Departments of 1 Anesthesiolgy and 3 Physiology and 2 Center for Cellular and Molecular Signaling, Emory University School of Medicine, Atlanta, Georgia 30322
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ABSTRACT |
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To test the hypothesis that intracellular Ca2+ activation of large-conductance Ca2+-activated K+ (BK) channels involves the cytosolic form of phospholipase A2 (cPLA2), we first inhibited the expression of cPLA2 by treating GH3 cells with antisense oligonucleotides directed at the two possible translation start sites on cPLA2. Western blot analysis and a biochemical assay of cPLA2 activity showed marked inhibition of the expression of cPLA2 in antisense-treated cells. We then examined the effects of intracellular Ca2+ concentration ([Ca2+]i) on single BK channels from these cells. Open channel probability (Po) for the cells exposed to cPLA2 antisense oligonucleotides in 0.1 µM intracellular Ca2+ was significantly lower than in untreated or sense oligonucleotide-treated cells, but the voltage sensitivity did not change (measured as the slope of the Po-voltage relationship). In fact, a 1,000-fold increase in [Ca2+]i from 0.1 to 100 µM did not significantly increase Po in these cells, whereas BK channels from cells in the other treatment groups showed a normal Po-[Ca2+]i response. Finally, we examined the effect of exogenous arachidonic acid on the Po of BK channels from antisense-treated cells. Although arachidonic acid did significantly increase Po, it did so without restoring the [Ca2+]i sensitivity observed in untreated cells. We conclude that although [Ca2+]i does impart some basal activity to BK channels in GH3 cells, the steep Po-[Ca2+]i relationship that is characteristic of these channels involves cPLA2.
antisense oligonucleotides; arachidonic acid; calcium-activated potassium channels
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INTRODUCTION |
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ONE IMPORTANT TYPE of central nervous system (CNS) ion channel is the large-conductance Ca2+-activated K+ (BK) channel, with at least six distinct functional types of BK channels having been described within the CNS (30). BK channels in the neurosecretory GH3 cell line have been well characterized (4-7, 10, 15, 16, 31). Briefly, BK channels are charybdotoxin and tetraethylammonium, but not apamin, sensitive (4, 15, 16, 31). BK channels in GH3 cells have a Hill coefficient for the intracellular Ca2+ concentration ([Ca2+]i)-open channel probability (Po) relationship of >2 (5, 10, 15, 31) and a unit conductance of 150-400 pS (10, 15, 31). BK channels in GH3 cells are much less sensitive to voltage than to [Ca2+]i, since a 20- to 30-mV change is required to produce an e-fold change in Po (4, 10, 15, 16, 31). These properties are shared by BK channels from a wide variety of tissues (12, 22). BK channels in the CNS are essential to maintain normal rhythmic activity of repetitively firing neuronal cells, and they are involved in regulating the level of secretion from neurosecretory cells (3, 32). BK channels are important in such widely diverse CNS 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 (3, 11, 21). Because of the physiological importance of BK channels, we are particularly interested in the cellular signaling mechanisms responsible for controlling the activity of BK channels. An important membrane-delimited signal transduction pathway that is involved in the regulation of a number of ion channels, including BK channels in several nonneuronal cell types, is the phospholipase A2 (PLA2)-arachidonic acid cascade (18, 25, 35). Channel regulation by this signal transduction pathway can involve arachidonic acid itself or its metabolic products (8, 24, 26, 27). We previously reported that BK channels in GH3 cells can be activated by exogenous arachidonic acid (and perhaps metabolites of arachidonic acid) and that pharmacological agents that increase cytosolic PLA2 (cPLA2) activity increase BK channel activity (6). Because pharmacological inhibitors of cPLA2 applied by themselves decrease endogenous BK channel activity, we also hypothesized that PLA2 (cPLA2) acting on membrane phospholipids could produce enough endogenous arachidonic acid to tonically activate BK channels (6). These observations are consistent with the well-described (9, 13) relationship between [Ca2+]i and the enzymatic activity of cPLA2, which has shown that physiologically relevant changes in [Ca2+]i result in translocation of cPLA2 to the plasma membrane and subsequent activation in a Ca2+ concentration-dependent manner (2). However, regulation of BK channel activity by cPLA2 is a novel observation if it is true. Therefore, relying only on pharmacological inhibitors of cPLA2 to establish its role in BK channel regulation is not ideal because of the possibility of incomplete inhibition or nonspecific effects of the inhibitors. Therefore, in an effort to obviate any nonspecific effects of pharmacological inhibitors, we incorporated antisense oligonucleotides into GH3 cells to inhibit the expression of cPLA2. We accomplished this by using antisense oligonucleotides directed at the two possible translation start sites in the rat cPLA2 sequence (RNU 38376, GenBank). Western blot analysis and a biochemical assay of cPLA2 functional activity were used to verify that expression of cPLA2 was decreased in cells treated with the antisense vs. the sense oligonucleotides. The electrophysiological responses of BK channels to changes in [Ca2+]i in cells treated with sense (as a control) or antisense oligonucleotides were then examined. Finally, the effects of exogenous arachidonic acid on the electrophysiological Ca2+ response of BK channels from antisense-treated cells were also determined.
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EXPERIMENTAL PROCEDURES |
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Cell culture. The GH3 cell line was obtained from American Type Culture Collection (Rockville, MD). GH3 cells were grown at 37°C in a 5% CO2 atmosphere in DMEM supplemented with 15% heat-activated horse serum, 2.5% fetal bovine serum, and 2 mM glutamine. Cells for electrophysiological experiments were plated on polylysine-coated petri dishes to which a polycarbonate recording chamber with a volume of 0.2 ml had been affixed with Sylgard. Cells were used 1-3 days after plating, and cells from passages 40-55 were used.
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. The phosphorothioate oligonucleotides were 22 bases
[5'-ATCAGGAGTGTCCAGCATATCG-3' (antisense) and
5'-CGATATGCTGGACACTCCTGAT-3' (sense)] or 24 bases
[5'-ATCTATGAAAGACATTTTGGTCCC-3' (antisense) and
5'-GGGACCAAAATGTCTTTCATAGAT-3' (sense)].
Oligonucleotides were stored at 20°C until used. Cells were
plated on 35-mm Falcon dishes at a density of 5 × 105 cells/dish in serum-free DMEM
alone (serum-free media), DMEM containing both sense oligonucleotides
at 5 µM each (sense) or DMEM containing both antisense
oligonucleotides at 5 µM each (antisense). After incubation at
37°C in a 5% CO2 atmosphere
for 24 h, the cells were collected for electrophysiological experiments
or cell lysates were obtained for Western blot analysis.
Western blot analysis. Cells (~107) were exposed to each of the following conditions for 24 h before harvest: control and treatment with serum-free media, sense oligonucleotides, and antisense nucleotides. Cells were incubated for 1 h at 4°C in RIPA buffer (150 mM NaCl, 10 mM NaPO4, pH 7.2, 1% NP-40, 0.1% SDS, 0.25% deoxycholate) containing the protease inhibitor Pefablock (1 mM; Centerchem, Stamford, CT). After cell lysis, the suspension was centrifuged at 2,000 g for 10 min to precipitate unlysed cells. Protein determinations on the supernatant were accomplished using a commercial protein assay kit (DC protein assay kit, Bio-Rad, Hercules, CA). Absorbance was measured on an Ultrospec 3000 (Pharmacia Biotech, San Francisco, CA). Samples were prepared for SDS-PAGE by diluting cell lysate with sample buffer (40 mM Tris, pH 6.8, 0.1% SDS, 10% glycerol, 0.025% bromphenol blue) and heating at 85°C for 2 min. Protein (30 µg) was loaded in each lane on a 7.5% polyacrylamide gel. After 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, Eli Lilly, Indianapolis, IN) according to the protocol provided by Amersham for enhanced chemiluminescence developing.
Assay of PLA2 activity.
Confluent T-75 flasks were exposed to each of the following conditions
for 24 h before harvest: control and treatment with serum-free media,
sense oligonucleotides, and antisense nucleotides. Cells were washed
twice with cold PBS (4°C), and the PBS was removed and replaced
with 1.5 ml of 250 mM sucrose buffer. Cells were removed from the
flasks by scraping. Cells were then lysed by sonication at 4°C.
After sonication, the suspensions were centrifuged at 160,000 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 the cytosol and
membrane fractions, as described above.
PLA2 activity was assessed using
the hydrolysis of
dipalmitoyl-[2-palmitoyl-1-14C]-L--phosphatidylcholine
to [14C]palmitic acid,
as described previously (28, 29). Each reaction mixture contained 20 µg of protein for cytosolic measurements and 10 µg of protein for
membrane measurements.
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 space volume of 1.0 ml. Previous experiments showed that
exchange was 90 ± 7% complete after 0.5 min (4). After a high-resistance (>25 G) seal was obtained, patches were excised in a 1 µM intracellular
Ca2+ solution. Control recordings
were obtained in K2EGTA-buffered solutions containing the desired
[Ca2+]i,
as described below. For Ca2+
dependence experiments, the patch was initially exposed to 0.1 or 1 µM intracellular Ca2+. After
control recordings (typically 2-5 min), the patch was perfused
with a second solution containing 1 or 100 µM intracellular Ca2+, and additional control
recordings were made for 2-5 min. For experiments involving
arachidonic acid, the patch was initially exposed to 0.1 or 1 µM
intracellular Ca2+. After control
recordings (typically 2-5 min), the patch was perfused with a
solution containing 1 or 100 µM intracellular Ca2+, and additional control
recordings were made for 2-5 min. This paradigm was then repeated
with the desired Ca2+ solutions
that also contained 2.5 µM arachidonic acid, and recordings continued
for an additional 10 min.
Electrophysiological recordings.
For electrophysiological measurements, cells were mechanically
disrupted after 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 affixed with Sylgard. All
experiments used the excised patch configuration of the patch-clamp
technique. Electrodes were fabricated from Corning 7052 glass (Garner
Glass, Fullerton, CA) in two steps on an electrode puller (model PP-83,
Narishige, Tokyo, Japan). Electrodes were fire polished to a final tip
resistance of 3-5 M. Recordings were performed at room
temperature with a patch-clamp amplifier (model 3900, Dagan,
Minneapolis, MN). All experiments were conducted with the patch
depolarized to +20 mV unless otherwise stated. Single-channel data were
stored on digital audio tape using a Sony model DAS-75 digital audio
tape recorder (Dagan).
Data analysis. Single-channel data were digitized using Axotape software (Axon Instruments) 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-time plots (where N is the number of channels in each patch) by using Fetchan and P-Stat software programs (Axon Instruments, Foster City, CA). NPo-time plots were used to determine the time course for reaching a stable maximum effect for each series of experiments. Po values were determined from the amplitude histograms by fitting each amplitude histogram to the appropriate sum of Gaussian distribution functions by using iterative nonlinear regression software (NONLIN algorithm in SigmaPlot for Windows version 3.03, Jandel Scientific, Corte Madera, CA) after correction of the baseline to make zero current coincident with the state in which all channels were closed. NPo values were first calculated from the amplitude histogram. Po was then calculated as NPo/N. N was estimated by dividing the total conductance obtained during exposure of the patch to 10 or 100 µM intracellular Ca2+ 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, data obtained from a patch that contained a single channel were binned logarithmically and analyzed according to the method of Sigworth and Sine (34). Michaelis-Menten constants and the Hill coefficient for the Po-[Ca2+]i relationship for BK channels from control cells and cells treated with antisense oligonucleotides in the presence and absence of arachidonic acid were obtained by fitting concentration-effect data by using the NONLIN algorithm in SigmaPlot for Windows version 3.03 (Jandel Scientific).
Solutions. The solutions used in all experiments were (mM) 150 KCl, 2 MgCl2, and 10 HEPES (pH 7.30) or 140 KCl, 5 HEPES, 5 K2EGTA, and 4.55 CaCl2 (10 µM free ionized Ca2+), pH 7.30, for the pipette and 140 KCl, 15 HEPES, 5 K2EGTA, and 0.1, 1, or 100 µM intracellular Ca2+, pH 7.4, for the bath.
Drugs and chemicals.
cPLA2 sense and antisense
oligonucleotides (described above) were synthesized by the
Microchemical Facility of Emory University. Dipalmitoyl[2-palmitoyl-1-14C]-L--phosphatidylcholine
(specific activity 55.5 mCi/mmol) was obtained from DuPont NEN (Boston,
MA). With the exceptions cited above, all chemicals were obtained from
Sigma Chemical (St. Louis, MO).
Statistical analysis. Within-group comparisons for two treatments were accomplished using a t-test for repeated measures. In all cases, P < 0.05 was required to reject the null hypothesis. Intergroup comparisons for a single treatment were made using an ANOVA followed by a post hoc Scheffé test for multiple comparisons. Values are means ± SD unless otherwise specified.
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RESULTS |
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Treatment of GH3 cells with
cPLA2 antisense oligonucleotides results in
a significant reduction in cPLA2 expression.
Equal numbers of plates of confluent
GH3 cells were incubated for 24 h
under four different conditions: Untreated cells were maintained in the
normal growth medium, i.e., DMEM supplemented with serum (as described
in EXPERIMENTAL PROCEDURES). The
serum-free group was incubated in DMEM without serum. The sense and
antisense groups were incubated in DMEM without serum, but with the
addition of sense or antisense oligonucleotides targeting the potential translation start codons of cPLA2.
Although there was a minimal decrease in
cPLA2 in cells grown in serum-free
media or exposed to sense oligonucleotides, Western blot analysis of
cell lysates prepared from cells grown under each condition shows that
only in the cells treated with antisense oligonucleotides
was there almost complete inhibition of the expression of
cPLA2 (Fig.
1A). The
results of the Western analysis were verified by assaying cells exposed
to each of the four conditions above for
PLA2 activity (Fig.
1B). Results of a biochemical assay
of cPLA2 activity confirm the Western analysis
results. PLA2 activities for control, serum-free, sense, and antisense groups were 67.3 ± 7.4, 70.7 ± 10.6, 53.6 ± 8.0, and 18.4 ± 2.7 pmol palmitic acid
released · min1 · mg
protein
1, respectively.
PLA2 activity was reduced
significantly in the antisense-treated cells
(P < 0.01) compared with all other
treatments.
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Single-channel kinetics for BK channels from cells exposed to cPLA2 antisense oligonucleotides are similar to those associated with BK channels from control cells exposed to the cPLA2 inhibitor aristolochic acid. In excised, cell-free patches with 0.1 µM Ca2+ on the cytosolic surface, single BK channels from cells treated with serum-free media or cPLA2 sense oligonucleotides are characterized by relatively long open times and relatively short closed times. This pattern is similar to the kinetics of BK channels from untreated cells. On the other hand, under the same recording conditions, BK channels in patches from cells treated with cPLA2 antisense oligonucleotides were characterized by relatively short open times and relatively long closed times. Similar kinetics have been observed in BK channel records obtained from cells treated with PLA2 inhibitors. In Fig. 2, A and B, representative single-channel characteristics for BK channels obtained from cells treated with cPLA2 sense and antisense oligonucleotides, respectively, and containing a single BK channel in the excised patch are compared. Single-channel records for BK channels from a control cell before and after treatment with the cPLA2 inhibitor aristolochic acid are shown in Fig. 2, C and D, respectively. Representative probability density histograms for open and closed times for cells treated with cPLA2 sense and antisense oligonucleotides are shown in Fig. 3, A and B, respectively. These data were derived from patches that contained a single BK channel. The open interval histograms are shifted toward shorter open times and the closed interval histogram is shifted toward longer closed intervals in the antisense- than in the sense-treated cells. Identical results were obtained from four patches containing a single BK channel in cells treated with sense oligonucleotides and in five patches from cells treated with antisense oligonucleotides and containing a single BK channel. These results are in excellent agreement with data previously reported for BK channels from control cells treated with the PLA2 inhibitor aristolochic acid. For comparative purposes, probability density functions for open and closed times for a control cell before and after treatment with aristolochic acid are shown in Fig. 3, C and D, respectively. As noted above for cells treated with cPLA2 sense or antisense oligonucleotides, BK channels treated with aristolochic acid are characterized by a shift toward shorter open times and longer closed times than untreated control cells.
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BK channels from cells treated with cPLA2 antisense oligonucleotides are insensitive to increases in [Ca2+]i. We knew from our previous work that inhibiting PLA2 altered the Ca2+ sensitivity of BK channels. Figure 4 summarizes the effects of changes in [Ca2+]i on Po for BK channels in excised patches from cells grown under normal conditions, cells exposed to serum-free media, cells exposed to cPLA2 sense oligonucleotides in serum-free media, or cells exposed to cPLA2 antisense oligonucleotides in serum-free media (antisense). The Po of patches from any of the groups of cells exposed to Ca2+-free saline was the same and very close to zero. However, when Ca2+ was increased above minimal levels, the response of antisense-treated cells was dramatically different from that of sense-treated cells (or any of the other control groups). Po for the cells exposed to cPLA2 antisense oligonucleotides in 0.1 µM intracellular Ca2+ was 0.14 ± 0.08, which was significantly lower than for all the other groups (P < 0.001). Po for cells grown under normal conditions, cells exposed to serum-free media, and cells exposed to cPLA2 sense oligonucleotides in 0.1 µM intracellular Ca2+ were 0.24 ± 0.05, 0.27 ± 0.04, and 0.25 ± 0.04, respectively. Po for the cells exposed to cPLA2 antisense oligonucleotides in 1 µM intracellular Ca2+ was 0.16 ± 0.07, which was not different from 0.1 µM intracellular Ca2+ but was much lower than all the other groups (P < 0.001); Po for control, serum-free media, and sense-treated cells in 1 µM intracellular Ca2+ were not significantly different from each other: 0.43 ± 0.06, 0.45 ± 0.06, and 0.37 ± 0.06, respectively. Even a 1,000-fold increase in [Ca2+]i from 0.1 to 100 µM had no significant effect on Po (0.16 ± 0.08) in cells exposed to cPLA2 antisense oligonucleotides (Fig. 4). It is important to note that because of the loss of sensitivity to [Ca2+]i in cells treated with cPLA2 antisense oligonucleotides, we could not employ our usual approach of increasing [Ca2+]i to activate all the channels in a patch so that the number of channels in a patch may be an underestimate and, therefore, Po may be an overestimate. The decrease in [Ca2+]i sensitivity observed in cells exposed to cPLA2 antisense oligonucleotides is not associated with alterations in the unit conductance of the BK channels. Unit conductances for control cells and cells treated with serum-free media, sense oligonucleotides, and antisense oligonucleotides (277 ± 31, 268 ± 45, 282 ± 24, and 275 ± 40 pS, respectively) were not statistically different from each other.
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Antisense treatment does not alter the voltage sensitivity of the channel. BK channels are sensitive to Ca2+ and voltage, which often makes it difficult to separate changes in Ca2+ sensitivity from changes in voltage sensitivity. The changes in Po described above could be due to a true change in the sensitivity of the channel to Ca2+ or a shift in the Po-[Ca2+]i curve to higher voltages. One measure of the voltage sensitivity of the channel is the slope of the Po-voltage curve at constant Ca2+ concentration. Even though changes in Ca2+ may shift the curves along the voltage axis, if the slopes of Po-voltage curves are the same under two treatment conditions, then the voltage gating components of the channel have not been altered. We evaluated the effect of increasing voltage on BK channels from cells treated with antisense oligonucleotides at 0.1 and 10 µM intracellular Ca2+ (Fig. 5A). These data show that a 100-fold increase in [Ca2+]i results in a small increase in Po (as expected from our results above), but the slope of the Po-voltage relationship is the same at the two different Ca2+ concentrations. For comparison, Fig. 5B shows BK channel activity from cells treated with sense oligonucleotides in 0.1 and 1 µM intracellular Ca2+. In these cells a 10-fold increase in [Ca2+]i results in a highly significant increase in Po (P < 0.001) without a change in the slope of the Po-voltage relationship (a result expected for untreated BK channels). Because the slopes of the Po-voltage relationship for sense- and antisense-treated cells were not significantly different, we conclude that antisense treatment is not altering the voltage sensitivity of the channel but, rather, is altering the intrinsic Ca2+ sensitivity.
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Exogenous arachidonic acid increases the Po of BK channels in antisense-treated cells but does not change the sensitivity to [Ca2+]i. Our hypothesis at this point was that the unusually low Po of BK channels in cells treated with cPLA2 antisense oligonucleotides was due to a decreased production of arachidonic acid secondary to decreased cPLA2 activity. If this hypothesis were correct, then addition of exogenous arachidonic acid should result in an increase in the Po for these channels. Exposure of patches from antisense oligonucleotide-treated cells to 2.5 µM arachidonic acid in 0.1 µM intracellular Ca2+ resulted in a significant increase in Po from 0.16 ± 0.04 to 0.36 ± 0.04 (P < 0.001). Exposure of patches from cells treated with cPLA2 antisense oligonucleotides to 2.5 µM arachidonic acid in 1.0 µM intracellular Ca2+ also resulted in a significant increase in Po from 0.16 ± 0.02 to 0.37 ± 0.02 (P < 0.001). Similarly, exposure of patches to 2.5 µM arachidonic acid in 100 µM intracellular Ca2+ resulted in a significant increase in Po from 0.22 ± 0.04 to 0.42 ± 0.02 (P < 0.001). Interestingly, increasing the [Ca2+]i from 0.1 or 1 to 100 µM in patches exposed to arachidonic acid did not result in any further increase in Po. The slopes of the lines describing the relationship between Po and [Ca2+]i for BK channels from cells treated with cPLA2 antisense oligonucleotides and for BK channels from cells treated with cPLA2 antisense oligonucleotides and arachidonic acid were not significantly different. Both slopes were significantly lower than control (Fig. 6).
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DISCUSSION |
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The major findings from this investigation are as follows: 1) cPLA2 antisense oligonucleotides effectively inhibit the synthesis of cPLA2 in GH3 cells. 2) Single-channel characteristics for BK channels from cells treated with cPLA2 antisense oligonucleotides are similar to BK channels from cells exposed to pharmacological inhibitors of cPLA2. 3) BK channels from cells treated with cPLA2 antisense oligonucleotides have a lower activity than BK channels from untreated cells. 4) BK channels from cells treated with cPLA2 antisense oligonucleotides are insensitive to [Ca2+]i compared with untreated cells (even very high concentrations). 5) Antisense treatment does not alter the voltage sensitivity of BK channels. 6) The addition of exogenous arachidonic acid results in a marked increase in BK channel activity in cells treated with cPLA2 antisense oligonucleotides but without any increase in the sensitivity to [Ca2+]i.
cPLA2 antisense oligonucleotides effectively inhibit the synthesis of cPLA2 in GH3 cells. Treatment of GH3 cells with antisense oligonucleotides directed against the two possible translation start sites for cPLA2 for 20-24 h resulted in a marked inhibition of protein expression as determined by Western blot analysis. Cells grown in serum-free media or in serum-free media containing sense oligonucleotides showed a modest decrease in the expression of cPLA2 compared with serum-treated control cells. The fact that the BK channels from cells treated under these two conditions behaved identically to control with respect to basal activity and increases in [Ca2+]i, despite the small decrease in cPLA2, demonstrates that the activity of cPLA2 itself is not normally the rate limiting step in determining channel kinetics. This effect may result from the withdrawal of serum from the cells, thus removing some of the elements required for optimal synthesis of cPLA2 (and other proteins). However, in the cells where serum was removed and antisense oligonucleotides were added, there was a significant reduction in cPLA2 protein synthesis (as shown by Western blots) and functional activity (as determined by a biochemical assay of PLA2-specific lipase activity). The results from our Western blot analysis were confirmed by a direct functional assay for PLA2 activity in cells exposed to each of the four experimental conditions. Results from this direct assay confirmed that membrane PLA2 activity was markedly decreased in cells treated with antisense oligonucleotides (78 ± 5%). A decrease in PLA2 activity of ~80% in the presence of 5 µM antisense oligonucleotides is in excellent agreement with previous reports (33).
As part of the cPLA2 assay, we determined the amounts of cPLA2 associated with the plasma membrane vs. the cytosolic amount. For untreated, serum-free, sense-treated, and antisense-treated cells, the membrane-associated cPLA2 values were 82.9 ± 16.8, 83.1 ± 22.8, 72.9 ± 16.9, and 67.4 ± 16.8% of total cPLA2, respectively. None of the treatments significantly alters the fraction of cPLA2 that is membrane associated, but the results do indicate that a large fraction of the cPLA2 is membrane associated and, therefore, potentially active in untreated cells. Thus there is cPLA2 available and presumably active that could produce arachidonic acid and tonically modulate BK channels in untreated cells.Single-channel characteristics for BK channels from cells treated with cPLA2 antisense oligonucleotides are similar to BK channels from cells exposed to pharmacological inhibitors of cPLA2. We previously reported that when BK channels are exposed to pharmacological inhibitors of cPLA2, their single-channel kinetics change from the long open times and short closed times of untreated cells to long closed times and short open times (6). Treatment of cells with antisense oligonucleotides produces a similar (but possibly more profound) change in kinetics with a concomitant large reduction in Po in 0.1 µM intracellular Ca2+. However, despite the clear qualitative difference in kinetics between sense- and antisense-treated cells, we did not attempt a quantitative description of the change because of the large number of open and closed states that have been well documented for BK channels (1, 19, 20, 23). It is important to note that even though antisense treatment reduced the channel activity, the unit conductance of the channels from antisense-treated cells was unchanged from control cells and was consistent with the values reported for BK channels in GH3 cells (4, 10, 15, 16, 31).
BK channels from cells treated with cPLA2 antisense oligonucleotides are relatively insensitive to changes in [Ca2+]i compared with untreated cells. Our previous results with cPLA2 inhibitors had suggested to us that at least part of the Ca2+ sensitivity of BK channels was not an intrinsic sensitivity of the channel itself but, rather, the sensitivity of cPLA2 to Ca2+, which subsequently altered the Po of the channel by increased arachidonic acid production. Nonetheless, the reduction of Po in BK channels blocked with inhibitors of cPLA2 could be partially restored by increases in [Ca2+]i (6), suggesting that some part of the Ca2+ sensitivity could have been intrinsic to the channel. Alternatively, Ca2+ and the inhibitors could compete for cPLA2 sites so that, even at extremely high concentrations of pharmacological inhibitors, high Ca2+ concentrations will activate residual cPLA2 activity. Thus one reason for performing the present experiments with antisense oligonucleotides was to investigate the properties of BK channels in the absence of any effects of cPLA2. The present data obtained using antisense oligonucleotides suggest that the BK channel-blocking effects of cPLA2 inhibitors were, indeed, due to an inhibition of cPLA2 and not to nonspecific effects of the inhibitors. Moreover, as noted above, BK channels from cells treated with antisense oligonucleotides exhibited a significantly lower Po in 0.1 µM intracellular Ca2+ than cells from any of the other control groups. In addition, BK channels from antisense-treated cells were relatively insensitive compared with untreated cells to large changes in [Ca2+]i (1,000-fold) in the normal range of activation of BK channels. These data suggest that a large part (but not all) of the previously described steep Po-[Ca2+]i relationship is due to a [Ca2+]i-cPLA2 interaction, rather than only binding of intracellular Ca2+ to multiple binding sites on the BK channel protein, as has been suggested by others (1, 5, 9, 13, 17, 20, 31). However, not all Ca2+ sensitivity of BK channels is due to the secondary effect of Ca2+ on cPLA2, since when [Ca2+]i is reduced to zero, BK channel activity is not observed in antisense-treated cells. Thus very low [Ca2+]i is responsible for some basal BK channel activity, presumably through direct interaction with the channel protein. This is consistent with BK channels from control cells (5, 10, 12, 15, 16, 20, 22, 31). The idea of a single Ca2+ binding site is also consistent with recent structural information (14).
Treatment with antisense oligonucleotides does not change the voltage sensitivity of BK channels. If our hypothesis is true, then a significant part of the Ca2+ sensitivity of BK channels arises because of Ca2+ activation of cPLA2. Because cPLA2 is a cytosolic or extrinsic membrane protein, it is unlikely that its Ca2+ activation is voltage sensitive. Therefore, any cPLA2-mediated changes in BK activity might not be expected to alter the voltage sensitivity of the channel. One accepted method for examining the voltage sensitivity of BK channels is to measure the slope of the Po-voltage curve at constant Ca2+ concentration. Even though changes in Ca2+ may shift the curves along the voltage axis, if the slopes of Po-voltage curves are the same under two treatment conditions, then the voltage gating components of the channel have not been altered. Our results showed that although removal of serum resulted in a small decrease in the voltage sensitivity from that in control cells, the voltage sensitivity was still similar to that previously described for GH3 cells (4, 10, 15, 16, 31). Also this small decrease in voltage sensitivity was not associated with any loss in [Ca2+]i sensitivity. On the other hand, treatment with sense or antisense oligonucleotides did not further reduce the voltage sensitivity, but treatment with antisense (but not sense) oligonucleotides did have a profound effect on the [Ca2+]i sensitivity. In fact, our results showed that the slopes of the voltage dependence were identical after sense or antisense treatment, so that inhibition of cPLA2 alone does not alter the intrinsic voltage sensitivity of BK channels.
The addition of exogenous arachidonic acid results in a marked increase in BK channel Po in cells treated with cPLA2 antisense oligonucleotides but no increase in the sensitivity to [Ca2+]i. If our hypothesis is correct, then a significant fraction of the Ca2+ dependence of native BK channels is due to Ca2+ activating cPLA2 to produce arachidonic acid, which then increases the Po of BK channels. Therefore, addition of exogenous arachidonic acid should restore the Po of BK channels after inhibition of cPLA2 but should not restore the Ca2+ sensitivity, which is conferred by cPLA2. We observed that addition of exogenous arachidonic acid to excised patches from normal GH3 cells results in at least a fourfold increase in BK channel Po, whereas inhibitors of arachidonic acid production decrease BK channel activity (6). If the majority of the Ca2+ sensitivity of BK channels arises because of the Ca2+ sensitivity of cPLA2 (and arachidonic acid production), then in antisense-treated cells, BK channels should be sensitive to exogenously added arachidonic acid but remain largely insensitive to changes in [Ca2+]i. In fact, addition of arachidonic acid to antisense-treated cells did result in an increase in Po, but even large changes in [Ca2+]i had little if any additional effect on Po. These data suggest that although arachidonic acid must be increasing BK channel activity by causing a thermodynamically favorable conformational change in the channel protein, a major part of the steep Po-[Ca2+]i relationship that is so characteristic of BK channels involves a [Ca2+]i-cPLA2 interaction. These results suggest that the antisense oligonucleotides are not affecting Ca2+ binding to the channel protein but are interfering with the Ca2+ binding or activation of some transduction element closely associated with the channel protein, which we believe to be cPLA2.
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ACKNOWLEDGEMENTS |
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The authors thank B. J. Duke for skillful technical assistance in the plating and maintenance of the cells and Dr. Sasanka Ramanadham (Div. of Metabolism, Dept. of Medicine, Washington University School of Medicine, St. Louis, MO) for numerous valuable suggestions and assistance regarding the functional assay for PLA2.
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FOOTNOTES |
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This work was supported in part by National Science Foundation Grant IBN-9603837 to D. D. Denson and D. C. Eaton and National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-37963.
A portion of this work was presented at the Annual Meeting of the Society for Neuroscience, New Orleans, LA, October 1997.
Address for reprint requests: D. D. Denson, Dept. of Anesthesiology, Emory University School of Medicine, 3B-South Emory University Hospital, 1364 Clifton Rd., Atlanta, GA 30322.
Received 4 November 1997; accepted in final form 29 September 1998.
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