Modulation of K+ channels by
arachidonic acid in T84 cells. II. Activation of a
Ca2+-independent
K+ channel
Daniel C.
Devor and
Raymond A.
Frizzell
Department of Cell Biology and Physiology, University of
Pittsburgh, Pittsburgh, Pennsylvania 15261
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ABSTRACT |
We used
single-channel recording techniques to identify and characterize a
large-conductance,
Ca2+-independent
K+ channel in the colonic
secretory cell line T84. In symmetric potassium gluconate, this channel
had a linear current-voltage relationship with a single-channel
conductance of 161 pS. Channel open probability
(Po) was
increased at depolarizing potentials. Partial substitution of bath
K+ with
Na+ indicated a permeability ratio
of K+ to
Na+ of 25:1. Channel
Po was reduced by
extracellular Ba2+. Event-duration
analysis suggested a linear kinetic model for channel gating having a
single open state and three closed states: C3
C2
C1
O.
Arachidonic acid (AA) increased the
Po of the
channel, with an apparent stimulatory constant
(Ks)
of 1.39 µM. Neither channel open time (O) nor the fast closed time
(C1) was affected by AA. In
contrast, AA dramatically reduced mean closed time by decreasing both
C3 and
C2. The
cis-unsaturated fatty acid linoleate increased Po
also, whereas the saturated fatty acid myristate and the
trans-unsaturated fatty acid elaidate
did not affect
Po. This channel
is activated also by negative pressure applied to the pipette during
inside-out recording. Thus we determined the effect of the
stretch-activated channel blockers amiloride and Gd3+ on the
K+ channel after activation by AA.
Amiloride (2 mM) on the extracellular side reduced single-channel
amplitude in a voltage-dependent manner, whereas
Gd3+ (100 µM) had no effect on
channel activity. Activation of this K+ channel may be important during
stimulation of Cl
secretion
by agonists that use AA as a second messenger (e.g., vasoactive
intestinal polypeptide, adenosine) or during the volume regulatory
response to cell swelling.
potassium channel; chloride secretion; fatty acids; intestine
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INTRODUCTION |
INTESTINAL CHLORIDE SECRETION is modulated by changes
in cellular adenosine 3',5'-cyclic monophosphate (cAMP)
[e.g., vasoactive intestinal polypeptide (VIP)], guanosine
3',5'-cyclic monophosphate (cGMP) (e.g., heat-stable
enterotoxin of Escherichia coli),
and Ca2+ (e.g., acetylcholine) in
response to secretory agonists (3, 4, 7, 24). Adenosine is released by
invading neutrophils and eosinophils during an inflammatory response
and thus may act as a stimulus for diarrhea in intestinal inflammatory
diseases (31, 32, 41). Although adenosine was shown to be a potent Cl
secretagogue, changes in
these classical second messengers were not detected (6, 12, 32).
However, adenosine was shown to increase cellular arachidonic acid (AA)
levels in T84 cells, and its secretory effects could be blocked by
inhibitors of both phospholipase
A2 and 1,2-diacylglycerol (DAG)
lipase (5, 6); both enzymes catalyze AA generation (36). More recently,
VIP (cAMP) and E. coli heat-stable
enterotoxin (cGMP) have been shown to increase AA in T84 cells (5), in
addition to their effects on cAMP production. Inhibition of DAG lipase
resulted in a partial inhibition of these
Cl
secretory responses (5).
These results suggest that AA may be an important second messenger in
modulating Cl
secretion in
intestinal diarrheal disease. However, the apical and basolateral ion
channels responsible for carrying the secretory current in response to
AA have not been identified.
AA is a well-known modulator of
K+,
Na+,
Ca2+, and
Cl
channels in a variety of
tissues (33, 38). Importantly, fatty acids have been shown to modulate
Cl
channels in secretory
epithelia. AA was shown to inhibit an outwardly rectifying
Cl
channel in airway
epithelia (2, 17) as well as a volume-sensitive Cl
conductance in
intestinal epithelium (23). Also, cAMP-mediated Cl
secretion is inhibited
by AA in airway epithelia (34). However, these inhibitory effects are
incongruent with the secretory response associated with elevated AA. In
our companion paper (10), we demonstrate that the basolateral membrane
K+ channel activated by
Ca2+-dependent agonists
(KCa) is potently inhibited by
AA. To our knowledge, neither an intestinal
K+ conductance
(GK) nor
Cl
conductance has been
shown to be upregulated directly by AA, as would be required if
elevated AA stimulates Cl
secretion under some conditions. During the course of our studies on
the role of AA in regulating KCa,
we identified a large-conductance K+ channel that is activated by
AA. We have characterized this channel's K+/Na+
selectivity, blocker sensitivity, and its regulation by fatty acids. We
speculate that this channel may be important in carrying K+ across the basolateral membrane
during Cl
secretory
responses in which AA serves as a stimulatory second messenger.
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METHODS |
Cell culture.
T84 cells were grown in Dulbecco's modified Eagle's medium and Ham's
F-12 (1:1) supplemented with 15 mM
N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), 14 mM NaHCO3, and
10% fetal bovine serum. The cells were incubated in a humidified
atmosphere containing 5% CO2 at 37°C. Patch-clamp experiments were performed on single cells plated onto glass coverslips 18-48 h before use.
Solutions.
During inside-out patch-clamp recordings, the bath contained (in mM)
145 potassium gluconate, 5 KCl, 1 MgCl2, 1 ethylene
glycol-bis(
-aminoethyl ether)-N,N,N',N'-tetraacetic
acid (EGTA), and 10 HEPES (pH adjusted to 7.2 with KOH). A free
Ca2+ concentration of <10 nM was
chosen for these experiments to eliminate the activity of the
KCa we previously described in
these cells (9). The pipette solution contained (in mM) 140 potassium
gluconate, 5 KCl, 1 CaCl2, 1 MgCl2, and 10 HEPES (pH adjusted
to 7.2 with KOH). For outside-out recordings, the bath contained 1 mM
CaCl2 in the absence of any added
EGTA, whereas the pipette solution Ca2+ was buffered to <10 nM with
EGTA (1 mM).
Single-channel recording.
Single-channel currents were recorded using a List EPC-7 amplifier
(Medical Systems) and recorded on videotape for later analysis as
described previously (9). Pipettes were fabricated from KG-12 glass
(Willmad Glass). All recordings were done at a holding voltage of
100 mV unless otherwise noted. The voltage is referenced to the
extracellular compartment, as is the standard method for membrane
potentials. Inward currents are defined as the movement of positive
charge from the extracellular compartment to the intracellular compartment and are presented as downward deflections from baseline in
all recording configurations.
Single-channel analysis was performed on records sampled at 8 kHz after
low-pass filtering at 2 kHz. The average length of record analyzed to
determine channel open probability
(Po) was 154 ± 4 s (n = 78).
Po was
calculated, using Biopatch software (version 3.11; Molecular Kinetics),
from the mean total current (I)
divided by the single-channel current amplitude
(i) and the maximum number of
channels observed in a patch (N),
such that Po = I/iN.
The i was determined from the
amplitude histogram of the current record, and
N was determined from a visual
inspection of the record after activation of the channel by a maximal
concentration of AA (3-10 µM).
For determining mean open and closed times, an open-closed transition
was considered valid if it remained in the state for at least two
sample periods (250 µs). Event-duration histograms for both the open
time (
o) and fast closed time
(
c1) were constructed by
binning at the sampling rate (125 µs) from 1.0 to 6-15 ms (i.e., >4 times the time constant). To determine the burst duration
(
burst) and the long closed
times (
c2 and
c3), the channel recordings were filtered at 100 Hz and sampled at 500 Hz to eliminate the contribution of
c1 from the
recordings. An open-closed transition was considered valid if it
remained in the state for at least two sample periods (4 ms). For
burst, the data were binned at 4-ms intervals for construction of the event-duration histogram. Because of the limited number of long closed events under control conditions, an exponential fit of the data could not be achieved from a
single record. Therefore, the event-duration histogram for
c2 and
c3 was constructed after the
concatenation of eight separate recordings with a
Po >3%
(average Po = 0.08). The additional five single-channel recordings had
Po
<3% such that these recordings would contribute very few long
closed events. The event-duration histogram was constructed by binning
at 10-ms intervals. These event-duration histograms were then fit to
exponential functions to determine the open
(
o) and closed
(
c) time constants.
Chemicals.
All fatty acids were obtained from Sigma Chemical, made as
>1,000-fold stock solutions in dimethyl sulfoxide (DMSO), and stored under N2 at
80°C. The
fatty acids were dissolved to the final working concentration just
before use, and all solutions were continuously bubbled with
N2 during perfusion through the
patch-clamp chamber. Charybdotoxin (CTX) was made as a 10 µM stock
solution in our bath solution for outside-out recording.
4-Aminopyridine (4-AP), quinine, glibenclamide, and
trans-6-cyano-4-(N-ethylsulfonyl-N-methylamino)-3-hydroxy-2,2-dimethyl-chromane (293B) were made as a 1,000-fold stock solution in DMSO. 293B was a
generous gift from Dr. Rainer Greger (Albert-Ludwigs-Universtat, Freiberg, Germany). Indomethacin and nordihydroguaiaretic acid (NDGA)
were obtained from Biomol. Cell culture medium was obtained from GIBCO.
Data analysis.
All data are presented as means ± SE, where
n indicates the number of experiments.
Statistical analysis was performed using the Student's
t-test. A value of
P < 0.05 was considered
statistically significant.
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RESULTS |
After excision of membrane patches from T84 cells into symmetric
K+ in the absence of bath
Ca2+ (<10 nM), a
large-conductance channel was observed in ~10-15% of
recordings. Single-channel currents for one such patch are shown in
Fig. 1 in the absence of AA
(A). The average current-voltage (I-V)
relationship for eight recordings is shown in Fig.
1B. The channel displayed a linear
I-V
relation with an average conductance of 161 ± 3 pS
(n = 8). The
K+/Na+
selectivity of this channel was assessed by equimolar substitution of
Na+ for
K+ in the bath solution;
K+-to-Na+
permeability ratio
(PK/PNa)
was calculated from the Goldman-Hodgkin-Katz relation. Substitution of
100 meq Na+ for
K+ resulted in a shift in the
reversal potential of 26 ± 1 mV
(n = 3). A shift of 27 mV is predicted
for a perfectly K+-selective
electrode. These data yield a calculated
K+/Na+
selectivity of ~25:1. As is apparent from Fig. 1, this channel exhibited modest voltage dependence, with the
Po increasing at depolarizing potentials. In five patches, the
Po at
100
mV was 0.17 ± 0.02, and this increased to 0.59 ± 0.05 at +100 mV.

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Fig. 1.
A: single-channel recording from an
excised, inside-out patch at several holding potentials
(Ei-o).
Recording was done in absence of arachidonic acid. Arrows indicate
closed state of channel. Pipette and bath solutions were symmetric
potassium gluconate. B: average
current-voltage relationship for excised patches in either symmetric
150 meq K+ ( ) or after
equimolar substitution of 100 meq bath
K+ with
Na+ ( ). Anion was gluconate
throughout.
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Effect of AA on
K+ channel
activity.
The effect of AA on this large-conductance,
Ca2+-independent
K+ channel is shown in Fig.
2A. Under
control conditions, the channel opened infrequently. However,
superfusion of AA (3 µM) resulted in a rapid increase in channel
activity. The average
concentration-Po curve is shown in Fig. 2B. These data
were fit to a Michaelis-Menten function with an apparent stimulatory
constant (Ks) of
1.39 µM. At a maximally effective concentration (10 µM), AA
increased the Po
from 0.04 ± 0.01 (n = 13) to 0.66 ± 0.03 (n = 4). On the basis of
this AA-dependent activation, this channel will be referred to as
KAA throughout this paper.

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Fig. 2.
A: effect of arachidonic acid (3 µM)
on K+ channel activity in an
excised, inside-out patch. Data are from a continuous recording.
Arachidonic acid entered patch-clamp chamber at asterisk. Patch was
voltage clamped to 100 mV in symmetric potassium gluconate.
Arrows indicate closed state of channel.
B: average open probability
(Po)-[arachidonic
acid] relation. Each point is average of at least 4 experiments.
Data were fitted to a Michaelis-Menten function with an apparent
stimulatory constant
(Ks) of 1.39 µM.
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As is apparent in Figs. 1 and 2,
KAA shows burst-type openings
punctuated by long-lived closed states. To determined the kinetic state
of the channel affected by AA, we constructed open and closed time
event-duration histograms, as shown for one patch in Fig. 3,
A-C.
In the absence of AA, the open time
(
o) is defined by a single
exponential (2.4 ms), indicating a single open state (Fig.
3A). In 13 patches,
o averaged 2.3 ± 0.2 ms.
The closed time (
c) of
KAA was described by three states:
a short-lived closed state
(
c1) and two long-lived
closed states (
c2 and
c3). As illustrated for one
patch (Fig. 3B), the short-lived
closed state was defined by a single exponential
(
c1 = 1.0 ms).1
In 13 patches,
c1 averaged 1.3 ± 0.2 ms. Because of the limited number of long closed events in
any one single-channel recording, we are unable to determine
c2 and
c3 from a single patch.
Therefore, we concatenated eight recordings that had a
Po of >3%
(average Po = 0.08; see METHODS). These data were
fit by two exponentials (Fig. 3C),
where
c2 = 14.9 ms and
c3 = 106 ms. These results indicate that KAA is kinetically
described by one open and three closed states in the absence of AA (see
DISCUSSION).

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Fig. 3.
Open- and closed-time event-duration histograms for arachidonic acid
(AA)-dependent K+ channel
(KAA) in absence
(A-C) and presence
(D and
E) of AA (3 µM). In absence of AA,
open time ( o;
A) was fit to a single exponential
(2.4 ms), whereas closed time was fit to 3 exponentials
( c1,
B;
c2,
c3,
C). Addition of AA did not affect
either o
(D) or
c1
(E). Because of a limited number of
long closed events, c2 and
c3 could not be determined in
presence of AA. A, B, D,
and E were all determined from same patch. For
determinations of o and
c1, data were filtered at 2 kHz, sampled at 8 kHz, and binned in 125-µs intervals from 1 to 15 ms
( o) or 1 to 6 ms
( c1). For determining
c2 and
c3, 8 control recordings were
concatenated after filtering data at 100 Hz and sampling at 500 Hz.
Data were binned at 10-ms intervals and fit to a multiexponential
function. In all event-duration analysis, to be considered a valid
event, channel needed to dwell in state to which it was moving for 2 sample periods (either 250 µs or 4 ms).
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The effect of AA on the open and closed times for one patch is
illustrated in Fig. 3, D and
E. Addition of 3 µM AA had no effect
on the
o (Fig.
3D) of
KAA (2.6 ms). In 11 experiments, the
o averaged 2.2 ± 0.2 ms
after activation by AA (3 µM), which is not different from control.
In contrast, AA induced a concentration-dependent decrease in mean
closed time from 266 ± 125 ms in control solutions to 2.6 ± 1.0 ms in the presence of 3 µM AA (n = 11; P < 0.05). As shown for one
patch in Fig. 3E,
c1 was independent of AA
concentration (1.2 ms). In 11 patches,
c1 averaged 1.5 ± 0.1 ms,
which is not different from control. Because of the limited number of
long closed events in the presence of AA, we are unable to determine
c2 and
c3 in these experiments. These
data demonstrate that AA increases the
Po of
KAA by reducing the long closed
times (
c2 and
c3) of the channel, thereby
increasing the channel opening rate (see
DISCUSSION).
The effect of AA on burst duration
(
burst) was determined after
filtering of the data at 100 Hz to eliminate the fast closed events
(
c1) that punctuate the open
channel burst (see METHODS). In one
patch,
burst was 16.7 ms (Fig.
4A), and
this increased to 35.2 ms in the presence of 3 µM AA (Fig.
4B). In eight experiments,
burst averaged 19.8 ± 4.3 ms, and this was increased to 46.7 ± 4.3 ms
(P < 0.01) in the presence of AA (3 µM).

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Fig. 4.
Burst event-duration histogram in absence
(A) and presence
(B) of AA (3 µM). Both control and
AA histograms were fit to a single exponential, where
burst was 16.7 and 35.2 ms,
respectively. Both histograms were generated from a single patch. Data
were filtered at 100 Hz and sampled at 500 Hz to eliminate contribution
of c1 from exponential fit. To
be considered a valid open-closed transition, channel needed to reside
in a given state for 2 sample periods (4 ms). Data were binned in 4-ms
intervals between 10 and 160 ms.
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Effect of cyclooxygenase, lipoxygenase, and cytochrome P-450
inhibitors.
AA is rapidly metabolized by the cyclooxygenase-, lipoxygenase-, and
cytochrome P-450-dependent oxidative
pathways. Thus we determined whether inhibitors of these pathways would
alter the ability of AA to activate
KAA. Neither phenidone (250 µM),
an inhibitor of both cyclooxygenase and lipoxygenase, a combination of
cyclooxygenase (indomethacin; 1 µM) and lipoxygenase (NDGA; 2 µM)
inhibitors, nor clotrimazole (1 µM), an inhibitor of cytochrome P-450, altered the ability of AA to
active the channel (data not shown). Thus these oxidative metabolites
are not responsible for the increased gating observed.
Effects of additional fatty acids.
We next determined whether the observed stimulatory effect of AA on
KAA was specific for AA or whether
additional fatty acids would also modulate its activity. For these
experiments, we used an additional
cis-unsaturated fatty acid, linoleic
acid (C18; cis,cis-
9,
12),
the trans-unsaturated fatty acid
elaidic acid (C18;
trans-
9),
and a saturated fatty acid, myristic acid
(C14). The results of one
experiment are shown in Fig.
5A.
Elaidic acid (3 µM) failed to increase
Po above control
levels. The subsequent addition of linoleic acid (3 µM) induced a
distinct increase in channel
Po, and the
further addition of AA (3 µM) resulted in an additional activation.
The data for these fatty acids plus myristic acid are summarized in
Fig. 5B. Similar to elaidic acid,
myristic acid failed to activate
KAA, whereas linoleic acid was
~40% as effective as AA in activating the channel.

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Fig. 5.
A: effect of elaidic, linoleic, and
arachidonic acids (all 3 µM) on
KAA in an excised, inside-out
patch. Patch was voltage clamped to 100 mV in symmetric
potassium gluconate. Arrows indicate closed state of channel. All
traces are from a single recording. B:
average effects of myristic (Myr), elaidic (Ela), linoleic (Lin), and
arachidonic (AA) acids on
Po of
KAA. Each panel shows average
response for n = 3 or 4 patches to a
given fatty acid followed by AA. Only linoleic acid and AA induced a
significant increase in
Po. Cont,
control. * P < 0.05.
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Effects of K+
channel blockers.
Although AA has been implicated in modulating
Cl
secretion in T84 cells,
no blocker pharmacology has been defined for the GK that
participates in this secretory response. Because of the very low
Po of this
channel under control conditions, all of our blocker studies were
carried out in the presence of 3 µM AA to activate the channel. In
initial experiments, we determined whether Ba2+ would block
KAA from the cytoplasmic side in
an inside-out patch. The results of one experiment are shown in Fig.
6. Initially, the patch voltage was held at
+80 mV. Application of Ba2+ (1 mM)
to the inside of the channel resulted in a nearly complete inhibition
of activity, which was subsequently relieved by changing the clamp
potential to
80 mV. This voltage-dependent block is typical for
Ba2+ inhibition of
K+ channel currents (47).

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Fig. 6.
Effect of Ba2+ (1 mM) on
KAA activity when applied to
cytoplasmic side of channel in an inside-out patch. Patch was voltage
clamped (Vc) to
either +80 or 80 mV in symmetric potassium gluconate.
Ba2+ produced a nearly complete
inhibition of channel activity at +80 mV. However, clamping patch to
80 mV relieved this block. Data are from a continuous recording.
Arrows indicate closed state of channel at both holding potentials.
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To relate our findings to results from intact epithelia, it is more
relevant to determine whether compounds that block
K+ channels in other systems will
inhibit KAA from the outside of the membrane. Therefore, we determined the effects of several blockers
during outside-out recordings. For these studies, AA was used to
activate the channel similar to our inside-out recordings (data not
shown). This indicates that KAA is
activated by AA from the outside of the membrane also. The effect of
Ba2+ (3 mM) on
KAA in an outside-out patch is
shown in Fig. 7.
Ba2+ induced a voltage-dependent
block of KAA from the
extracellular side, inhibiting the channel at
100 mV while
having no apparent effect at +60 mV. In four patches, 3 mM
Ba2+ reduced mean current
(I) by 90 ± 1% at
100 mV
(P < 0.01). Although cytoplasmic
Ba2+ blocked
KAA by inducing a long-lived
closed state (Fig. 6), the inhibition of
KAA by extracellular
Ba2+ was accompanied by an
apparent reduction in single-channel amplitude (i; Fig. 7), although this could not
be clearly resolved at this high concentration of
Ba2+. Therefore, we determined the
effect of 1 mM Ba2+ in three
additional patches. Ba2+ (1 mM)
reduced Po from
0.54 ± 0.02 to 0.25 ± 0.02 at
100 mV (n = 3), and this was accompanied by
an apparent reduction in i from 17.6 ± 0.6 to 14.9 ± 1.0 pA (n = 3;
P < 0.05). These results suggest
that Ba2+ blocks
KAA by interacting with two
distinct binding sites from the intra- and extracellular side of the
channel.

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Fig. 7.
Effect of Ba2+ (3 mM) on
KAA activity when applied to
extracellular side of channel in an outside-out patch. Patch was
voltage clamped
(Vc) to either
100 mV (top) or +60 mV
(bottom) in symmetric potassium
gluconate. In this patch, Ba2+
reduced mean current by 91% at 100 mV. Voltage clamping to +60
mV relieved Ba2+-induced block.
Arrows indicate closed state of channel.
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In addition to Ba2+, we evaluated
the effects of tetraethylammonium (TEA; 10 mM), CTX (50 nM), 293B (30 µM), 4-AP (4 mM), glibenclamide (300 µM), and quinine (300 µM) on
channel activity in outside-out patches. TEA induced a small,
voltage-dependent reduction in i. At
100 mV, i was reduced 21.8 ± 0.2% (P < 0.01),
whereas at +100 mV, this inhibition was 7.2 ± 1.1%
(n = 4;
P < 0.01). We previously demonstrated that CTX was a potent blocker of KCa
(inhibition constant = 3.5 nM) in both single-channel and Ussing
chamber studies (9, 11). In contrast to this property of
KCa, CTX (50 nM) failed to
inhibit KAA (data not shown).
Lohrmann et al. (28) characterized a novel inhibitor of the
cAMP-dependent K+ channel in
rabbit colon, 293B. We recently demonstrated that this
compound inhibited the cAMP-mediated
Cl
secretion across T84
monolayers while not affecting
Cl
secretion dependent on
KCa (11). In three outside-out
patches, 293B (30 µM) failed to inhibit
KAA, suggesting it is not the
cAMP-activated K+ channel.
Additionally, 4-AP (4 mM; n = 2),
glibenclamide (300 µM; n = 3), and
quinine (300 µM; n = 5)
all failed to inhibit channel activity.
Effect of membrane stretch on
K+ channel
activity.
During the course of our studies, we observed that negative pressure
applied to the pipette during inside-out recordings resulted in the
activation of a large-conductance channel that appeared to have
identical characteristics to the channel activated by AA (Fig.
8, top).
Therefore, we wished to determine whether this stretch-activated
channel was in fact the same as the AA-dependent K+ channel we have characterized.
Application of a submaximal concentration of AA (2 µM) to the same
patch that was shown to possess the stretch-activated channel resulted
in the activation of KAA (Fig. 8,
bottom), suggesting they are the
same channel. In the presence of AA, application of negative pressure
resulted in the further activation of the same channel rather than an
additional conductance state. This result demonstrates that both AA and
stretch are capable of activating this large-conductance,
Ca2+-independent
K+ channel.

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Fig. 8.
Top: effect of negative pressure
applied to pipette (solid line) on
K+ channel activity in an excised,
inside-out patch. Bottom: after
activation of KAA with AA (2 µM), negative pressure (solid line) applied to pipette resulted in a
further activation of KAA. Data
are from a single recording. Patch was voltage clamped to 100 mV
in symmetric potassium gluconate. Arrows indicate closed state of
channel.
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Effect of stretch-activated channel blockers on
KAA.
The above results led us to determine whether the known inhibitors of
stretch-activated ion channels, amiloride (25, 44) and
Gd3+ (49), would block
KAA in excised, outside-out
patches. For these experiments, we used AA to first activate the
channel. Gd3+ (100 µM) had no
effect on KAA
(n = 3; data not shown). In contrast, amiloride induced an apparent reduction in
i at both +100 and
100 mV. The
effect of 2 mM amiloride on KAA in
an outside-out patch is shown in Fig. 9. In
this patch, i was reduced from 15.2 to
7.0 pA at +100 mV and from 17.0 to 3.6 pA at
100 mV. In four patches, amiloride (2 mM) reduced i at
100 mV from 18.5 ± 0.6 to 3.6 ± 0.1 pA
(P < 0.001). This inhibition of
i resulted in an 80 ± 3.5%
reduction in I with no apparent
reduction in channel Po (control = 0.43 ± 0.04; amiloride = 0.43 ± 0.06). These results suggest
that amiloride induces a voltage-dependent reduction in i of
KAA. Unfortunately, we were not
able to routinely maintain patch seal integrity at positive voltages,
so the voltage dependence of this inhibition could not be
quantitatively determined.

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Fig. 9.
Effect of amiloride (2 mM; right) on
KAA activity when applied to
extracellular side of channel in an outside-out patch. Patch was
voltage clamped to either +100 mV
(top) or 100 mV
(bottom) in symmetric potassium
gluconate. In this patch, amiloride reduced single-channel current
(i) at +100 mV from 15.2 to 7.0 pA
(A) while reducing
i from 17.0 to 3.6 pA at 100 mV
(B). Arrows indicate closed state of
channel.
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DISCUSSION |
We have characterized a large-conductance (160 pS), voltage-dependent,
highly K+-selective channel from
the colonic cell line T84. We demonstrate that this channel gates
independently of changes in cytoplasmic Ca2+. However, we cannot rule out
the possibility that Ca2+ might
modulate this channel's activity. Our experiments were done in a
nominally free Ca2+ solution to
inhibit the activity of the inwardly rectifying
KCa that we previously described
(9). Because KCa is observed in >90% of all patches, we could not obtain patches in which
KAA was present in the absence of
KCa. Several investigators have described a "maxi" K+
channel from the surface cells of intestinal epithelium (22, 35, 46).
Although these channels were inhibited by CTX, the channel described
here is not. Large-conductance (130-190 pS) K+ channels have also been
described in crypts from rabbit distal colon (8, 29). Similar to our
findings, the 190-pS channel described by Burckhardt and Gogelein (8)
from distal colonic crypts was observed in only 10% of patches and
exhibited a similar blocker pharmacology; the channel was not sensitive
to block by CTX, although extracellular
Ba2+ did inhibit the channel. Thus
a channel in native epithelium studied in vitro expresses many of the
same characteristics as described here.
Mechanism of AA-induced activation.
Our results demonstrate that the activation of
KAA by AA is not due to an
oxidative metabolite of AA; neither phenidone, indomethacin, NDGA, nor
clotrimazole inhibited the AA-induced activation. Rather, we speculate
that AA itself activates KAA. In
addition, the levels of AA required to activate
KAA
(Ks = 1.4 µM)
are likely to be physiologically relevant, since both cyclooxygenase
and lipoxygenase have Michaelis constants of 3-5 µM for AA
oxidation in vitro (36).
Based on event-duration analysis data, we demonstrate that
KAA is minimally described by a
single open state (O), a fast closed state
(C1), and two long-lived closed
states (C2,
C3). We predict a linear state
diagram of the form
The rate constant for the
O
C1 transition
(k0,1) can be
directly determined from the inverse of the time constant
(1/
o = 435 s
1). An initial
approximation of the additional rate constants was obtained from their
respective time constants. Two of these can be reliably approximated.
The opening rate
k1,0 (769 s
1) can be determined
from
c1, since this transition
constitutes the majority of closed events. This is especially true in
the presence of AA, where
Po is defined by
the open time divided by the open plus fast closed times
(
o/
o +
c1).
c1 is independent of AA
concentration. In addition, we can approximate the
C1
C2 transition rate from the burst duration analysis. In this case, C1 was eliminated by filtering the
data at a low frequency such that the transition rate of interest,
O
C2, can be determined from 1/
burst (50.5 s
1). Using channel
simulation software (Biopatch, version 3.11), we determined that these
first approximations did not reliably predict channel behavior under
control conditions; these rate constants predicted a
Po of 0.35 (the
channel records used to determine
c2 and
c3 had a
Po of 0.08). With
k0,1,
k1,0, and k1,2 held
constant, the additional rate constants were determined based on their
ability to reliably predict the observed channel behavior. The rate
constants shown above predict a
Po of 0.08, a
o of 2.6 ms, and
c1,
c2, and
c3 of 1.3, 15.8, and 200 ms, respectively. Eliminating events shorter than 4 ms (3 times
c1) from the simulated data
indicated a
burst of 31 ms.
Thus this kinetic scheme reliably predicts the observed channel
behavior.
We used this kinetic scheme to predict which state was being affected
by AA. If AA interacts with C3 to
increase the opening rate
k3,2, then we can
approximate the AA-dependent on-rate
(Kd = koff/kon)
at 18 µM
1 · s
1.
At 10 µM AA, this would predict a
Po of 0.38. Similarly, with the assumption that AA interacts with
C2 to increase
k2,1 with a
predicted on-rate of 36 µM
1 · s
1,
our model predicts a
Po of 0.42 at 10 µM AA. Neither of these accurately reflects the observed gating of
KAA. Because AA does not affect
C1 or the open state of the
channel, this suggests that AA interacts with both
C3 and
C2. Using our model, we evaluated this possibility. If AA induced a 10-fold increase in both
k3,2 and
k2,1, this would
predict a Po of
0.61, similar to that observed at 10 µM AA. In addition, this model
accurately predicts the increased burst duration observed during AA
stimulation (Fig. 4). In the simulated model, with 10 µM AA
interacting with both C3 and
C2,
burst increased to 45 ms.
Finally, our results suggest that the on-rates for these two reactions
must be different. Identical on-rates would predict a
concentration-Po
curve exhibiting negative cooperativity, whereas our data were best fit
using a Hill coefficient of 1.4, i.e., slight positive cooperativity.
Thus we conclude that AA interacts with both long-closed states of the
channel to increase the opening rate and hence
Po. Clapham et
al. (48), studying an AA-activated
K+ channel in cardiac tissue,
predicted a similar kinetic scheme, comprising one open and three
closed states. Similar to our findings, the open time of this channel
was not affected by AA (48).
Although we are able to identify which kinetic state of the channel is
being modulated by AA, our results do not allow us to discern whether
AA is acting at the channel protein itself or interacting with a
closely associated protein or with the membrane bilayer. Fatty acids
are known to interact directly with purified protein kinase C (43) as
well as with fatty acid binding proteins. Indeed, a putative fatty acid
binding domain has been identified in the
N-methyl-D-aspartate
receptor (40). Thus a similar direct interaction with
KAA is possible. We demonstrate
also that stretch activates KAA.
Although a kinetic analysis has not been undertaken on the
stretch-dependent activation, it appears qualitatively to be similar to
that induced by AA, i.e., the channel clearly displays bursting
behavior that is consistent with an open state punctuated by fast
closed events (Fig. 8). This, coupled with an increased
Po, suggests that
stretch decreases the long-closed time(s) of the channel. The
KAA described in cardiac tissue is also activated by stretch; in both cases a similar kinetic scheme describes this activation (18). A similar kinetic has previously been
described for stretch-activated channels of chick skeletal muscle (13)
and amphibian proximal tubule (42) as well. In both cases, it was the
interburst interval that was shortened to increase
Po with no effect
on
o or the fast closed time(s) (13, 42). This suggests a similar mechanism of action for these two
modulators or that a common modulator, AA, activates the channel in
both instances.
Effects of K+
channel blockers on KAA.
We evaluated the effects of several known
K+ channel blockers on
KAA activity, including CTX, 4-AP,
293B, TEA, glibenclamide, quinine, and
Ba2+. Of these, only TEA and
Ba2+ blocked the channel from the
extracellular side. However, neither of these blocked with high
affinity. TEA blocked in a voltage-dependent fashion, similar to its
effect on KCa (9). Our results
suggest KAA expresses two binding
sites for Ba2+. From the
cytoplasmic side of the channel,
Ba2+ induced a long-lived,
voltage-dependent closed state (Fig. 6) as previously described (47).
In contrast, application of Ba2+
to the extracellular side of the channel resulted in an apparent reduction in single-channel amplitude that was also voltage dependent (Fig. 7). Recently, Gray and co-workers (45) demonstrated a similar
blocker pharmacology for Ba2+ in
epithelial cells from human vas deferens, i.e., cytoplasmic Ba2+ induced long closed events,
whereas extracellular Ba2+ caused
a voltage-dependent flickery block. Similarly, Hurst et al. (16)
demonstrated that extracellular
Ba2+ induced both long- and
short-lived blocked states in the Shaker K+ channel. In both cases, these
results were interpreted as indicating the channel expressed two
distinct Ba2+ binding sites.
Effect of stretch-activated ion channel blockers on
KAA.
Our results demonstrate that Gd3+,
a known inhibitor of nonselective, stretch-activated cation channels
(49), had no effect on KAA,
suggesting it is unrelated to these channels. Amiloride has also been
shown to inhibit both stretch-activated
K+ channels (44) and nonselective
cation channels (25). We demonstrate that amiloride (2 mM) dramatically
reduced the i of
KAA (Fig. 9). This reduction in
i by amiloride is similar to what has
previously been reported for amiloride block of both the
Xenopus oocyte nonselective cation
channel and Lymnaea neuron
K+ channel (25, 44). Although our
results on the voltage dependence of this block are preliminary, they
suggest that the amiloride-induced reduction in
i is voltage dependent, i.e., the
block is partially relieved at positive voltages (Fig. 9). The
amiloride block of the mechanosensitive channel in
Xenopus oocytes was highly voltage dependent, being completely relieved by positive holding potentials (25), whereas the amiloride block of the
K+ channel of
Lymnaea neurons was voltage
independent (44). Thus our results fall between these two extremes. Our
results with amiloride further support the notion that
KAA and
Kstretch are indeed the same
protein.
The elucidation of the role of KAA
in either transepithelial
Cl
secretion or volume
regulation (see below) will require the identification of high-affinity
blockers that distinguish between
KAA,
KCa, and the cAMP-activated
K+ channel,
KcAMP. Lane et al.
(25) have demonstrated a structure-activity relationship
for the block of the Xenopus oocyte
mechanosensitive channel by amiloride analogs. It will be important to
determine whether high-affinity analogs can be identified for
KAA in T84 cells, thus allowing
its physiological role to be elucidated.
Does AA modulate intestinal Cl
secretion?
Cl
secretion requires the
coordinate regulation of both an apical
Cl
conductance and
basolateral GK.
Minimally, if AA is to modulate Cl
secretion, it must
increase basolateral
GK, thus
hyperpolarizing both membranes and increasing the driving force for
Cl
exit across the apical
membrane through constitutively open
Cl
channels, a paradigm
proposed previously for the mechanism of action of
Ca2+-dependent agonists (4). We
have now characterized a K+
channel that is potently activated by AA,
KAA. Recently, Barrett (5)
demonstrated that the response of T84 cells to VIP and forskolin was
attenuated by a DAG lipase inhibitor and that VIP increased AA. These
results suggest that AA may be an important modulator of
Cl
secretion in intestinal
tissue. Although our results demonstrate that
KAA has a distinct pharmacological
profile from the cAMP-activated GK (it is not
blocked by 293B, an inhibitor of
KcAMP), a selective blocker for
KAA will be required to further
explore its role in this response.
Inflammatory diseases of the intestine, including Crohn's disease and
ulcerative colitis, are characterized by the migration of eosinophils
and neutrophils into the intestinal lumen (15). It is now known that
both of these cell types release AMP, which is converted by an apical
ecto-5'-nucleotidase to produce the secretagogue adenosine (32,
41). Several investigators found no increase in cAMP, cGMP, or
Ca2+ during an adenosine-mediated
Cl
secretory response (6,
12, 32), suggesting a novel signaling pathway. Subsequently, Barrett
and Bigby (6) demonstrated a tight correlation between changes in
cellular AA generation and the resultant
Cl
secretory response to
apical adenosine, suggesting that AA may serve this role.
Ulcerative colitis is also characterized by elevated active kallikrein,
which will release kinin (50). The kinins are known to activate
phospholipase A2 and therefore
increase AA levels (1). Also, the proinflammatory cytokine tumor
necrosis factor-
, which is thought to play a role in the
pathogenesis of inflammatory bowel disease, was recently shown to
potentiate the phospholipase A2-stimulated release of AA (14,
27). Clearly then, the inflamed intestine is characterized by elevated
levels of secretory agonists that likely increase cellular AA levels.
In fact, the inflamed intestine is primed for an exaggerated response;
the AA composition of phospholipids is increased in both Crohn's
disease and ulcerative colitis (37, 39). The oxidative conversion of AA
to the eicosanoids is known to be a key step in the etiology of
inflammatory bowel disease. Because the initial step in this cascade is
the liberation of AA from phospholipids, and AA is a well-known
modulator of ion channels in a variety of tissues (33, 38), including
epithelia (2, 17, 34), it is likely that AA itself may play an
important role in the diarrhea associated with inflammatory bowel
disease. We speculate that KAA is
the GK
responsible for carrying K+
current across the basolateral membrane during an AA-mediated Cl
secretory response.
Does KAA play a role in volume
regulation?
In addition to being activated by AA, we demonstrate that
KAA is similarly activated by
changes in membrane tension. Thus KAA may be activated by the
mechanical stimuli associated with haustral contractions required to
move the colonic contents toward the rectum or with the regulatory
volume decrease associated with cell swelling. The regulatory volume
decrease response of jejunum (30) is associated with activation of a
Ba2+-sensitive
GK. In our
studies, Ba2+ inhibited the
channel while other known K+
channel blockers did not. Both AA and stretch have previously been
shown to activate K+ channels in
smooth muscle (21), neurons (20), and cardiac tissue (18, 19). One
hypothesis that has been proposed to link these two separate modulators
is that the physical deformation of the patch during stretch activates
a phospholipase causing the release of free AA and activation of the
channel (18).
Differential modulation of two
K+ channels in
the T84 cell line.
In our companion paper we characterized the effect of AA on the
basolateral membrane K+ channel
activated by Ca2+-dependent
agonists, KCa, in intestinal
epithelia (10). Although KCa was
potently inhibited by AA (inhibition constant = 425 nM), KAA is potently activated
(Ks = 1.39 µM).
Although KCa was inhibited by
fatty acids in general, the activation of
KAA was very selective for
cis-unsaturated fatty acids, with AA
being more effective than linoleic acid at the same concentration. The
differential modulation of two distinct
K+ channels in the same cells has
previously been described in cardiac myocytes (19) and stomatal guard
cells (26). Thus the role that AA will play in the
Cl
secretory response of
the cell will depend on the preexisting regulatory context in which it
acts.
In conclusion, we have characterized a large-conductance,
Ca2+-independent
K+ channel with a linear
I-V
relationship whose activity is increased by AA, stretch, and membrane
depolarization. We speculate that this
K+ channel is important in the
modulation of Cl
secretion
by agonists employing AA as a second messenger (e.g., VIP, adenosine)
during both physiological and pathophysiological responses. As such,
this channel may represent a novel pharmacological target in the
treatment of diarrheal disease. The development of selective blockers
of this channel (perhaps amiloride analogs) will be required to confirm
its role in the Cl
secretory response, particularly in inflammatory states.
 |
ACKNOWLEDGEMENTS |
We thank Dr. Bruce Schultz for help in data simulation and in
interpreting the kinetic data. We gratefully acknowledge the excellent
technical assistance of Cheng Zhang Shi in tissue-culture work.
 |
FOOTNOTES |
This work was supported by Cystic Fibrosis Foundation Grant DEVOR 96PO
(to D. C. Devor) and National Institute of Diabetes and Digestive and
Kidney Diseases Grant DK-31091 (to R. A. Frizzell).
1
If the closed-time histograms are fit starting
at 0.625 ms (the limit of resolution using a filter cut-off frequency
of 2 kHz), an additional exponential was required to fit the data that averaged 0.21 ± 0.02 ms
(n = 19). Because this is below the
limit of resolution filtering at 2 kHz, the first three bins were
excluded from the fit such that we began binning at 1 ms. Because this is five times the rapid time constant, it will contribute little to the
remaining fit.
Address for reprint requests: D. C. Devor, Dept. of Cell Biology and
Physiology, S312 BST, 3500 Terrace St., University of Pittsburgh,
School of Medicine, Pittsburgh, PA 15261 (E-mail: dd2+{at}pitt.edu).
Received 7 February 1997; accepted in final form 20 September
1997.
 |
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AJP Cell Physiol 274(1):C149-C160
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