTP-bound state and an inactive
GDP-bound state. GDP
S is a nonhydrolyzable analog of GDP that
competes with GTP or GTP analogs for the nucleotide binding sites on G
proteins, rendering them inactive. To examine further the role of G
proteins in whole cell anion current activation, 10 mM GDP
S was
included in the pipette solution. In the presence of 10 mM GDP
S,
only three of six cells (50%) activated spontaneously with GTP
S
compared with six of six cells in the paired control group. GDP
S
also inhibited the rate of GTP
S- induced current activation and
decreased the amplitude of the peak current by 82% and 71%,
respectively (Fig. 2).

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Fig. 2.
Guanosine 5'-O-(2-thiodiphosphate) (GDP S)
inhibits both the rate of activation and peak GTP S-induced current.
For these experiments, the concentration of CsCl in the pipette
solution was reduced to 110 mM to osmotically compensate for the
GDP S that was added. The control pipette solution contained 110 mM
CsCl and 10 mM Li3 citrate to control for the
Li+ that was added with GDP S. Values are means ± SE. *P < 0.05; ***P < 0.001. Number
of observations (n) is shown in parentheses.
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The outwardly rectifying current could be due to activation of
ICl,swell or Ca2+-dependent
Cl
channels (ICl,Ca). G protein
stimulation has been reported to activate ICl,Ca
in several (28, 36), but not all (30, 45), cell types. All experiments presented in this paper, however, were
carried out using nominal Ca2+ in the bath solution (0.4 mM) and a pipette solution containing the highly selective fast
Ca2+ buffer BAPTA (10 mM), demonstrating that
ICl,Ca are not responsible for the
GTP
S-induced current.
Intracellular Ca2+ actually appeared to exert an inhibitory
effect on the GTP
S current activation. During pilot studies, cells were patch clamped in the presence of 1.3 mM bath Ca2+ and
a pipette solution containing 1 mM EGTA instead of 10 mM BAPTA. Peak
GTP
S-induced currents and rates of current activation and
inactivation were unaffected by reduced Ca2+ buffering
(data not shown). However, in the presence of 1 mM EGTA,
GTP
S-induced current activation occurred in <50% of patch-clamped cells. A much more consistent activation of the GTP
S current was
observed when 10 mM BAPTA was used to buffer intracellular Ca2+. Current activation was detected in 93%
(n = 92) of control cells dialyzed with BAPTA-buffered
pipette solutions. The reason increased Ca2+ buffering
increases the frequency of current activation is unknown. It is
conceivable that a Ca2+-dependent process antagonizes the
stimulatory effect of GTP
S.
To determine whether the GTP
S-induced current is due to the activity
of the ICl,swell channel, we examined its
biophysical characteristics and volume sensitivity. As shown in Table
1, the rectification ratio and relative
anion permeability of the channel responsible for the GTP
S current
are the same as those observed for ICl,swell.
Furthermore, neither current exhibited significant voltage-dependent
activation or inactivation (Fig. 3A).

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Fig. 3.
Characteristics of GTP S- and swelling-induced anion
currents. A: whole cell currents elicited by stepping
membrane voltage from 100 to +100 mV in 20-mV steps from a holding
potential of 0 mV. ICl,swell was activated by
exposing cells to 0.5 mM GTP S or by swelling with a hypotonic bath
solution (200 mosmol/kgH2O). The arrows indicate
zero-current levels. B: cell shrinkage inhibits the
GTP S-induced current. Current and volume measurements shown are for
a single patch-clamped cell. Exposure of the cell to a hypertonic bath
solution (400 mosmol/kgH2O or 400 mOsm) caused the
GTP S-induced current to rapidly inactivate. The mean rate of current
inactivation for all experiments was ~8 times faster than spontaneous
inactivation observed in the absence of cell shrinkage (see Fig.
1A).
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The rate of spontaneous current inactivation (see Fig. 1A)
during GTP
S stimulation was
0.7 ± 0.2 pA · pF
1 · min
1
(n = 18). To determine whether the GTP
S-induced
current was volume sensitive, the current was allowed to reach a stable
plateau level and cells were then shrunken by exposure to a hypertonic bath solution (400 mosmol/kgH2O). Cell shrinkage inhibited
the peak GTP
S-induced current by 68 ± 6% (n = 5) at a rate of
5.5 ± 2.3 pA · pF
1 · min
1
(n = 5; see Fig. 3B). The rate of
shrinkage-induced inactivation is nearly eight times faster than the
rate of spontaneous current inactivation, demonstrating that the
GTP
S-activated channel is sensitive to cell volume. On the basis of
results shown in Figs. 1-3 and Table 1, we conclude that
stimulation of G proteins with GTP
S activates
ICl,swell in the absence of cell swelling.
GTP
S-induced ICl,swell activation is not modulated
by ATP or phosphorylation reactions.
Intracellular ATP and nonhydrolyzable ATP analogs modulate but are not
essential for swelling-induced activation of
ICl,swell in N1E115 cells (6). In a
variety of cell types, nonhydrolyzable ATP analogs support normal
ICl,swell activity, indicating that phosphorylation events are not involved in channel activation (6,
49). However, the results of a number of studies have also
suggested that protein kinases and phosphatases modulate channel
activity (14, 61, 64). Recently, Voets et al.
(64) proposed that the stimulatory effect of GTP
S on
ICl,swell in endothelial cells is mediated by
tyrosine phosphorylation.
Given these findings, we examined the effect of tyrosine kinase
inhibitors on GTP
S-induced activation of
ICl,swell in N1E115 cells. Cells were treated
with the broadly selective tyrosine kinase inhibitors genistein (100 µM) or tyrphostin A51 (100 µM). Two experimental protocols were
used. Cells were patch clamped with inhibitors present only in the
pipette solution or with the inhibitors present in both the pipette and
bath solutions. In the latter case, cells were preincubated for
7-21 min with inhibitors before patch clamping. Pipette solutions
were kept on ice and bath and pipette solutions were remade every hour
to minimize problems associated with breakdown of the inhibitors
(64). Results using the two protocols were not
significantly different for either genistein (P > 0.7)
or tyrphostin A51 (P > 0.3), and the data were
therefore averaged and are presented in Fig.
4. GTP
S activated ICl,swell in all cells treated with tyrphostin
A51 and DMSO and in 9 of 10 cells treated with genistein. Neither
inhibitor significantly altered the rate of current activation or peak
current (Fig. 4).

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Fig. 4.
Tyrosine kinase inhibitors have no significant
(P > 0.5) effect on GTP S-induced current
activation. Genistein (100 µM) and tyrphostin A51 (100 µM) were
dissolved in DMSO and added to the patch pipette and bath solutions at
a final DMSO concentration of 0.1%. Control solutions contained 0.1%
DMSO. Values are means ± SE. Number of observations
(n) is shown in parentheses.
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To corroborate the inhibitor studies and to further test for the
involvement of phosphorylation events in the GTP
S-induced activation
of ICl,swell, cells were metabolically poisoned
and patch clamped with Mg2+-free pipette solutions
containing 1 mM EDTA. Cellular ATP production was blocked by incubating
cells for 10-30 min in bath solution containing 5 mM
2-deoxyglucose and 100 nM rotenone. In addition, the pipette solution
contained 40 µM oligomycin, 20 µM rotenone, and 5 µM iodoacetate.
In metabolically poisoned cells, removal of ATP from the pipette
solution or replacement with 2 mM AMP-PNP had no significant effect on
the rate of GTP
S-induced ICl,swell activation
or peak current (Fig. 5). These results
demonstrate clearly that ATP hydrolysis does not play a role in the
GTP
S signaling pathway. The number of metabolically poisoned cells
in which GTP
S triggered current activation was 9 of 9 in the
presence of 2 mM ATP, 17 of 18 with 0 mM ATP, and 16 of 16 cells with 2 mM AMP-PNP in the pipette solution.

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Fig. 5.
GTP S-induced current activation is not modulated by
intracellular ATP or phosphorylation reactions. All cells were patch
clamped with a Mg2+-free pipette solution containing the
metabolic inhibitors oligomycin (40 µM), rotenone (20 µM), and
iodoacetate (5 µM) and were also preincubated for 10-30 min in a
bath solution containing 2-deoxyglucose (5 mM) and rotenone (100 nM).
ATP removal or substitution with 5'-adenylylimidodiphosphate (AMP-PNP)
had no significant (P > 0.1) effect on the rate of
activation and peak GTP S-induced current. Values are means ± SE. Number of observations (n) is shown in parentheses.
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Cholera toxin inhibits GTP
S-induced activation of
ICl,swell via cAMP-independent mechanisms.
G proteins are categorized into three families: heterotrimeric,
low-molecular-weight (small) monomeric, and high-molecular-weight (large) monomeric (3). The heterotrimeric G proteins are
composed of three subunits termed
,
, and
. Pertussis and
cholera toxins are commonly used to determine whether a heterotrimeric
G protein family is involved in a specific signaling pathway.
Pertussis toxin catalyzes the ADP-ribosylation and inactivation of
members of the G
i/o subfamily. To test for the
involvement of G
i/o G proteins in
ICl,swell regulation, N1E115 cells were preincubated with 100 ng/ml pertussis toxin for 6-10 h before patch-clamp measurements were taken. Pretreatment with 1 µg/ml of
pertussis toxin for >4 h is sufficient to completely ADP-ribosylate the
-subunit of Gi in N1E115 cells (7).
Pertussis toxin had no significant (P > 0.5) effect on
ICl,swell activation. Rates of GTP
S-induced
current activation and peak currents (means ± SE) in the presence
and absence of pertussis toxin were 1.8 ± 0.4 pA · pF
1 · min
1
(n = 8) and 4.9 ± 0.8 pA/pF (n = 7), and 1.4 ± 0.4 pA · pF
1 · min
1
(n = 10) and 5.0 ± 0.9 pA/pF (n = 5), respectively. These results indicate that that G
i/o
proteins do not mediate the effect of GTP
S.
Cholera toxin catalyzes the ADP-ribosylation of the G
s
subfamily of G proteins, rendering them constitutively active.
Overnight incubation with 100 ng/ml cholera toxin reduced
GTP
S-induced ICl,swell activation and peak
current by ~70% (Fig. 6).

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Fig. 6.
G s signaling pathways inhibit
GTP S-induced activation of ICl,swell via
cAMP-independent mechanisms. Incubation of cells overnight with 100 ng/ml cholera toxin (CTX) inhibited GTP S-induced current activation
~70%. Exposure of cells to the adenylyl cyclase inhibitor
2'-5'-dideoxyadenosine (DDA; 100 µM) had no effect on the inhibitory
action of CTX. Overnight incubation of cells with 500 µM
8-bromoadenosine 3',5'-cyclic monophosphate (8-BrcAMP) did not mimic
the effects of CTX treatment. Values are means ± SE.
*P < 0.05; **P < 0.01;
***P < 0.001. Number of observations (n) is
shown in parentheses.
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Activation of G
s activates adenylyl cyclase and elevates
intracellular cAMP. This suggests that cAMP might mediate the
inhibitory effect of cholera toxin. To test whether inhibition occurred
via a cAMP-dependent mechanism, cells were incubated overnight with cholera toxin and 2'-5'-dideoxyadenosine (DDA; 100 µM), an inhibitor of adenylyl cyclase. As shown in Fig. 6, the inhibitory effect of
cholera toxin was unaltered by DDA.
In an effort to mimic the inhibitory action of cholera toxin, we
incubated cells overnight with 500 µM 8-bromoadenosine 3',5'-cyclic monophosphate (8-BrcAMP) and included it in the patch pipette solution.
Current activation was not significantly different in 8-BrcAMP-treated
cells (Fig. 6). We conclude that G
s inhibits GTP
S-induced activation of ICl,swell by
directly inhibiting the channel and/or channel regulatory mechanisms.
GTP
S-induced activation of ICl,swell is mediated by
Rho GTPases.
The low-molecular-weight monomeric G proteins include the Ras, Rho,
Rab, Arf, and Ran families (3). Ten classes of mammalian Rho GTPases have been identified (5): Rho (A, B, C
isoforms), Rac, Cdc42, Rnd1/Rho6, Rnd2/Rho7, Rnd3/RhoE, Rho D, RhoG,
TC10, and TTF. To test for the involvement of Rho G proteins in
ICl,swell regulation, N1E115 cells were
incubated with 1 ng/ml C. difficile toxin B for 19-24
h. Toxin B catalyzes the UDP-glucosylation of the Rho subfamily of
monomeric G proteins including Rho, Rac, and Cdc42 (1).
Incubation with toxin B inhibited the rate of GTP
S-induced current
activation and peak current ~70% (Fig.
7). These results demonstrate that Rho
GTPase signaling pathways regulate GTP
S-induced activation of
ICl,swell.

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Fig. 7.
GTP S-induced activation of
ICl,swell is regulated by Rho GTPases.
Incubation of cells overnight with 1 ng/ml C. difficile
toxin B inhibited the rate of current activation and peak current.
Values are means ± SE. *P < 0.05. Number of
observations (n) is shown in parentheses.
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Swelling-induced activation of ICl,swell is modulated
by Rho signaling pathways.
To determine whether G protein signaling pathways modulate
swelling-induced activation of ICl,swell, cells
were dialyzed with GTP
S and exposed to a hypotonic bath (100 mosmol/kgH2O reduction in bath osmolality) after the
GTP
S-induced current had inactivated (Fig.
8A). Spontaneous inactivation
of the GTP
S-induced current did not preclude further activation of
ICl,swell with a swelling stimulus. The
mean ± SE volume set points for current activation in the
presence and absence of GTP
S were 1.13 ± 0.02 (n = 22) and 1.18 ± 0.03 (n = 9),
respectively, and were not significantly (P > 0.1)
different. However, GTP
S stimulated the rate of swelling-induced current activation two- to threefold (Fig. 8A).

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Fig. 8.
G protein signaling pathways regulate swelling-induced
activation of ICl,swell. A: effect of
GTP S treatment on swelling-induced current activation in a single
N1E115 cell. After decay of the GTP S current, cell was swollen by
exposure to a hypotonic (200 mosmol/kgH2O) bath solution.
Inset: mean rates of swelling-induced current activation in
cells dialyzed with 0.5 mM GTP or GTP S. GTP S increased rate of
swelling-induced current activation 2- to 3-fold. B:
dialysis of cells with 10 mM GDP S or overnight exposure to 10 ng/ml toxin B inhibited swelling-induced
ICl,swell activation. For experiments with
GDP S, CsCl concentration in the pipette solution was reduced to 110 mM to maintain osmolality. The control pipette solution contained 110 mM CsCl and 10 mM Li3 citrate to control for the
Li+ that was added with GDP S. Values are means ± SE. *P < 0.03; ***P < 0.001. Number
of observations (n) is shown in parentheses.
|
|
To determine whether G proteins are required for swelling-induced
current activation, 10 mM GDP
S was included in the pipette solution
in the absence of GTP
S. GDP
S inhibited swelling-induced ICl,swell activation by ~80% (Fig.
8B) and significantly (P < 0.002) increased
mean ± SE channel volume set point from 1.1 ± 0.02 (n = 4) to 1.22 ± 0.01 (n = 4).
Overnight exposure of cells to 10 ng/ml toxin B inhibited current
activation ~70% (Fig. 8B) without altering channel volume
set point (control = 1.1 ± 0.02, n = 7;
toxin B = 1.1 ± 0.03, n = 5). Taken
together, these data suggest strongly that Rho GTPase signaling
pathways regulate swelling-induced activation of
ICl,swell.
 |
DISCUSSION |
ICl,swell is a swelling-activated anion
current that appears to be expressed ubiquitously in mammalian cells
(43, 50, 57). This current is outwardly rectifying,
exhibits an Eisenman type I anion permeability sequence, and is
inhibited by a wide variety of pharmacological agents. The
ICl,swell channel plays an important role in
cell volume regulation (reviewed in Refs. 43,
50, 57) and may participate in the control of
other physiological processes such as cell metabolism, membrane
excitability, and cell growth, proliferation, and apoptosis
(32, 38, 47).
The molecular identity of the channel responsible for
ICl,swell is uncertain and controversial.
P-glycoprotein and pICln have both been suggested to
function as the ICl,swell channel. However, most
workers in the field no longer consider these proteins to be viable
channel candidates (21, 50, 56). More recently, ICl,swell was proposed to be due to the activity
of ClC-3, a member of the ClC superfamily of voltage-gated anion
channels (19). The findings of Duan and co-workers
(19) on ClC-3 have not yet been reproduced by other
laboratories, and a variety of recent observations have begun to raise
doubts about a widespread role for this channel in
ICl,swell function (35, 48, 65).
Regulation of ICl,swell.
Cell swelling and reduced intracellular ionic strength activate
ICl,swell (8, 22, 44, 50, 57). The
signal transduction mechanisms responsible for channel activation are
unclear. Recently, Nilius and co-workers (46) demonstrated
that ICl,swell in endothelial cells is activated
transiently by GTP
S in a pertussis toxin-insensitive manner. A
similar outwardly rectifying anion current exhibiting many of the basic
properties of ICl,swell was originally shown by
Doroshenko and colleagues (15, 16) to be triggered by
GTP
S in bovine chromaffin cells.
ICl,swell in N1E115 neuroblastoma cells is also
activated transiently by GTP
S (Fig. 1). Activation of
ICl,swell by swelling is dramatically stimulated
by GTP
S (Fig. 8A) and inhibited by GDP
S (Fig.
8B). Taken together, these results indicate that cell swelling is transduced into channel activation at least in part via G
protein signaling pathways.
GTP
S current activation occurs via a pertussis toxin-insensitive
mechanism. However, cholera toxin significantly inhibited GTP
S-induced current development, suggesting that G
s
regulates ICl,swell (Fig. 6). Activation of
G
s stimulates adenylyl cyclase. Several studies have
recently demonstrated that ICl,swell is
modulated by cAMP. Du and Sorota (17) observed both
inhibitory and stimulatory effects of cAMP in dog atrial cells. They
showed that inhibition of ICl,swell is due to
cAMP-induced activation of protein kinase A (PKA) and increased
protein phosphorylation, whereas the stimulatory effect of cAMP occurs
in a phosphorylation-independent fashion. More recently, Shimizu et al.
(55) demonstrated that cAMP enhances ICl,swell activation in Intestine 407 cells by a
PKA-independent mechanism.
We tested for the involvement of adenylyl cyclase and cAMP in mediating
the effect of cholera toxin on GTP
S-induced activation of
ICl,swell. The inhibitory action of cholera
toxin was not mimicked by overnight exposure to 8-BrcAMP and was not
blocked by the adenylyl cyclase inhibitor DDA (Fig. 6), indicating that
G
s functions through cAMP-independent pathways.
Recent studies have demonstrated that G
s is capable of
modulating ion channel activity in the absence of adenylyl cyclase and
PKA function (e.g., Refs. 31, 37).
G
s may directly inhibit the
ICl,swell channel and/or may act on signaling
pathways that regulate channel activation. G
may also inhibit
ICl,swell in a cAMP-independent manner. It has
been demonstrated that G
subunits can directly modulate ion
channel activity in a stimulatory or inhibitory fashion (10,
52). It is interesting to speculate that spontaneous
inactivation of ICl,swell (Fig. 1) may be
mediated by GTP
S stimulation of G
s or G
signaling mechanisms. Extensive molecular biological studies are
required to fully determine which heterotrimeric subunit inhibits
ICl,swell activity and to delineate the
mechanism by which this inhibition occurs.
Regulation of ICl,swell is mediated at least in
part by small monomeric Rho GTPases. C. difficile toxin B
inhibited both GTP
S- and swelling-induced channel activation (Figs.
7 and 8). Toxin B inhibits Rho, Rac, and Cdc42 Rho GTPases
(1). Recently, Nilius et al. (46)
demonstrated that ICl,swell in endothelial cells is inhibited by Clostridium C3 exoenzyme. The C3 exoenzyme
is a selective inhibitor of Rho A, B, and C (1). C3
exoenzyme does not readily permeate plasma membranes, and, in our
study, we were unable to ensure that it was loaded effectively into
N1E115 cells. However, assuming that ICl,swell
in endothelial and N1E115 cells are controlled by similar mechanisms,
our findings in conjunction with those of Nilius et al.
(46) argue that Rho A, B, and/or C are important
regulators of this current.
The molecular details of how Rho GTPase signaling pathways regulate
ICl,swell are unknown. Rho GTPases have been
implicated in a variety of cellular processes including actin
cytoskeletal organization, membrane trafficking, transcriptional
activation, cell growth, motility, and morphogenesis (24, 53,
63). Alterations in cytoskeletal organization regulated by Rho
GTPases have been studied extensively and include the formation of
focal adhesions, actin stress fibers, lamellipodia, membrane ruffles,
and filopodia (24, 42, 53, 60).
Changes in the organization of the actin cytoskeleton have long been
implicated in regulating volume-sensitive transport pathways (32,
41, 50, 57), including the ICl,swell
channel (34, 54, 62). Mechanical forces have been shown to
directly modulate G protein activity (23) as well as
cytoskeletal architecture (25, 26). It is attractive to
postulate then that swelling-induced alterations in Rho GTPase activity
may alter cytoskeletal structure, which in turn triggers
ICl,swell activation. Alternatively, cell swelling may directly alter the organization of the cytoskeleton. Cytoskeletal changes could conceivably activate Rho G protein signaling
pathways that regulate ICl,swell.
Role of protein phosphorylation in ICl,swell
regulation.
Phosphorylation has emerged as an extremely confounding variable in
understanding how ICl,swell is regulated. In
numerous cell types, nonhydrolyzable ATP analogs support normal
swelling-induced current activation (6, 49, 50, 57), an
observation that argues strongly against a role for phosphorylation
events in channel regulation. However, serine/threonine phosphorylation
(e.g., Refs. 9, 40), serine/threonine
dephosphorylation (18, 19), tyrosine phosphorylation
(e.g., Refs. 12, 33), and tyrosine dephosphorylation (14, 61) have been proposed to play
roles in channel activation.
Even experiments from the same laboratory have generated confounding
results. Szücs et al. (59) failed to detect an
inhibitory effect of the tyrosine kinase inhibitor genistein on
swelling-induced ICl,swell activation in bovine
endothelial cells. However, more recently, Voets et al.
(64) demonstrated that this compound inhibited activation
of the current by both GTP
S and swelling. Voets et al.
(64) suggested that the discrepant findings may have been
due to low solubility and stability of genistein.
In our study, we were unable to detect any inhibitory action of
genistein or tyrphostin A51 on GTP
S-induced activation of ICl,swell (Fig. 4). Because of potential
problems associated with the use of these drugs (see Ref.
64), we examined the combined effects of intracellular ATP
and Mg2+ removal on current activation. As shown in Fig. 5,
ATP and Mg2+ removal or replacement of ATP with the
nonhydrolyzable analog AMP-PNP in metabolically poisoned cells has no
effect on GTP
S-induced current activation. Similarly, we have shown
previously that swelling-induced ICl,swell
activation in neuroblastoma cells occurs normally in the absence of
hydrolyzable ATP (6). Indeed, even dialysis of
metabolically poisoned cells with Mg2+- and ATP-free
pipette solutions containing AMP-PNP and alkaline phosphatase to
dephosphorylate proteins has no effect on current activation
(6). On the basis of these results, we conclude that
phosphorylation signaling pathways do not regulate
ICl,swell in N1E115 neuroblastoma cells during
GTP
S- or swelling-induced activation.
The requirement for phosphorylation observed in other cell types may
reflect the existence of distinct channel types. Alternatively, it may
reflect the existence of multiple signaling/regulatory pathways
involved in channel activation. These pathways could be cell specific,
they may reflect the physiological status of the cell, and/or they may
be sensitive to experimental parameters such as the mechanism or rate
of cell swelling (see Ref. 6). It is also distinctly
possible that pharmacological agents used to inhibit kinases and
phosphatases may directly block the channel, such as has been shown for
inhibitors of arachidonic acid metabolism (39), or they
may have other nonspecific effects. Conclusions drawn from
pharmacological studies of phosphorylation-dependent regulation of
ICl,swell should be corroborated where possible by metabolic inhibition and Mg2+ and ATP removal
experiments such as those shown in Fig. 5 and described by Bond et al.
(6).
Downstream effectors of Rho GTPases include various protein and lipid
kinases (2, 5), a finding consistent with the postulated
role of Rho kinases in regulating
ICl,swell in endothelial cells
(46). However, Rho GTPases can also regulate
cellular processes in a phosphorylation-independent manner. For
example, Rho GTPase-regulated actin polymerization and cross-linking in vitro occur in the absence of ATP and phosphorylation reactions (27). The nonkinase effectors of Rho GTPases include
various scaffolding proteins that play important roles in actin
cytoskeletal organization (2, 5).
Phosphorylation-independent activation of
ICl,swell in N1E115 cells may be mediated by
changes in the interaction of Rho-regulated scaffolding proteins with
the ICl,swell channel and/or associated
regulatory machinery.
To conclude, we have demonstrated that ICl,swell
in neuroblastoma cells is regulated by G protein signaling pathways.
Swelling- and GTP
S-induced channel activation are mediated at least
in part by Rho GTPases. Extensive additional studies utilizing
molecular and electrophysiological approaches are required to fully
elucidate the mechanisms by which G protein-dependent regulation occurs.
 |
ACKNOWLEDGEMENTS |
This work was supported by National Institutes of Health Grants
NS-30591 and DK-51610. A.Y. Estevez was supported by a National Science
Foundation postdoctoral fellowship. T. Bond was supported by a Stroke
Investigator Award from the Heart and Stroke Foundation of Ontario.
 |
FOOTNOTES |
Address for reprint requests and other correspondence: K. Strange, Vanderbilt Univ. Medical Center, Anesthesiology Research Division, T-4202 Medical Center North, Nashville, TN 37232-2520 (E-mail: kevin.strange{at}mcmail.vanderbilt.edu).
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 10 October 2000; accepted in final form 31 January 2001.
 |
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