Department of Molecular and Cellular Physiology, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267-0576
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ABSTRACT |
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An HEK-293 cell line stably expressing the human
recombinant ClC-2 Cl channel was used in patch-clamp
studies to study its regulation. The relative permeability
Px/PCl calculated from
reversal potentials was I
> Cl
= NO3
= SCN
Br
. The
absolute permeability calculated from conductance ratios was
Cl
= Br
= NO3
SCN
> I
. The channel was activated
by cAMP-dependent protein kinase (PKA), reduced extracellular pH, oleic
acid (C:18 cis
9), elaidic acid (C:18
trans
9), arachidonic acid (AA; C:20
cis
5,8,11,14), and by inhibitors of AA metabolism,
5,8,11,14-eicosatetraynoic acid (ETYA; C:20
trans
5,8,11,14),
-methyl-4-(2-methylpropyl)benzeneacetic acid (ibuprofen), and
2-phenyl-1,2-benzisoselenazol-3-[2H]-one (PZ51, ebselen). ClC-2
Cl
channels were activated by a combination of forskolin
plus IBMX and were inhibited by the cell-permeant myristoylated PKA
inhibitor (mPKI). Channel activation by reduction of bath pH was
increased by PKA and prevented by mPKI. AA activation of the ClC-2
Cl
channel was not inhibited by mPKI or staurosporine and
was therefore independent of PKA or protein kinase C activation.
pH-activated ion channels; gastric HCl secretion; lung chloride channels; cystic fibrosis; nonsteroidal anti-inflammatory agents; ibuprofen; ebselen; 5,8,11,14-eicosatetraynoic acid
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INTRODUCTION |
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IN EPITHELIAL AND
NONEPITHELIAL tissues (2, 42) including
stomach (3, 21, 38) and lung
(1, 26, 36, 38), ClC-2 Cl channels are widely distributed.
Activation by voltage and pH have been documented in a variety of
recombinant systems (2, 7, 15,
21, 36, 38, 41,
42). ClC-2 Cl
channels exhibit volume
regulation (7, 9, 15,
47) and contain consensus phosphorylation sites for
cAMP-dependent protein kinase (PKA) activation (2,
38). AA (arachidonic acid) and inhibitors of AA metabolism
have been shown to affect other channels (20,
23, 29, 32), including
epithelial Cl
channels (5, 13,
14, 24). Whole cell patch clamp was used to
study the effects of pH, PKA, and AA on the recombinant ClC-2
Cl
channel. Understanding the mechanisms of regulation of
this channel will help elucidate the mechanisms of control of the
physiological processes in which the Cl
channels
participate. In the present study, the human recombinant form of the
ClC-2 Cl
channel was studied in a stably transfected
HEK-293 cell line. HEK-293 cells have low endogenous Cl
currents and have been well characterized for electrophysiological studies of ClC Cl
channels (31).
The rabbit and human forms of the recombinant ClC-2 Cl
channel have been previously shown to be activated by PKA catalytic subunit in planar lipid bilayer studies (21,
38, 41). In contrast, IB3-1 cells, a
bronchial epithelial cell line isolated from a cystic fibrosis patient
that expresses the ClC-2 Cl
channel, and IB3-1 cells
overexpressing human recombinant ClC-2, did not exhibit PKA-dependent
activation of net 36Cl
flux
(36). The rat ClC-2 Cl
channel also did not
exhibit PKA-activated Cl
currents (15).
Precise details of previous attempts to demonstrate PKA activation of
ClC-2 in cells have not appeared (15, 36). Differences in expression systems, the use of different activators of
PKA, and the lack of use of phosphodiesterase inhibitors by others may
underlie the reported lack of activation by PKA. In the present study,
a cell-permeant form of the PKA inhibitor, myristoylated PKI (mPKI),
was used to inhibit PKA and thus to determine whether there was
endogenous PKA activation of Cl
currents. Forskolin was
employed to activate adenylate cyclase, and IBMX was used to inhibit
phosphodiesterases. The interdependence of PKA, pH, and AA effects was
studied using PKA inhibitors and activators. The effect of the protein
kinase C (PKC) inhibitor staurosporine was also studied.
AA and its metabolites serve as lipid first or second messengers
(29) in regulation of ion channels. AA is released from membrane phospholipids through the action of phospholipase
A2 and phospholipase C. Nonsteroidal anti-inflammatory
agents (NSAIDS) such as ibuprofen
[-methyl-4-(2-methylpropyl)benzeneacetic acid] are inhibitors of
cyclooxygenase 1 and 2 (27, 43,
46) and would be expected to increase levels of AA
(23). ETYA (5,8,11,14-eicosatetraynoic acid, C:20
trans
5,8,11,14) is a nonmetabolized analog of AA that inhibits cyclooxygenase and lipoxygenase (23). Ebselen is
also an inhibitor of cyclooxygenase and lipoxygenase (10,
12, 44). These drugs were tested to determine
whether inhibition of metabolism of AA also increased Cl
currents.
Cis-unsaturated fatty acids including oleic acid (C:18
cis9), AA (C:20 cis
5,8,11,14), and
ETYA(C:20 trans
5,8,11,14) and trans-unsaturated fatty acids including elaidic acid (C:18
trans
9) activate K+ channels, whereas saturated fatty
acids are without effect (32). The effects of these fatty
acids on ClC-2 Cl
channels were examined to compare with
the more thoroughly studied effects of fatty acids on K+ channels.
The present study confirms and extends studies of PKA and pH activation
of the human ClC-2 Cl channel using patch-clamp studies,
reports expected differences between ion selectivity obtained from
current measurements (15, 36,
42) and those determined from zero current reversal
potentials (3, 21), and demonstrates for the
first time that AA activates ClC-2 Cl
channels. The
present study allows a comparison between the effects of AA and other
fatty acids on ClC-2 Cl
channels and previous studies on
other channels, including K+ channels (23,
32), and studies with cyclooxygenase and lipoxygenase inhibitors suggest new mechanisms of action of NSAIDS. These results may be important for understanding regulation of these channels in the
physiological processes in which they participate.
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MATERIALS AND METHODS |
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Transfection of HEK-293 cells.
Frozen aliquots of HEK-293 cells were obtained from American Type
Culture Collection (ATCC), thawed, and maintained at 37°C in 5%
CO2 in MEM supplemented with 5% heat-inactivated horse
serum, 0.1 mM nonessential amino acids, 2 mM L-glutamine, 1 mM sodium pyruvate, 100 U/ml penicillin G, and 100 µg/ml streptomycin
sulfate. Cells were transfected with 15 µg of His- and T7-tagged
human ClC-2 cDNA in the mammalian expression vector pcDNA3.1 using
Lipofectamine for 5 h at 37°C in serum-free medium. Cells were
returned to serum-containing medium, maintained for 2 wk in
serum-containing medium, and then grown in the presence of 300 µg/ml
G418. The surviving cells were then tested for expression of
Cl currents and ClC-2 mRNA. Mock transfected cells were
prepared using the same vector but expressing a different protein.
Positive cell lines were grown and kept frozen as stocks.
Measurement of whole cell Cl currents.
Currents were elicited by voltage-clamp pulses (1,500-ms duration)
between +40 and
140 mV in 20-mV increments from a beginning holding
potential of
30 mV. Currents were measured 50-100 ms after start
of the pulse. The external solution was normal Tyrode solution
containing (in mM) 135 NaCl, 1.8 CaCl2, 1 MgCl2, 5.4 KCl, 10 glucose, and 10 HEPES (pH 7.35 or 6.5, as indicated). The pipette solution was (in mM) 130 CsCl, 1 MgCl2, 5 EGTA, and 10 HEPES (pH 7.35). Freshly prepared AA,
ETYA, and ibuprofen were dissolved in DMSO and diluted into the bath
solution resulting in a final concentration of 1% DMSO. The free acid
form of AA was used. This was shipped under inert gas. All precautions
recommended by the manufacturer were taken to prevent oxidation of AA,
including use of solutions in organic solvent, storage of stock
solutions in organic solvent at
20°C in sealed containers, and
protection from light. In some cases, DMSO was bubbled with nitrogen
before preparation and immediate use of the solutions. All measurements with compounds in DMSO were compared with controls containing 1% DMSO
alone. Pipettes were prepared from borosilicate glass and pulled by a
two-stage Narashige puller to give 1-1.5 M
resistance. Data was
acquired with an Axopatch CV-4 headstage with a Digidata 1200 digitizer
and an Axopatch 1D amplifier. Data was analyzed using pCLAMP 6.04 software (Axon Instruments, Foster City, CA), Lotus 123 (Microsoft),
and Origins (Microcal). Statistical significance of the difference
between two means was determined with the Student's t-test
using n, the number of cells. I-V
(current-voltage relationship) curves were fit using the least-squares
method to obtain slopes and reversal potentials. Shifts of reversal
potential were used to calculate relative permeabilities according to
the relationship (11)
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Materials.
ETYA: C:20 trans5,8,11,14; mPKI: protein kinase A
inhibitor 14-22 amide myristoylated
Myr-N-Gly-Arg-Thr-Gly-Arg-Arg-Asn-Ala-Ile-NH2, trifluoroacetate salt; AA: ETYA 20.4 (C:20 cis
5,8,11,14);
oleic acid: cis 9-octadecanoic acid 18:1 (C:18
cis
9); palmitic acid; hexadecanoic acid; ibuprofen:
-methyl-4-(2-methylpropyl)benzeneacetic acid; and staurosporine were
obtained from Calbiochem. Elaidic acid: trans 9 octadecanoic acid 18:1 (C:18 trans
9), IBMX, ATP, HEPES,
inorganic and organic salts were from Sigma Chemical. Sylgard Isomer
184 was from Dow Corning. HEK-293 cells were obtained from ATCC. MEM,
Lipofectamine, and G418 were obtained from GIBCO. pcDNA3.1 was from
Invitrogen. Borosilicate glass (#7052) was obtained from Garner Glass.
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RESULTS |
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PKA and pH activation of human ClC-2 Cl channels.
Figure
1A
shows typical whole cell Cl
currents of nontransfected
HEK-293 cells in the basal state (left), with 5 µM
forskolin plus 20 µm IBMX (center), and with subsequent
reduction of bath pH from pH 7.35 to pH 6.5 (right). As
summarized in Fig. 1E, there was no significant effect of
forskolin plus IBMX or reduction of bath pH on Cl
currents of nontransfected cells. In contrast, Fig. 1B shows that typical whole cell Cl
currents of transfected cells
in the basal state (left) are increased after treatment with
forskolin plus IBMX (center) and further increased after
subsequent reduction of the pH of the bath solution from pH 7.35 to pH
6.5 (right). As summarized in Fig. 1, D and E, forskolin plus IBMX significantly increased
(P < 0.001) Cl
currents at
140 mV from
135 ± 14 pA to
569 ± 42 pA (n = 19). In
a separate set of experiments to test the effect of extracellular pH,
basal currents of
146 ± 35 pA were significantly increased (P < 0.001) to
499 ± 76 pA with forskolin plus
IBMX at pH 7.4, and these currents further significantly increased
(P < 0.001) to
811 ± 73 pA (n = 7) with subsequent reduction of bath pH to pH 6.5. Cl
currents of mock transfected cells were not increased by forskolin plus
IBMX (
66 ± 17 pA vs.
73 ± 34 pA, n = 3), not shown.
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Effect of pH on basal Cl currents.
Figure 1B, left, shows typical Cl
currents of
transfected cells in the basal state, and Fig. 1C, left,
shows currents after reduction of bath pH to pH 6.5 without prior
treatment with forskolin plus IBMX. As shown in the summarized data of
Fig. 1E, there was a small but significant increase
(P < 0.001) in Cl
currents at
140 mV
from
87 ± 13 pA to
301 ± 22 pA (n = 11) upon reduction of bath pH to pH 6.5 in the absence of stimulation with
forskolin plus IBMX. These small pH effects are in contrast to the
large effects of pH on forskolin plus IBMX activated channels (Fig. 1,
D and E). The effect of bath pH on
Cl
currents after treatment with PKA inhibitors was
therefore investigated to determine whether pH effects required prior activation.
Effect of pH with inhibition of PKA.
Figure 1C, center and right, show
typical Cl currents of cells treated with 0.4 µM mPKI
and forskolin plus IBMX and measured at bath pH 7.35 or pH 6.5, respectively. Treatment with forskolin plus IBMX was initiated at pH
7.35 and was continued upon reduction of pH. As summarized in Fig. 1,
D and E, 0.4 µM mPKI prevented activation by 5 µM forskolin plus 20 µM IBMX, and there was no significant increase
in Cl
currents upon reduction of pH when PKA was
inhibited by mPKI. In this expression system, endogenous or exogenous
activation of PKA is required for pH activation of ClC-2
Cl
channels.
Effect of sodium nitroprusside.
Sodium nitroprusside is an activator of guanylate cyclase. Ten
micromolar sodium nitroprusside increased Cl currents in
transfected cells at all tested holding potentials (Fig.
2A). As summarized in Fig.
2B, 10 µM sodium nitroprusside had no significant effect
on currents of nontransfected cells. Cl
currents measured
at
140 mV significantly (P < 0.01) increased from
124 ± 33 pA to
557 ± 60 pA (n = 4)
after treatment of transfected cells with 10 µM sodium nitroprusside.
Pretreatment of the cells with 0.4 µM mPKI prevented activation at
all potentials tested (Fig. 2, A and B). These
studies suggest that cGMP acting through PKA also activates ClC-2
Cl
channels.
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Ion selectivity of human ClC-2 Cl channels from
current measurements.
I-V curves for transfected cells treated with forskolin plus
IBMX were generated with symmetrical Cl
and with nearly
bi-ionic substitution of bath Cl
with test anions for
three cells and are shown in Fig.
3A. Ratios of slopes and
differences in reversal potentials (MATERIALS AND METHODS)
after changing from Cl
to test anions in the bath were
used to determine absolute and relative permeabilities, respectively.
Conductance ratios (absolute permeabilities) for the halides were
Br
(1.03 ± 0.02) = Cl
(1.0) > I
(0.68 ± 0.06); n = 3. Conductance ratios for the nonhalides were Cl
(1.0) > SCN
(0.85 ± 0.06) = NO3
(0.74 ± 0.22) > I
(0.68 + 0.06);
n = 3. SO42
(0.3 ± 0.05) gave
the lowest conductance ratio. The ratios of conductance are similar to
those reported by others (7, 42). The
relative permeabilities,
PX
/ PCl
for the halides were: I
(1.11 ± 0.04) > Cl
(1.0) = Br (1.0); n = 3. PI
/PCl
was significantly greater (P < 0.01) than for
Cl
. This is the same sequence for
PX
/PCl
that was previously reported for the native and recombinant rabbit channel (3, 21).
PX
/PCl
for the other anions anions were I
> NO3
= SCN
Br
. Figure
3B shows a portion of an I-V curve for a typical
cell that was expanded to more clearly show shifts in
reversal potentials for I
, Cl
, and
Br
.
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Effect of AA.
Figure 4, A and B,
respectively show Cl currents of transfected cells before
and after treatment with 1 µM AA. As shown in Fig. 4C, AA
increased Cl
currents at all holding potentials tested.
The increase in Cl
currents was dose dependent with a
K0.5 of 0.35 µM (Fig. 4D). As
summarized in Fig. 4E for current measurements at
140 mV, 1 µM AA significantly increased (P < 0.01)
Cl
currents from
106 ± 15 pA to
2,639 ± 389 pA (n = 6). Cl
currents of
nontransfected cells were not affected by 1 µM AA (Fig.
4E). Currents at
140 mV in mock transfected cells were also not affected by 1 µM AA (
34 ± 11 pA vs.
27 ± 6.4 pA, n = 3), not shown.
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Independence of PKA and AA effects.
Figure 4E shows that Cl currents of
mPKI-treated cells were also activated (P < 0.05) by 1 µM AA from
296 ± 23 pA to
2,311 ± 504 pA
(n = 4), a current level that was not significantly
different from that in the absence of mPKI (
2,639 ± 426 pA;
Fig. 4E). Thus AA activation of ClC-2 Cl
currents is independent of PKA action. Cl
currents at
140 mV of nontransfected cells were not affected by 1 µM AA (Fig.
4E) or 0.4 µM mPKI (
81.8 ± 43 pA,
n = 3), not shown.
Effect of PKC inhibition on AA effects.
PKC has been implicated in some studies of AA effects on ion channels
(23). AA activates PKC with a K0.5
of ~100 µM (28). Control currents at 140 mV of
251 ± 52 pA were not significantly different after addition of
10 nM of the PKC inhibitor staurosporine,
334 ± 102 pA
(n = 3). Subsequent treatment with 1 µM AA
significantly increased (P < 0.001) currents to
1,736 ± 134 pA (n = 3; data not shown), a level
not significantly different from the level of activation by 1 µM AA
in the presence or absence of mPKI (data not shown). Inhibition of PKC
and other protein kinases by staurosporine did not prevent activation
by AA, suggesting that PKC is not involved in AA activation of ClC-2.
Effect of ETYA.
ETYA is a nonmetabolized analog of AA that inhibits cyclooxygenase and
lipoxygenase (23). ETYA increased Cl
currents at all holding potentials tested (Fig.
5A). The effect of ETYA was
dose dependent with a K0.5 of 3 µM (Fig.
5B). Basal Cl
currents of transfected cells at
140 mV were significantly increased (P < 0.01) from
300 ± 87 pA (n = 16) to
3,594 ± 410 pA
(n = 4) with 10 µM ETYA (Fig. 5E), but
currents of nontransfected cells were not affected (Fig.
5E). These results suggest that the effect of AA (or ETYA
acting as an analog of AA) may be due to direct effects on the channel.
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Effect of ibuprofen and ebselen.
Ibuprofen is an inhibitor of cyclooxygenase 1 and 2 (27, 43, 46). As shown in Fig.
5C, ibuprofen activated Cl currents in
transfected cells at all holding potentials tested. The effect of
ibuprofen was dose dependent with a K0.5 of 300 µM (Fig. 5D). As summarized in Fig. 5E,
Cl
currents at
140 mV significantly increased
(P < 0.02) from
91 ± 12 pA (n = 16) to
518 ± 57 pA (n = 3) with 200 µM
ibuprofen and increased (P < 0.05) to
3,101 ± 676 pA (n = 3) with 400 µM ibuprofen. There was no
effect of ibuprofen on nontransfected cells (Fig. 5E).
Ebselen (PZ51) is a peroxide scavenger that is structurally and
chemically unrelated to the NSAIDS but that inhibits cyclooxygenase
and lipoxygenase (12, 19). One hundred
micromolar ebselen significantly increased (P < 0.01)
currents at
140 mV from
130 ± 32 pA to
1,485 ± 147 pA
(n = 3, not shown). This supports the view that
inhibition of AA metabolism pathways can lead to activation of ClC-2
Cl
channels.
Effect of other fatty acids.
K+ channels have been shown to be activated by AA, ETYA,
and other saturated fatty acids (32). Oleic acid (100 µM), a cis-saturated C:18 fatty acid, significantly
increased (P < 0.001) Cl currents at
140 mV from
300 ± 87 pA to
2,120 ± 310 pA
(n = 4), not shown. Elaidic acid (100 µM), a
trans-unsaturated C:18 fatty acid, significantly increased
(P < 0.01) currents from
195 ± 94 pA to
1,891 ± 433 pA (n = 4), not shown. Palmitic
acid (100 µM), a saturated C:16 fatty acid, did not affect currents
(n = 3), not shown. ClC-2 Cl
channels
exhibit the same pattern of activation with fatty acids as the TREK-1
K+ channel (32). Oleic acid is an activator of
PKC at these high concentrations (25), but elaidic acid
does not affect PKC (39). The activation of ClC-2 by both
cis- and trans-unsaturated C:18 fatty acids,
together with a lack of effect of staurosporine on AA activation,
strengthens the argument that PKC is not involved in the activation by
fatty acids.
Effect of intracellular Ca2+.
Ten micromolar ionomycin plus 2.8 mM Ca2+ failed to cause
an increase in Cl currents in either nontransfected or
transfected cells (n = 3), not shown. The lack of
Ca2+ dependence has been previously noted for ClC-2
expressed in oocytes (3, 7, 21,
38, 41).
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DISCUSSION |
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An HEK-293 cell line stably expressing human recombinant ClC-2
mRNA and function was used in patch-clamp studies to investigate the
effects of PKA activators and inhibitors, pH, and AA. The relative
permeability,
PX/PCl
, from reversal potentials were I
> Cl
= NO3
= SCN
Br
. The absolute permeability from conductance ratios was
Br
= Cl
= NO3
= SCN
> I
> SO42
. The channel was
activated by a combination of forskolin plus IBMX, and this activation
was inhibited by the cell-permeant form of the PKA inhibitor mPKI. The
channel exhibited activation by pH that was increased with forskolin
plus IBMX and abolished by mPKI. Sodium nitroprusside, an activator of
guanylate cyclase, was shown to be an activator, but Cl
current activation was inhibited by mPKI, suggesting crossover activation of PKA by cGMP. Increased intracellular Ca2+ was
without effect on human ClC-2 Cl
currents. AA at
submicromolar concentrations activated Cl
currents, and
this activation was not inhibited by mPKI or staurosporine. Inhibition
of cyclooxygenase with ibuprofen, or both cyclooxygenase and
lipoxygenase with ETYA or ebselen, mimicked the effect of AA.
Patch-clamp studies of transfected cells are widely used for studies of
the regulation of ion channels. HEK-293 cells exhibit a low intrinsic
Cl current and have been widely utilized for studies of
ion channels, including the ClC-2 Cl
channel
(31). Previous studies of human ClC-2 in HEK-293 cells have employed a transient expression system. The stably transfected cell line used in the present studies gave reproducible
Cl
currents in the basal state and after treatment with
various agents, facilitating comparisons between various treatments.
The Cl
currents in this cell line were activated by
treatment with forskolin plus IBMX, as expected from the presence of
numerous consensus phosphorylation sites on human ClC-2 and from
previous studies of the rabbit (3, 21,
41) and human (38) recombinant ClC-2 channels
reconstituted in planar lipid bilayers. Forskolin was combined with
IBMX to inhibit phosphodiesterase action. Previous attempts in IB3-1
clonal cell lines transfected with human recombinant ClC-2
Cl
channels failed to show
8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate plus
forskolin stimulated 36Cl
efflux
(36). Similar failures were reported when cAMP levels were
raised in Xenopus laevis oocytes expressing human ClC-2
under conditions sufficient to stimulate cystic fibrosis transmembrane conductance regulator (CFTR) (15). In the former study,
phosphodiesterase inhibitors were not used, and in the latter study,
the agents or conditions employed were not reported. Inhibitors of PKA
were employed in the present study to determine whether there was basal activation of the channels by endogenous PKA. The rationale for the use
of the PKA inhibitor in the present study was similar to that used in
recent studies of physiologically relevant cAMP stimulation of
Ca2+ channel
-subunits in both Xenopus laevis
oocytes and HEK-293 cells. The Ca2+ channel is fully
phosphorylated in the basal state (33, 34) in
both of these expression systems. It was, however, possible to study
reversible regulation of Ca2+ channels by PKA after first
inhibiting endogenous PKA with protein kinase inhibitors. The results
with the cell-permeant PKA inhibitor, mPKI, shows that endogenous basal
PKA activity is low in the HEK-293 cell line used here. The level of
endogenous activation of PKA in the basal state was not established in
other expression studies (7, 9,
31, 36, 42).
Block of PKA activation by mPKI did not affect subsequent AA
activation. This shows that AA activation is independent of PKA activation and that myristoylated PKI did not have deleterious effects
on expression of ClC-2 Cl currents. In contrast to the
lack of mPKI effect on AA stimulation, prior treatment of the cells
with mPKI in the presence of forskolin plus IBMX abolished
Cl
channel activation by reduced extracellular pH. In
other studies where only pH activation of ClC-2 currents have been
demonstrated (15, 36), one possibility is
that endogenous basal activation by PKA or other activators may have
been higher than in the present studies.
In our previous studies (3, 21), zero current
reversal potentials were obtained from single channel studies under
nearly bi-ionic conditions using the channel from native tissues or the recombinant channel expressed in oocytes and reconstituted into planar
lipid bilayers. The reversal potential measurements were then used in
the zero current form of the Goldman-Hodgkin-Katz equation (in Ref. 11)
to determine the permeability ratios, PX/PCl
(11), which were I
> Cl
Br
> NO3
(3, 21). In the present study, the relative
permeability ratios were identical to those obtained in bilayer
studies. The ratios of the slopes of the I-V curves
(conductance ratios) gave ion selectivity as absolute ionic
permeabilities (11) of Cl
= Br
= NO3
SCN
> I
> SO42
. This sequence is
similar to that found for rat ClC-2 by others (42). The
difference between permeability ratios defined by currents and
permeability ratios determined by zero current reversal potentials is
not unexpected. To quote Hille, it is "... a quite common
finding that absolute permeabilities determined from conductances or
sizes of currents do not agree with permeability ratios determined from
reversal potentials" (11). This is because the two
different measurements depend on different properties of the pore.
Absolute permeabilities obtained from measurements of currents are
sensitive to block and partial saturation, whereas those obtained from
zero current reversal potentials are not.
AA effects on ion channels are sometimes mediated through the activation of PKC (23). PKC inhibition of recombinant rat ClC-2 currents in neurons has been demonstrated (40). ETYA inhibits PKC in vitro (28) and ETYA prevents activation of PKC in vivo by inhibition of the lipoxygenase pathway mediators that activate PKC in vivo (8, 30).
The cis-unsaturated C:18 fatty acid, oleic acid, activated ClC-2 and also activates PKC isozymes at high concentrations (K0.5 50 µM) (16, 25). The trans C:18 unsaturated fatty acid, elaidic acid, and the saturated C:16 fatty acid, palmitic acid, do not activate PKC (39). The activation of ClC-2 by elaidic acid suggests that PKC is not involved in fatty acid activation of ClC-2. In intact cells, PKC pathways can be activated by metabolites of palmitic acid, but these actions occur at the transcriptional level and require long periods of incubation. AA does not act through that pathway (35). Staurosporine did not affect activation of ClC-2 by AA. A similar lack of involvement of PKC in AA activation of a K+ channel was demonstrated using staurosporine (4). ETYA activates ClC-2 (present study) and inhibits PKC (28). Elaidic acid (present study) activates ClC-2 and is without effect on PKC (39). Staurosporine inhibits PKC without affecting AA activation. Taken together, these results demonstrate that activation of ClC-2 by fatty acids does not involve PKC.
The TREK-1 K+ channel responds similarly to AA, ETYA, and oleic, elaidic, and palmitic acids (32). This suggests a similar mechanism of action of fatty acids in activation of these two types of channels. Patel et al. (32) have suggested that activation of the TREK-1 K+ channel by fatty acids involves preferential insertion of negatively charged amphiphiles into the external leaflet of the bilayer, causing a change in the structure of the membrane. The structural change in the membrane activates the channel through a charged sensor region on the K+ channel. It is tempting to speculate that fatty acid activation of ClC-2 might occur through a similar mechanism.
AA and inhibitors of AA metabolism have been reported to inhibit or activate a large variety of ion channels (5, 13, 14, 20, 23, 24, 29, 32). AA may either bind directly to ion channels (23, 29) or indirectly affect ion channels through local modification of membrane structure (20, 32, 37). When the metabolism of endogenous AA was blocked by ETYA (23) or the cyclooxygenase inhibitor ibuprofen (27, 43, 46), the channel was activated, suggesting an indirect action of ibuprofen on channel activity.
An alternate possibility is that ibuprofen binds directly to the
channel. NSAIDS, including flufenamic acid, mefenamic acid, and
niflumic acid (but not ibuprofen) inhibit nonspecific cation channels
(6). At least one compound that is chemically related to
NSAIDS, 5-nitro-2-(3-phenylpropylamino)benzoic acid, potently inhibits
Cl channels (45). All NSAIDS are
structurally related because they fit into the binding groove of
cyclooxygenase isozymes and exclude substrate from binding
(18, 22). A different class of inhibitor of
cyclooxygenase and lipoxygenase unrelated in structure to the NSAIDS
was required to test the hypothesis that inhibition of AA metabolism
was responsible for ClC-2 activation by ibuprofen. Cyclooxygenase
requires hydroperoxides for initiation of the cyclooxygenase reaction.
Peroxide scavengers such as glutathione peroxidase block the activity
of cyclooxygenase 1 and 2 (19) and also block lipoxygenase (12, 44). Ebselen is a glutathione peroxide
mimetic that has been previously shown to block cyclooxygenase and
lipoxygenase activity by reducing the reactive oxygen species required
for the activity of these enzymes (10). As such, ebeselen
is not an NSAID, acts by a different mechanism than ibuprofen, and
inhibits the metabolism of AA. In the present studies, ebselen
increased ClC-2 Cl
channel activity, strengthening the
argument that inhibition of AA metabolism will affect ClC-2 and other
channels (23) such as K+ channels,
(32) which are affected by AA and other fatty acids.
The activating effects of fatty acids are in sharp contrast to the
inhibitory effect of AA on the 45-pS outward rectifier of normal airway
and T84 cells (13, 14) and the inhibitory effect of ibuprofen on CFTR (5). Given that the ClC-2
channel is present in the lung and has been suggested to be an
alternative pathway for Cl transport in the lung
(1, 26, 38), this channel,
unlike CFTR (5) or the outward rectifier (13,
14), may be activated during ibuprofen therapy of cystic
fibrosis patients (17). AA activation of ClC-2
Cl
channels may have other larger implications in NSAID
treatment. The present results suggest that NSAID effects on ion
channels may have physiological and pathophysiological consequences in addition to effects on pain and inflammation.
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ACKNOWLEDGEMENTS |
---|
We thank Dr. John W. Hanrahan and Dr. Robert Bridges for helpful discussions regarding ion selectivity.
![]() |
FOOTNOTES |
---|
This work was supported by National Institute of Health Grants DK-43816 and HL-58399.
Address for reprint requests and other correspondence: J. Cuppoletti, Dept. of Molecular and Cellular Physiology, Univ. of Cincinnati College of Medicine, PO Box 670576, 231 Bethesda Ave., Cincinnati, OH 45267-0576 (E-mail: John.Cuppoletti{at}uc.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. §1734 solely to indicate this fact.
Received 20 September 1999; accepted in final form 26 January 2000.
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