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INTRODUCTION |
Long-term modulation of ion channels is beginning to be
appreciated. This concept suggests that changes in ion channel
properties are not dependent on continued occupation of a receptor by
an agonist, but rather arise via some long-lasting metabolic
modification such as protein phosphorylation (1-3). For this process
to occur, the receptor and ion channel do not have to be intimately
associated, but communicate some signal transduction pathway activated
by occupancy of the receptor. Such a signal transduction pathway, which
may involve several steps, ultimately results in a modification that
alters the activity and persists until this modification is reversed
(1). Protein phosphorylation and dephosphorylation have been shown to
be important in the modulation of a number of ion channels,
particularly those in the central nervous system (1-4). One important
type of central nervous system ion channel that has been shown to be
modulated by phosphorylation/dephosphorylation is the large conductance
Ca2+-activated potassium (BK) channel, with at least six
functionally distinct types having been described within the central
nervous system (5). BK channels are important to such widely diverse central nervous system functions as neural regulation of the heart originating in the nucleus tractus solitarius and sleep, which is
dependent on repetitive rhythmic activity originating in the reticular
formation of the thalamus (6-8). BK channels are also involved in
neuropeptide secretion, regulation of presynaptic calcium signals, and
neurotransmitter release (9). Because of the physiologic importance of
BK channels, we have been particularly interested in the cellular
signaling mechanisms responsible for controlling the activity of BK
channels (10, 11).
It is well documented that BK channels that have been reconstituted in
lipid bilayers can be either activated or inhibited by the addition of
ATP, protein kinases, or protein phosphatases (1, 4, 9, 12, 13). In
fact, BK channels have been classified as either type I or II based on
their response to the catalytic subunit of the
cAMP-dependent protein kinase (4). Type II BK channels in
mammalian brain reconstituted in lipid bilayers are activated by ATP
and ATP analogs via an endogenous protein kinase activity intimately
associated with the channel. For these channels, it appears the kinase
involved is protein kinase C
(PKC)1 since activators of
PKC enhance the response to ATP, whereas inhibitors of PKC reverse the
response to ATP (1). BK channels in GH3 cells appear to
belong to the class that is activated in the presence of ATP with or
without the addition of exogenous protein kinase, whereas BK channels
in GH4 cells appear to be inactivated by ATP or protein
kinases and activated by protein phosphatases (14, 15). Investigators
examining BK channels that are activated in response to ATP in lipid
bilayers concluded that there must be an endogenous protein kinase
activity intimately associated with these channels (1, 4, 9, 13).
However, because BK channels in lipid bilayers are at infinite
dilution, the phosphorylation target could be either the channel
protein itself or a regulatory protein that is intimately associated
with the ion channel. The possibility that the action of the kinase may
be on a regulatory protein rather than (or in addition to) the channel
protein itself is intriguing. One important regulatory protein for BK
channels in GH3 cells is cytosolic phospholipase A2 (cPLA2) (10, 11). Until recently, it has
been thought that optimal activation of cPLA2 requires both
Ca2+i and phosphorylation (16-19). It was
also thought that cPLA2 resided in the cytosol and
translocated to the cell membrane only in response to large increases
in [Ca2+]i. It is now clear that the mechanisms
involved in the regulation of cPLA2, including the relative
importance of phosphorylation, [Ca2+]i, and the
site of subcellular localization, show considerable variability in
different cell types and even between different agonists in the same
cell type. However, in most cells, there is a significant pool of
cPLA2 constitutively associated with the membrane capable
of producing arachidonic acid (17, 20). In addition, recent evidence
suggests that there is more than one isoform of cPLA2 (21)
that responds to intracellular calcium and other stimuli differently.
In this investigation, we sought to determine whether cPLA2
could be a potential target for phosphorylation, resulting in subsequent activation of BK channels. To test this hypothesis, we
studied the response of wild-type BK channels in GH3 cells to ATP, to the poorly hydrolyzable analog ATP
S, and to the
non-hydrolyzable substrate AMP-PNP. These results were compared with
responses from BK channels that had been exposed to either aristolochic acid or antisense oligonucleotides to cPLA2 prior to the
addition of ATP. Since PKC has been reported to be involved in the
activation of reconstituted BK channels as well as the phosphorylation
of cPLA2, we examined the possibility that PKC could be
associated with the ATP response of BK channels in GH3
cells. Finally, since two isoforms of cPLA2,
viz. cPLA2-
(86 kDa) and
cPLA2-
(60 kDa), have now been identified, we also
sought to verify that the cPLA2-
isoform is the one
closely associated with BK channels in GH3 cells (21).
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EXPERIMENTAL PROCEDURES |
Cell Culture--
The GH3 cell line was obtained
from American Type Culture Collection (Manassas, VA). GH3
cells were grown at 37 °C in a 5% CO2 atmosphere in
Dulbecco's modified Eagle's medium supplemented with 15%
heat-activated horse serum, 2.5% fetal bovine serum, and 2 mM glutamine. Cells for electrophysiologic experiments were plated on polylysine-coated Petri dishes to which a polycarbonate recording chamber with a volume of 0.2 ml had been previously affixed
with Sylgard®. Cells were used 1-3 days after plating,
and cells from passages 23 to 40 were used in the experiments described
in this study.
cPLA2 Sense and Antisense
Oligonucleotides--
Sense and Antisense oligonucleotides targeting
two possible translation start codons of rat cPLA2-
were
synthesized by the Microchemical Facility of Emory University as
previously reported (11). A GenBankTM/EBI Data Bank search
using the sequences for the sense and antisense oligonucleotides used
in this investigation resulted in only one full match, viz.
cPLA2-
, and no partial matches. Since inhibition by
antisense oligonucleotides usually requires a perfect match between
oligonucleotide and target mRNA, it is extremely unlikely that our
antisense oligonucleotides bound to cPLA2-
mRNA (if it were present) or to any other mRNA present in GH3 cells.
Western Blot Analysis--
Approximately 107 cells
were washed twice with phosphate-buffered saline without
Ca2+ or Mg2+. Cells were lysed at 4 °C in
the buffer described by Leslie and co-workers (17). Following cell
lysis, the suspension was centrifuged at 2000 × g for
10 min to precipitate unlysed cells. Protein determinations on the
supernatant were accomplished using a commercial protein assay kit
(Bio-Rad DC Protein Assay Kits®). Absorbance
measurements were made on an Ultrospec® 3000 (Amersham
Pharmacia Biotech). Samples were prepared for SDS-polyacrylamide gel
electrophoresis by diluting cell lysate with sample buffer (Tris (pH
6.8), 0.1% SDS, 10% glycerol, and 0.025% bromphenol blue) and
heating at 85 °C for 2 min. 30 µg of protein were loaded in each
lane on a 7.5% polyacrylamide gel. Following electrophoresis (~1 h
at 150 V), the proteins were transferred to nitrocellulose. The
nitrocellulose blots were then probed with a specific
anti-cPLA2 antibody (a generous gift from Dr. Ruth Kramer
(Lilly) following the protocol provided by Amersham Pharmacia Biotech
for ECL developing.
Assay of PLA2 Activity--
cPLA2
activity was determined by direct biochemical analysis as previously
reported (11). Briefly, cells from confluent T-75 flasks were washed
twice with cold phosphate-buffered saline (4 °C), and the
phosphate-buffered saline was removed and replaced with 1.5 ml of 250 mM sucrose buffer. Cells were removed from the flasks by
scraping and then lysed by sonication at 4 °C. Following sonication,
the suspensions were centrifuged at 100,000 × g for 1 h. The cytosolic supernatant was removed, and the membrane
fraction was resuspended in 0.5 ml of sucrose buffer. Protein
determinations were made on both the cytosolic and membrane fractions
as described above. PLA2 activity was assessed using the
hydrolysis of
L-
-[2-palmitoyl-1-14C]dipalmitoyl
to [14C]palmitic acid as described previously (22, 23).
Each reaction mixture contained 20 µg of protein for cytosolic
measurements and 10 µg of protein for membrane measurements.
Identification of cPLA2 Isoforms in GH3
Cells--
To identify which cPLA2 isoform (
or
) is
present in GH3 cells, we prepared mRNA from
GH3 cells (FasTrack, Invitrogen). 1 µg of mRNA was
used to synthesize double-stranded cDNA (Marathon cDNA
amplification kit, CLONTECH). Two degenerate
oligonucleotide PCR primers based on conserved regions of rat
cPLA2-
, human cPLA2-
, and human
cPLA2-
(5'-GGAAGCAAATT(T/C)(T/A)(A/T)(G/T)A(A/T)GGGAA-3' and 5'-A(G/C)(T/A)(A/G)AAGTC(A/G)AAGGA(G/A)A(G/T)GATGAG) were used in
an attempt to amplify fragments of cPLA2-
or
cPLA2-
from the GH3 double-stranded cDNA
(HotStartTaq Master Mix kit, QIAGEN Inc.). The PCR products were
separated by electrophoresis on 1% agarose gel. A single band was
purified from the gel (QIAquick gel extraction kit, QIAGEN Inc.),
ligated into the pGEM-T-Easy vector (Promega), and transformed into
JM109 (Promega). Plasmid DNA was isolated from the resulting colonies
using an alkaline extraction miniprep kit (QIAGEN Inc.) and sequenced
at the Emory University Sequencing Facility.
Drug Exposure Paradigm--
For all experiments, drug exposure
was effected using a gravity perfusion/suction removal technique with a
perfusion rate of 2.0 ml/min and a dead volume of 1.0 ml. Previous
experiments showed that exchange was 90 ± 7% complete after 0.5 min (24). After obtaining a high resistance (>25 gigaohms) seal,
patches were excised in a 1 µM Ca2+i
solution. Control recordings were obtained in
K2EGTA-buffered solutions containing the desired
Ca2+i concentration as described below. For calcium
dependence experiments, the patch was initially exposed to 0.1 µM Ca2+i. After control recordings
(typically 2-5 min), the patch was perfused with a second solution
containing 0.1 µM Ca2+i and either 2 mM MgATP or 200 µM ATP
S, and recordings were continuously made for 10-15 min. For experiments involving aristolochic acid, the patch was initially exposed to either 0.1 or 1 µM Ca2+i. After control recordings
(typically 2-5 min), the patch was perfused with a solution containing
250 µM aristolochic acid, and recordings were made for 10 min. This paradigm was then repeated with either 2 mM MgATP
or 200 µM ATP
S, and recordings were continued for an
additional 10-15 min. All solutions containing ATP, ATP
S, phorbol
esters, or GF 109203X were made immediately prior to use.
Electrophysiologic Recordings--
For electrophysiologic
measurements, cells were gently suspended in phosphate-buffered saline
following the 24-h treatment with oligonucleotides and plated on
polylysine-coated Petri dishes to which a polycarbonate recording
chamber with a volume of 0.2 ml had been previously affixed with
Sylgard®. All experiments in this study used the excised
patch configuration of the patch-clamp technique. Electrodes were
fabricated from Corning 7052 glass (Garner Glass Co., Fullerton, CA) in
two steps on a Narishige PP-83 electrode puller. Electrodes were
fire-polished to a final tip resistance between 3 and 5 megaohms.
Recordings were performed at room temperature with a Dagan Model 3900 patch-clamp amplifier. All experiments were conducted with the patch
depolarized to +20 mV. Single channel data were stored on digital audio
tape using a Sony Model DAS-75 digital audio tape recorder (Dagan
Corp., Minneapolis, MN).
Data Analysis--
Single channel data were digitized using
Axotape software (Axon Instruments, Inc., Foster City, CA) at a
sampling rate of 5 kHz and filtered at 2 kHz using a four-pole low-pass
Bessel filter. The digitized single channel data were analyzed in 1-min segments to generate NPo versus time
plots using Fetchan and P-Stat software programs (Axon Instruments,
Inc.). NPo versus time plots were used to
determine the time course for reaching a stable maximum effect for each
series of experiments. Open probabilities were determined from the
amplitude histograms by fitting each amplitude histogram to the
appropriate sum of gaussian distribution functions using iterative
nonlinear regression software (PeakFit Version 4, SPSS Inc., Chicago,
IL) after correction of the base line to make zero current coincident
with the state in which all channels were closed.
NPo values were first calculated from the amplitude
histogram. The open probability (Po) was then
calculated as NPo/N. The number of
channels in each patch (N) was estimated by dividing the
total conductance obtained during exposure of the patch to 10 or 100 µM Ca2+i by the unit conductance
associated with a one-state change. Because the duration of open and
closed intervals varied from very short to very long durations, data
obtained from a patch that contained a single channel were
binned logarithmically and analyzed according to the method of
Sigworth and Sine (25).
Solutions--
The solutions used in all experiments were as
follows: 150 mM KCl, 2 mM MgCl2,
and 10 mM HEPES (pH 7.30) or 140 mM KCl, 5 mM HEPES, 5 mM K2EGTA, and 4.55 mM CaCl2 (10 µM free ionized
Ca2+) (pH 7.30) for the pipette and 140 mM KCl,
15 mM HEPES, 5 mM K2EGTA, and 0.1, 1, or 100 µM Ca2+i (pH 7.4) for the bath.
Drugs and
Chemicals--
L-
-[2-palmitoyl-1-14C]dipalmitoyl
(specific activity, 55.5 mCi/mmol) was obtained from PerkinElmer Life
Sciences. GF 109203X was obtained from BIOMOL Research Labs Inc.
(Plymouth Meeting, PA). With the exceptions cited above, all other
chemicals were obtained from Sigma.
Statistical Analysis--
Within-group comparisons for two
treatments were accomplished using a t test for repeated
measures. In all cases, a p value of <0.05 was required to
reject the null hypothesis. Inter-group comparisons for a single
treatment were made using analysis of variance followed by a post
hoc Scheffe test for multiple comparisons. All data are presented
as means ± S.D. unless otherwise specified.
 |
RESULTS |
BK Channels in GH3 Cells Are Activated by MgATP and
ATP
S, but Not by AMP-PNP--
All of these experiments were done
using the excised patch configuration of the voltage-clamp technique
with the patch depolarized to +20 mV. Both the bath and pipette
solutions contained 140 mM K+, and the bath
solution also contained 0.1 µM Ca2+i.
After control recordings had been made, 1 mM MgATP, 200 µM ATP
S, or 1 mM AMP-PNP (a non-substrate
analog) was added, and recordings were continued until any effects had
stabilized. MgATP and ATP
S caused the Po to
increase significantly in all cells studied (p < 0.001) (Fig. 1, A and
B). MgATP caused the Po to increase from
0.14 ± 0.06 to 0.19 ± 0.07, whereas ATP
S resulted in an
increase in Po from 0.10 ± 0.04 to 0.24 ± 0.10. The magnitude of the effect noted for ATP
S was significantly larger than that noted for MgATP (p < 0.001) presumably because there was a phosphorylation/dephosphorylation
equilibrium established in the MgATP experiments, whereas the reaction
of ATP
S is only poorly reversible. On the other hand, AMP-PNP (Fig. 1C) did not result in any significant change in
Po (0.14 ± 0.02 versus 0.14 ± 0.03), suggesting that the effects of ATP were not simply
ligand-stimulated increases in channel activity.

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Fig. 1.
BK channels in GH3 cells are
activated by MgATP and ATP S, but not by
AMP-PNP. All of these experiments were done using the excised
patch configuration of the voltage-clamp technique with the patch
depolarized to +20 mV. Both the bath and pipette solutions contained
140 mM K+, and the bath solution also contained
0.1 µM Ca2+i. After control
recordings had been made, 1 mM MgATP, 200 µM
ATP S, or 1 mM AMP-PNP was added, and recordings were
continued until any effects had stabilized. MgATP (A) and
ATP S (B) caused the Po to increase
significantly in all cells studied (p < 0.001). MgATP
caused the Po to increase from 0.14 ± 0.06 to
0.19 ± 0.07, whereas ATP S resulted in an increase in
Po from 0.10 ± 0.04 to 0.24 ± 0.10. AMP-PNP (C) did not result in any significant change in
Po (0.14 ± 0.02 versus 0.14 ± 0.03).
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|
Aristolochic Acid Can Either Inhibit or Reverse the Activation of
BK Channels by MgATP--
Because cPLA2 is an important
element in the regulation of BK channels in GH3 cells and
since cPLA2 can be activated by phosphorylation, we
examined the effects of MgATP in the presence of the pharmacologic cPLA2 inhibitor aristolochic acid. All of the following
experiments were done using the excised patch configuration of the
voltage-clamp technique with the patch depolarized to +20 mV. Both the
bath and pipette solutions contained 140 mM K+,
and the bath solution also contained 1 µM
Ca2+i. Two sets of experiments were conducted.
First, after control single channel recordings had been made on excised
patches, a solution containing 250 µM aristolochic acid
was introduced. After a 5-min exposure, single channel recordings were
again made. Finally, a solution containing both 250 µM
aristolochic acid and 1 mM MgATP was introduced, and
recordings were continued for an additional 10 min. (The second set of
experiments started with 1 mM MgATP being added to the bath
solution after control single channel recordings had been made.) After
the effects of MgATP had stabilized, a solution containing 1 mM MgATP and 250 µM aristolochic acid was
introduced, and single channel recordings were made for an additional
10 min. In the first set of experiments, the addition of aristolochic
acid caused a significant reduction in Po from
0.36 ± 0.05 to 0.06 ± 0.04 (p < 0.001).
Subsequent addition of MgATP caused the Po to change
from 0.06 ± 0.04 to 0.04 ± 0.03. These data are summarized
in Fig. 2A. In the
second set of experiments, the addition of MgATP resulted in a
significant increase in Po from 0.15 ± 0.05 to
0.22 ± 0.06. Subsequent addition of aristolochic acid resulted in
a significant decrease in Po to 0.05 ± 0.03 (p < 0.001). These data suggested that
cPLA2 was a target for phosphorylation by ATP.

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Fig. 2.
Aristolochic acid and cPLA2
antisense oligonucleotides can inhibit the activation of BK channels by
MgATP. For the following experiments, the excised patch
configuration of the voltage-clamp technique was used with the patch
depolarized to +20 mV. Both the bath and pipette solutions contained
140 mM K+, and the bath solution also contained
1 µM Ca2+i. A, after
control single channel recordings had been made on excised patches, a
solution containing 250 µM aristolochic acid was
introduced. After a 5-min exposure, single channel recordings were
again made. Finally, a solution containing both 250 mM
aristolochic acid and 1 mM MgATP was introduced, and
recordings were continued for an additional 10 min. The addition of
aristolochic acid caused a significant reduction in
Po from 0.36 ± 0.05 to 0.06 ± 0.04 (p < 0.001). Subsequent addition of MgATP caused the
Po to decrease from 0.06 ± 0.04 to 0.04 ± 0.03 (not significant). B, the solutions and
conditions described for A were used for these experiments,
except that the cells were treated with cPLA2 antisense
oligonucleotides prior to use. After control single channel recordings
had been made on excised patches, the patches were exposed to 1 mM MgATP, and recordings were continued for 10 min. The
Po decreased from 0.13 ± 0.06 to 0.08 ± 0.06 (not significant) following treatment with MgATP.
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|
Treatment of Cells with cPLA2 Antisense
Oligonucleotides Eliminates the Activation of BK Channels by
MgATP--
Because aristolochic acid is a pharmacologic inhibitor of
cPLA2 and could possibly be producing its effect on the
MgATP response via some other mechanism, we examined the effects of
MgATP on excised patches derived from cells treated with
cPLA2 antisense oligonucleotides. For the following
experiments, the excised patch configuration of the voltage-clamp
technique was used with the patch depolarized to +20 mV. Both the bath
and pipette solutions contained 140 mM K+, and
the bath solution also contained 1 µM
Ca2+i. After control single channel recordings for
antisense oligonucleotide-treated cells had been made, the patches were exposed to 1 mM MgATP, and recordings were continued for 10 min. The Po decreased from 0.13 ± 0.06 to
0.08 ± 0.06 (p < 0.05) following treatment with
MgATP. These data are presented in Fig. 2B. Both biochemical
and Western blot analyses verified that the expression of
cPLA2 had been reduced by >90% from control levels, as we
have previously reported (11). These data further support the
hypothesis that ATP phosphorylation of cPLA2 is an important pathway for the activation of BK channels by ATP.
PKC Is Involved in the Activation of BK Channels by ATP--
PKC
(or PKC-like kinase) activity has been reported to be associated with
BK channels that have been reconstituted in lipid bilayers. It is
likely that such kinase activity could be preserved in excised patches
containing BK channels. To test this hypothesis, we conducted three
sets of experiments. For all of the following experiments, the excised
patch configuration of the voltage-clamp technique was used with the
patch depolarized to +20 mV. Both the bath and pipette solutions
contained 140 mM K+, and the bath solution also
contained 0.1 µM Ca2+i for wild-type
cells and 1 µM Ca2+i for experiments
in which cells treated with cPLA2 antisense oligonucleotides were used. In the first set of experiments, after control single channel recordings had been made, the patches were exposed to 1 mM MgATP, and recordings were continued for 10 min. After the effects had stabilized, a solution containing 1 mM MgATP and either 1 µM PMA or
-phorbol
(as an inactive control) was added, and recordings were made for an
additional 10 min. The addition of 1 mM MgATP resulted in a
significant increase in Po from 0.22 ± 0.04 to
0.29 ± 0.04 (p < 0.001). Subsequent addition of
PMA resulted in a further significant increase in Po to 0.41 ± 0.04 (p < 0.001) (Fig.
3A). On the other hand, the
addition of
-phorbol did not result in any additional increase in
Po over the increase resulting from MgATP. In the
second set of experiments, the above paradigm was repeated on cells
that had been treated with cPLA2 antisense
oligonucleotides. As expected from experiments described earlier, the
addition of MgATP resulted in little change in Po
from control levels. The Po for control conditions
was 0.10 ± 0.04 and 0.08 ± 0.05 after treatment with MgATP.
The addition of PMA also produced little change, with a Po following treatment with PMA of 0.079 ± 0.06 (Fig. 3B). In the third set of experiments, after
control single channel recordings had been made, 20 µM GF
109203X, a potent PKC inhibitor, was introduced, and recordings were
continued for 10 min. A solution containing both GF 109203X and 1 mM MgATP was then introduced, and recordings were again
made for 10 min. Finally, the patches were exposed to a solution once
again containing only GF 109203X, and recordings were made for a final
10 min. The addition of GF 109203X did not result in a significant
change in Po from control levels (0.27 ± 0.03 to 0.28 ± 0.03). However, the addition of 1 mM MgATP
in the presence of GF 109203X caused the Po to
significantly decrease from 0.28 ± 0.03 to 0.17 ± 0.06 (p < 0.005). The washout of MgATP resulted in a
significant increase (p < 0.0025) and restored the
Po to a control value of 0.28 ± 0.02. Similar
results were obtained in three cells using the PKC pseudosubstrate
inhibitor residues 19-31. These data further support the
hypothesis that PKC can be involved in the activation of BK channels by
ATP and that, in the absence of PKC, other kinases appear to
phosphorylate additional elements in the regulatory pathway that reduce
channel activity (Fig. 3C).

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Fig. 3.
PKC is involved in the activation of BK
channels by ATP. For all of the following experiments, the excised
patch configuration of the voltage-clamp technique was used with the
patch depolarized to +20 mV. Both the bath and pipette solutions
contained 140 mM K+, and the bath solution also
contained 0.1 µM Ca2+i for wild-type
cells and 1 µM Ca2+i for experiments
in which cells treated with cPLA2 antisense
oligonucleotides were used. A, after control single channel
recordings had been made, the patches were exposed to 1 mM
MgATP, and recordings were continued for 10 min. Next, a solution
containing 1 mM MgATP and 1 µM PMA was added,
and recordings were made for an additional 10 min. The addition of 1 mM MgATP resulted in a significant increase in
Po from 0.22 ± 0.04 to 0.29 ± 0.04 (p < 0.001). Subsequent addition of PMA resulted in a
further significant increase in Po to 0.41 ± 0.04 (p < 0.001). B, the above paradigm was
repeated on cells that had been treated with cPLA2
antisense oligonucleotides. The addition of MgATP resulted in a
non-significant change in Po from control levels.
The Po for control conditions was 0.10 ± 0.04 and 0.08 ± 0.05 after treatment with MgATP. Subsequent addition
of PMA also produced a non-significant change in activity with a
Po following treatment with PMA of 0.079 ± 0.06. C, after control single channel recordings had been
made in wild-type GH3 cells, 20 µM GF 109203X
(a potent PKC inhibitor) was introduced, and recordings were continued
for 10 min. A solution containing both GF 109203X and 1 mM
MgATP was then introduced, and recordings were again made for 10 min.
Finally, the patches were exposed to a solution once again containing
only GF 109203X, and recordings were made for a final 10 min. The
addition of GF 109203X did not result in a significant change in
Po from control levels (0.27 ± 0.03 to
0.28 ± 0.03). However, the addition of 1 mM MgATP in
the presence of GF 109203X caused the Po to
significantly decrease from 0.28 ± 0.03 to 0.17 ± 0.06 (p < 0.005). The washout of MgATP resulted in a
significant increase (p < 0.0025) and restored the
Po to a control value of 0.28 ± 0.02.
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|
cPLA2 Is Associated with Membranes in GH3
Cells--
A PLA2 assay (described under Experimental
Procedures") was conducted on both the cytosolic and membrane
fractions of GH3 cell lysates prepared using the procedure
described by Osterhout and Shuttleworth (26). In wild-type
GH3 cells, 81 ± 19% of the PLA2 was
found in the membrane fraction, which was significantly greater than
the 19 ± 5% found in the cytosolic fraction (p < 0.01) (Fig. 4). It should be
noted that although the PLA2 assay employed here does not
distinguish between secretory and cytosolic PLA2, the
Western blot analysis and PLA2 amplification data described earlier suggest that cytosolic PLA2 is the major form of
the enzyme present in GH3 cells. These data support the
idea that, in GH3 cells as well as other cells (first
suggested by Osterhout and Shuttleworth (26)), a portion of the
PLA2 pool resides in the cell membrane.

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Fig. 4.
A majority of the PLA2
in GH3 cells is membrane-associated.
Cells grown under control conditions were isolated from a confluent
T-75 flask, and PLA2 assays were conducted on both the
cytosolic ( ) and membrane ( ) fractions of the cell lysate as
described in detail under "Experimental Procedures." Values are
means ± S.D. (n = 6). These data show that a
majority of the PLA2 found in wild-type GH3
cells is located in the membrane fraction (81 ± 19%), with the
remaining 19 ± 5% in the cytosol. It should be noted that
although the PLA2 assay employed here does not distinguish
between secretory and cytosolic PLA2, the Western blot
analysis and PLA2 amplification data described earlier
suggest that cytosolic PLA2 is the major form of the enzyme
present in GH3 cells.
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|
cPLA2-
Appears to Be the Major Isoform Found in
GH3 Cells--
Western blot analysis of GH3
cell lysates revealed two bands at ~86 kDa that were the
phosphorylated and unphosphorylated forms of cPLA2-
.
There was no band in the 60-kDa range, which would be the size expected
for cPLA2-
, a cPLA2 isoform usually found in
skeletal muscle, but not in brain. To confirm that there was only
cPLA2-
and no cPLA2-
in GH3
cells, we designed and synthesized PCR primers that would amplify both
isoforms. Amplification of cDNA derived from GH3 cell
mRNA produced products that ran as a single 606-base pair band on a
1% agarose gel. This is the size expected for cPLA2-
.
When the amplified cDNA was cloned and sequenced, it had 100%
identity to the previously reported rat cPLA2-
. Although
we repeated the amplification four times, we never observed any PCR
product that corresponded to the
-isoform and therefore concluded,
as others have reported (21), that cPLA2-
is and
cPLA2-
is not present in brain-derived cells like GH3.
 |
DISCUSSION |
The major findings in this investigation are as follows. 1) ATP
results in a significant activation of BK channels in wild-type GH3 cells. 2) Inhibition of cPLA2 by
aristolochic acid blocks the activation of BK channels by ATP in
wild-type GH3 cells. 3) Reducing the expression of
cPLA2 with antisense oligonucleotides also suppresses the
activation of BK channels by ATP. 4) Activation of PKC results in a
significant potentiation of the activation in BK channels produced by
ATP. 5) Inhibition of PKC in wild-type GH3 cells results in
a significant decrease in BK channel activity when ATP is added. 6)
Inhibition of cPLA2 blocks the activation of BK channels by
PKC. 7) cPLA2-
appears to be the isoform associated with
BK channels in GH3 cells.
Protein phosphorylation and dephosphorylation are important in the
modulation of central nervous system BK channels. (1-4). However,
modulation of BK channels by phosphorylation is complicated. BK
channels reconstituted in lipid bilayers can be either activated or
inhibited by the addition of ATP, protein kinases, or protein phosphatases (1, 4, 12). In fact, BK channels have been classified as either type I or II based on their response to
phosphorylation (4). Type II BK channels in mammalian brain
reconstituted in lipid bilayers are activated by ATP and ATP analogs
via an endogenous protein kinase activity intimately associated with
the channel. For these reconstituted channels, it appears that the
kinase involved is similar to protein kinase C since activators of PKC
enhance the response to ATP, whereas inhibitors of PKC reverse the
response to ATP (1, 4, 9, 13). Besides a PKC-like protein, Levitan and
co-workers (27-29) have shown a close association between BK channels
and a several other regulatory proteins that directly alter BK
activity. BK channels in GH3 cells appear to belong to the
class that is activated in the presence of ATP with or without the
addition of exogenous protein kinase, whereas BK channels in
GH4 cells (a subclone of GH3 cells) appear to
be inactivated by ATP or protein kinases and activated by protein
phosphatases (9, 14, 15). To test this hypothesis, we studied the
responses of wild-type BK channels to ATP in GH3 cells.
BK Channels in GH3 Cells Are Activated by MgATP and
ATP
S, but Not by AMP-PNP--
MgATP and ATP
S caused the
Po of BK channels in wild-type GH3 cells
to increase significantly. The non-substrate ATP analog AMP-PNP did not
result in any significant change in Po, further
suggesting that the effects produced by ATP require ATP hydrolysis for
channel activation (30). This is different from the ATP-sensitive ion
channels found in a variety of tissues in which ATP functions as a
ligand to alter channel properties by binding reversibly to an
allosteric site, but without ATP hydrolysis (30). The magnitude of the
increase noted for ATP
S was significantly larger than that noted for
MgATP presumably because there was a phosphorylation/dephosphorylation
equilibrium established in the MgATP experiments, whereas the reaction
of ATP
S is only slowly reversible (9, 13). This activation could be
due to a direct kinase-mediated phosphorylation of the channel by ATP.
Alternatively, since PLA2 production of arachidonic acid is
also a potent activator of BK channels in excised patches and since
PLA2 can be activated by phosphorylation, ATP activation of
BK channels could be due to kinase-mediated phosphorylation of
PLA2 rather than direct phosphorylation of BK channels.
Although these data clearly show that ATP hydrolysis and subsequent
protein phosphorylation are required for activation of BK channels in GH3 cells, the target for phosphorylation is still unclear.
Inhibition of cPLA2 Blocks the Activation of BK
Channels by ATP in Wild-type GH3 Cells--
Investigators
examining BK channels that are activated in response to ATP in lipid
bilayers concluded that there must be an endogenous protein kinase
activity intimately associated with these channels (1, 4, 9, 13).
However, the phosphorylation target could be either the channel protein
itself or a regulatory protein that is intimately associated with the
ion channel (1, 2, 4, 9, 13). We have shown that one important
regulatory protein for BK channels in GH3 cells is
cPLA2 (11). Optimal activation of cPLA2
requires both Ca2+i and phosphorylation (16-19).
In this investigation, we sought to determine whether cPLA2
could be a potential target for phosphorylation, resulting in
subsequent activation of BK channels. To test this hypothesis, we used
a pharmacologic and an antisense approach to lower cPLA2
activity in wild-type GH3 cells. Aristolochic acid caused a
significant reduction in Po for BK channels. This
observation is consistent with our previous reports (11). Subsequent
addition of MgATP caused the Po to decrease,
although this decrease was not statistically significant. The addition
of aristolochic acid to patches in which BK channels were activated by
ATP resulted in a significant decrease in activity. These data
suggested that cPLA2 was one target for phosphorylation by
ATP. Because aristolochic acid is a pharmacologic inhibitor of
cPLA2 and could possibly be producing its effect on the
MgATP response via some other mechanism, we examined the effects of MgATP on excised patches derived form cells treated with
cPLA2 antisense oligonucleotides. As previously reported,
treatment of wild-type GH3 cells in the present
investigation with antisense oligonucleotides resulted in an ~90%
decrease in the expression of cPLA2 by both biochemical and
Western blot analyses (11). Exposure of excised patches from antisense
oligonucleotide-treated cells resulted in a modest, albeit significant
decrease in BK channel activity. These data support the hypothesis that
ATP phosphorylation of cPLA2 is one important pathway for
the activation of BK channels by ATP. Our data further suggest that, in
the absence of cPLA2, there may be additional targets for
phosphorylation that act to inhibit BK channel activity like the
inhibition seen in GH4 cells (14, 15).
PKC Is Involved in the Activation of BK Channels by ATP--
Type
II BK channels in mammalian brain reconstituted in lipid bilayers are
activated by ATP and ATP analogs via an endogenous protein kinase
activity intimately associated with the channel. For these channels, it
appears that the kinase involved is similar to protein kinase C since
activators of PKC enhance the response to ATP, whereas inhibitors of
PKC reverse the response to ATP (1, 4, 9, 13). Since PKC could activate
cPLA2 either directly or via an alternative kinase pathway,
we studied the effect of MgATP on BK channels in the presence of the
specific PKC inhibitor GF 109203X. Treatment of excised patches with GF 109203X had no effect on the Po of BK channels.
However, the addition of MgATP to cells treated with GF 109293X
resulted in a significant decrease in Po. This
decrease was similar to that observed in cells treated with antisense
oligonucleotides, where the addition of MgATP also decreased the
Po. On the other hand, treatment of cells that had
been exposed to MgATP with PMA, an activator of PKC, resulted in a
significant increase in Po. On the other hand, for
BK channels in cells treated with cPLA2 antisense
oligonucleotides, exposure to PMA did not result in any significant
change in Po. These results suggest that 1) PKC is
necessary for ATP activation of BK channels; 2) in the absence of PKC,
other kinases appear to phosphorylate additional elements in the
regulatory pathway that reduce channel activity; 3) PLA2 is
partially phosphorylated and active in GH3 cells under
basal conditions; and 4) PLA2 is a target for PKC phosphorylation. (However, our data cannot exclude the possibility that
mitogen-activated protein kinase also plays a role in the activation of
cPLA2, as suggested by Leslie and co-workers (16, 31).)
cPLA2-
Rather than cPLA2-
Appears to
Be the Isoform Associated with BK Channels in GH3
Cells--
Recently, a novel membrane-associated cPLA2
isoform (cPLA2-
) has been described (21) (in contrast to
the original isoform, termed cPLA2-
).
cPLA2-
is associated with the plasma membrane, but it
lacks the [Ca2+]i-dependent
phospholipid-binding domain found in cPLA2-
, so its
activation is entirely [Ca2+]i-independent.
Although cPLA2-
shares significant sequence homology
with cPLA2-
, the absence of the phospholipid-binding domain reduces the size to ~60 kDa, so cPLA2-
can be
easily distinguished on Western blots from the "classical" type IV
cPLA2 (cPLA2-
). Several lines of evidence
argue against cPLA2-
being the isoform associated with
BK channels in GH3 cells. First, a
GenBankTM/EBI Data Bank search using the sequences for the
sense and antisense oligonucleotides used in this study did not
identify cPLA2-
(or any other sequence besides
cPLA2-
). Second, Western blots do not have a band at 60 kDa, where cPLA2-
should run. Finally, PCR amplification
of GH3 cell mRNA using primers that would amplify any
cPLA2 isoforms in GH3 cells resulted in the
amplification of only cPLA2-
. This is entirely
consistent with the tissue distribution of cPLA2-
, which
showed very little of this isoform in brain or brain-derived tissues
(21). These data support our claim that the results with cells treated
with antisense oligonucleotides were, in fact, due solely to the
depletion of cPLA2-
.
A Portion of the cPLA2-
Pool in GH3
Cells Resides in the Cell Membrane of Wild-type Cells--
Until
recently, it has been thought that cPLA2 resides almost
exclusively in the cytosol and translocates to the cell membrane (and
other membranes) only when stimulated by large increases in
[Ca2+]i. If this were true, then it would be
difficult to reconcile with our observation that cPLA2
activity appears to be constitutively associated with the small patches
of plasma membrane excised with our patch pipettes. An explanation of
this apparent discrepancy involves the observation by Osterhout and Shuttleworth (26) that at least a portion of the cellular pool of
cPLA2-
resides in the cell membrane and appears to be
activated without increases in [Ca2+]i. Our data
showing that the relative amount of cPLA2-
is higher in
GH3 cell membranes than in the cytosol are consistent with
this previous observation. It should be noted that although the
PLA2 assay employed here does not distinguish between
secretory and cytosolic PLA2, the Western blot analysis and
PLA2 amplification data described earlier suggest that
cytosolic PLA2 is the major form of the enzyme present in
GH3 cells. Some investigators have suggested that the
membrane-associated cPLA2-
is stabilized in the membrane
by interaction with certain anionic phospholipids such as
phosphatidylinositol 4,5-bisphosphate (32, 33). We do not question the
validity of the widely reported
[Ca2+]i-dependent activation of some
fraction of the type IV cPLA2 that is known to be
associated with the [Ca2+]i-dependent
translocation of the enzyme to the membrane. Our data suggest, however,
that activation of PLA2 associated with BK channels may
specifically involve a pool of cPLA2 that is already
located at or near its substrate in the membrane and is bound to
phosphatidylinositol 4,5-bisphosphate or other phospholipids.
In summary, our data point to cPLA2-
as one target
protein for phosphorylation that is intimately associated with the BK channel protein. Although we cannot unequivocally rule out the BK
channel protein itself as an additional target for phosphorylation, the
data that show a decrease in BK channel activity in the presence of ATP
and the absence of cPLA2 suggest that direct
phosphorylation of the channel protein would serve to decrease
rather than increase activity.